372
I myo-Inositol metabolism in Rhizobium leguminosarum JUDITH FRY PH.D. THESIS 2000
I
myo-Inositol metabolism
in Rhizobium leguminosarum
JUDITH FRY PH.D. THESIS 2000
II
ABSTRACT
myo-Inositol is a sugar/polyol that is present in soil (Sulochana, 1962, Wood
and Stanway, 2000), plant tissues and nodules (Skøt and Egsgaard, 1984,
Streeter, 1987, Streeter and Salminen, 1985). Rhizobium leguminosarum
biovar viciae has an inducible pathway for myo-inositol utilisation as the sole
carbon source but bacteroids do not utilise myo-inositol (Poole et al., 1994). In
this study, the pathway of myo-inositol catabolism was further investigated.
Three discrete loci involved in myo-inositol utilisation in R. leguminosarum bv.
viciae were mutated, cloned and characterised. Genes with homology to iolA
and iolD, which encode proteins involved in the final steps of myo-inositol
degradation in Bacillus subtilis (Yoshida et al, 1997) and a putative transport
system for myo-inositol were identified. The catabolic mutants RU360 (iolD)
and RU361 (iolA) were not able to induce the first two enzymes in the
proposed catabolic pathway for myo-inositol, which suggests end-product
induction of the pathway. The pathway is subject to catabolite repression by a
range of carbon compounds.
The mutants nodulated peas (Pisum sativum) and vetch (Vicia sativa) at the
same rate as the wild type. Acetylene reduction rates and overall plant dry
weights were also the same. When the wild type, 3841, and the catabolic
mutants, RU360 and RU361, were co-inoculated onto plants, nodules were
predominantly infected with the wild type. However, a transport mutant
(RU307) was not at a competitive disadvantage. Therefore, the ability to
III
utilise myo-inositol is essential for competitive ability of the wild type, but the
advantage of the wild type is unlikely to be due to myo-inositol being used
purely as a carbon source. Instead, myo-inositol might be involved in
signalling between host plants and rhizobia or myo-inositol catabolism might
be important in the early steps of the developing infection process.
IV
ACKNOWLEDGEMENTS
I would like to thank my supervisors Dr Philip Poole and Dr Martin Wood for
their guidance and assistance throughout my PhD.
I am indebted to Mrs Mary Leonard and Mrs Kim Carter for their invaluable
support, both in and out of the laboratory. I should also like to thank Dr David
Allaway and the people of Lab 160 for their help. Thank you also to the
people of the Central Services Section and the electrical workshop in the
School of Animal and Microbial Sciences for technical assistance.
This work would not have been possible without the University of Reading
studentship awarded to me, for which I am very grateful.
Finally, a big thank you to Mazda, my parents and friends for their unwavering
support and encouragement.
V
abbreviations
3-O-MSI L-3-O-methyl-scyllo-inosamine
ADP adenosine diphosphate
AMA/AMS acid minimal agar/acid minimal salts
ATP adenosine triphosphate
bp base pairs
cfu colony forming units
CoA coenzyme A
CO2 carbon dioxide
DNA deoxyribonucleic acid
GFP green fluorescent protein
HPLC high performance liquid chromatography
kb kilobase pairs
LCO lipo-chito-oligosaccharide
NAD nicotinamide adenine dinucleotide
N/T not tested
ORF open reading frame
PBM peribacteroid membrane
PCR polymerase chain reaction
PHB polyhydroxybutyrate
SD Shine-Dalgarno
SI scyllo-inosamine
TY tryptone yeast medium
VI
Contents
CHAPTER 1 - INTRODUCTION....................................................................... 1
1.1 Introduction .............................................................................................. 2
1.2 Rhizobium and Symbiosis ...................................................................... 2
1.2.1 Nodulation ......................................................................................................4
1.2.2 Nitrogen Fixation by Bacteroids .....................................................................9
1.2.3 Carbon Metabolism by the Bacteroid ...........................................................11
1.2 Soil Carbon............................................................................................. 14
1.3 myo-Inositol............................................................................................ 15
1.3.1 Synthesis of myo-Inositol .............................................................................16
1.3.2 myo-Inositol Utilisation by Bacteria ..............................................................17
1.3.2.1 myo-Inositol Uptake by Bacteria............................................................21
1.3.3 myo-Inositol in the Environment...................................................................22
1.3.3.1 Plant myo-Inositol..................................................................................22
1.3.3.2 Soil myo-Inositol ....................................................................................25
1.3.4 myo-Inositol Derivatives ................................................................................27
1.3.4.1 Auxins....................................................................................................28
1.3.4.2 Methyl Esters of myo-Inositol ................................................................28
1.3.4.3 Di-myo-inositol 1,1’-phosphate..............................................................30
1.3.4.4 Phosphatidylinositol...............................................................................30
1.3.4.5 myo-Inositol Hexaphosphate.................................................................31
1.3.4.5.1 Phytase ..............................................................................................33
1.3.6 Rhizopines ....................................................................................................34
VII
1.3.6.1 Rhizopine Synthetic and Catabolic Genes of S. meliloti ...........................36
1.3.6.2 Agrobacterium Opines...........................................................................38
1.4 Rhizosphere Growth and Competition for Nodulation ...................... 40
1.4.1 Competition for Rhizosphere Colonisation...................................................40
1.4.2 Competition for Nodulation...........................................................................42
1.4.2.1 Utilisation of Secondary Metabolites .....................................................45
1.4.3 Improving Nodulation Competitiveness........................................................48
1.5 Research Objectives ............................................................................. 50
CHAPTER 2 – MATERIALS AND METHODS .............................................. 52
2.1 Bacterial Strains and Plasmids ............................................................ 53
2.2 Culture Conditions ................................................................................ 56
2.2.1 Calculation of Mean Generation Time..........................................................56
2.3 DNA and Genetic Manipulations .......................................................... 57
2.3.1 PCR Amplification ........................................................................................58
2.3.2 DNA Sequencing..........................................................................................59
2.4 Analysis of Sequence Data................................................................... 61
2.5 Statistical Analysis of Data................................................................... 61
2.6 Transport Assay..................................................................................... 62
2.7 Enzyme Assays...................................................................................... 62
2.7.1 myo-Inositol Enzyme Assay .........................................................................62
2.7.2 β-Galactosidase Assay ................................................................................63
2.7.3 Alkaline Phosphatase Assays ......................................................................64
VIII
2.8 GFP-UV Assay........................................................................................ 64
2.9 Plant Assays........................................................................................... 66
2.9.1 Seed Sterilisation .........................................................................................66
2.9.2 Nodulation Competition................................................................................66
2.9.3 Nodule Harvesting........................................................................................68
2.9.4 Acetylene Reduction ....................................................................................68
2.9.5 Plant Dry Weight ..........................................................................................68
2.9.6 Nodule Mass ................................................................................................69
2.9.7 Rhizosphere Growth ....................................................................................69
CHAPTER 3 - MYO-INOSITOL CATABOLIC MUTANTS OF RHIZOBIUM
LEGUMINOSARUM BIOVAR VICIAE ........................................................... 70
3.1 Introduction ............................................................................................ 71
3.2.1 Growth Characteristics of the myo-Inositol Mutants.................................... 72
3.2.2 Cosmids Carrying myo-Inositol Catabolic Genes........................................ 76
3.2.3 Southern Hybridisation................................................................................ 79
3.2.4 Transduction of RU307 ............................................................................... 85
3.2.5 Sequencing of myo-Inositol Catabolic Genes ............................................. 86
3.2.5.1 RU360 .................................................................................................. 86
3.2.5.1.1 Complementation of the iol region of RU360 ...................................... 102
3.2.5.2 RU361 ................................................................................................ 104
3.2.3.3 RU307 ................................................................................................ 119
3.2.5 Uptake of Glucose and myo-Inositol ......................................................... 125
3.3 Discussion............................................................................................ 133
IX
CHAPTER 4 - NODULATION AND RHIZOSPHERE GROWTH ........ ERROR!
BOOKMARK NOT DEFINED.
4.1 Introduction ..................................................Error! Bookmark not defined.
4.2 Results ..........................................................Error! Bookmark not defined.
4.2.1. Rate of Nodulation ....................................... Error! Bookmark not defined.
4.2.1.1 Dry Weight of Vetch Plants .................... Error! Bookmark not defined.
4.2.2 Acetylene Reduction Assay on Pea Nodules Error! Bookmark not defined.
4.2.3 Plant Dry Weights for Pea............................. Error! Bookmark not defined.
4.2.4 Nodule Number and Mass for Pea................ Error! Bookmark not defined.
4.2.5 Competition for Nodulation............................. Error! Bookmark not defined.
4.2.5.1 Competitive Ability of the Complemented Mutants Error! Bookmark not
defined.
4.2.6 Measurement of Nodule Co-Residence ........ Error! Bookmark not defined.
4.2.7 Average Number of Nodules per Vetch Plant Error! Bookmark not defined.
4.2.9 Rhizosphere Growth Assay....................... Error! Bookmark not defined.
4.2.10 Analysis of a nodC-phoA Fusion................. Error! Bookmark not defined.
4.3 Discussion.....................................................Error! Bookmark not defined.
CHAPTER 5 - REGULATION OF MYO-INOSITOL CATABOLISM ... ERROR!
BOOKMARK NOT DEFINED.
5.1 Introduction ..................................................Error! Bookmark not defined.
5.2 Results ..........................................................Error! Bookmark not defined.
5.2.1 Isolation of a myo-inositol Specific Promoter Error! Bookmark not defined.
X
5.2.2 Sequence Analysis of the myo-Inositol Inducible Promoter Error! Bookmark
not defined.
5.2.3 Induction of the myo-Inositol Inducible Promoter ......... Error! Bookmark not
defined.
5.2.4 Catabolite Repression................................... Error! Bookmark not defined.
5.2.5 Relationship to myo-Inositol Mutants ............ Error! Bookmark not defined.
5.2.6 Expression in the Rhizosphere and Nodules Error! Bookmark not defined.
5.2.7 Stability of pRU601 in the Rhizosphere ........ Error! Bookmark not defined.
5.2.8 Construction of a Specific myo-Inositol Inducible GFP-UV Promoter Probe
................................................................................ Error! Bookmark not defined.
5.2.9 RU360 β-Galactosidase Assay ..................... Error! Bookmark not defined.
5.2.10 myo-Inositol Catabolic Enzyme Assays ...... Error! Bookmark not defined.
5.3 Discussion.....................................................Error! Bookmark not defined.
CHAPTER 6 - FINAL DISCUSSION ................................................................. 249
6.1 Conclusions .............................................................................................. 250
6.2 Future Work............................................................................................... 256
BIBLIOGRAPHY ............................................................................................... 260
APPENDIX………………………………………………………………………...316
1
Chapter 1 - Introduction
2
1.1 Introduction
There has been extensive study of the Rhizobium-legume symbiosis and
many of the rhizobial genes required for nodulation and nitrogen fixation have
been elucidated. However, the circumstances enabling growth and survival of
free-living Rhizobium in soil and the rhizosphere are still something of an
enigma. Identifying molecules that have effects on bacterial growth in the
rhizosphere and elucidating the genes that are involved in responding to
these factors is essential to understanding how communities of micro-
organisms develop in the rhizosphere. This could also lead to a greater
understanding of the mechanisms of the Rhizobium-plant symbiosis and the
relationship between the two distinct growth states. Ultimately, this may lead
to the construction of strains that increase yields of agricultural crops.
1.2 Rhizobium and Symbiosis
Rhizobia are Gram negative, motile, aerobic bacteria that are capable of fixing
atmospheric nitrogen. They are ubiquitous in the soil where they live
saprotrophically, but they can also live in association with leguminous plants
(family Leguminosae) (Jordan, 1984) and some members of the genus
Parasponia (family Ulmaceae) (Trinick, 1988). Several of the specific
rhizobial-plant symbioses are listed in Table 1.1.
3
Table 1.1 Some Rhizobium Species and Their Plant Hosts
Rhizobium species plant host
species name
common name
Bradyrhizobium japonicum Glycine max soybean
R. leguminosarum bv. phaseoli Phaseolus vulgaris common bean
R. etli Phaseolus vulgaris common bean
R. leguminosarum bv. trifolii Trifolium species clover
R. leguminosarum bv. viciae Pisum sativum
Vicia sativa
pea
vetch
Mesorhizobium loti Lotus species
Sinorhizobium melilotil Medicago sativa alfalfa
Rhizobium NGR234 most legume genera
and Parasponia
van Rhijn and Vanderleyden, 1995.
Biological processes account for approximately 60% of the biosphere’s fixed
nitrogen. It is difficult to quantify the amount of nitrogen fixed globally by
rhizobia, but legume symbioses are estimated to contribute at least 70 million
tonnes of fixed nitrogen per annum (Brockwell et al., 1995). This represents
approximately 50% of the total nitrogen that enters terrestrial ecosystems from
the process of biological nitrogen fixation (Tate, 1995). The association is of
major agronomic importance as many food crops can be grown without
requiring nitrogenous fertiliser to give economically viable yields (reviewed in
Zahran, 1999).
4
1.2.1 Nodulation
As stated in Section 1.1, nodulation and nitrogen fixation have been
extensively studied. These topics are not the subject of this thesis, so instead
of an exhaustive review, an overview follows (detailed reviews include Poole
and Allaway, 2000, van Rhijn and Vanderleyden, 1995, Rosendahl et al.,
1991, Schultze and Kondorosi, 1998, Whitehead and Day, 1997).
Rhizobium-legume interactions begin with the exchange of signals between
the plant and microbe. Plants release signalling molecules such as flavonoids
and chalcone into the rhizosphere. There are several classes of flavonoids,
including flavones, flavonols, flavanones and isoflavones, which are produced
from the phenylpropanoid pathway of plants (van der Meer et al., 1993). The
compounds secreted differ according to the plant species. Some are even
released by non-legumes. Many flavonoids are constantly released by plants,
but when inoculated with a rhizobial symbiont, the composition and quantities
often change (Dakora et al., 1993, Lawson et al., 1996, Recourt et al., 1991,
Schmidt et al., 1994, Schmidt et al., 1991).
Flavonoids act as chemoattractants (Aguilar et al., 1988, Caetano-Anollés et
al., 1992, Kape et al., 1991) and in conjunction with the transcriptional
activator protein NodD, they induce expression of nodulation (nod) genes in
Rhizobium (Schlaman et al., 1992). The effects of flavonoids are both species
and strain specific. For example, the flavone apigenin is able to induce the
NodD proteins of many species, whereas the isoflavone daidzein is only a
5
strong activator of NodD B. japonicum and Rhizobium NGR234 (van Rhijn
and Vanderleyden, 1995). Some flavonoids can even inhibit nod gene
activation by inhibiting inducers (Djordjevic et al., 1987, Firmin et al., 1986,
Kosslak et al., 1990, Peters and Long, 1988).
Transcriptional activation of nod genes occurs when NodD binds to nod
boxes. These are conserved DNA sequences upstream of the nod operon
(Rostas et al., 1986). The NodD proteins belong to the LysR family of
transcriptional activators, which means that they share common features such
as a characteristic helix-turn-helix motif (Goethals et al., 1990, Henikoff et al.,
1988). They are highly conserved between rhizobial species, although the
numbers of copies of the nodD gene varies between species. NodD is one of
the factors responsible for host specificity, as mutations in nodD cannot
usually be complemented by a nodD gene from another species (Horvath et
al., 1987, Spaink et al., 1987). The NodD proteins of narrow host range
rhizobia such as S. meliloti, R. leguminosarum and R. trifolii only respond to a
few flavonoids, whereas that of the broad host range Rhizobium NGR234
responds to most flavonoids (Györgypal et al., 1991).
The products of nod genes, Nod-factors, are lipo-chito-oligosaccharides
(LCOs), which stimulate root hair curling and deformation, through which
infection occurs in most legumes (Bhuvaneswari and Solheim, 1985, van
Brussel et al., 1986, Yao and Vincent, 1969). In addition to nodulation genes,
there is another class of rhizobial genes involved in infection and nodulation.
These encode compounds such as exopolysaccharides (EPS), which are
6
necessary for proper formation of infection threads and root hair curling on
plants that form indeterminate nodules, such as vetch (V. sativa) (Leigh et al.,
1985, Rolfe et al., 1996, van Workum et al., 1998). It is thought that EPS
either inhibit plant defence mechanisms that prevent damage of plant root hair
cells or that they accelerate plant hair curling and infection processes so that
root penetration by rhizobia precedes the plant defence response (van
Workum et al., 1998).
After root hair deformation has occurred, plant root hair walls are degraded by
hydrolysis, rhizobia enter the roots (Newcombe et al., 1979, Turgeon and
Bauer, 1982) and an infection thread is formed. The thread is comprised of
plant cell wall material, which surrounds the rhizobia within. (Callaham and
Torrey, 1981, Turgeon and Bauer, 1985). Plant cortical cells divide to form
nodule primordia, towards which the infection threads grow (Libbenga et al.,
1973, Newcombe, 1981, Vasse and Truchet, 1984, Wood and Newcombe,
1989). Specialised structures called nodules are formed, whereupon rhizobia
are released from the infection threads and differentiate into bacteroids. The
peribacteroid membrane (PBM), which is derived from plant material,
encloses the bacteroids (Brewin et al., 1985, Mellor and Werner, 1987
Robertson et al., 1978, Verma et al., 1978). The PBM is thought to have a
vital role in regulating nutrient flow between the plant and the bacteroids and
may also help maintain low oxygen conditions (reviewed in Rosendahl et al.,
1991, Whitehead and Day, 1997).
7
Depending on the plant species, there are two major types of nodule
development. Temperate legumes such as alfalfa, pea and vetch form
indeterminate nodules. These nodules maintain an apical meristem and are
elongated in shape. Most tropical legumes such as soybean form determinate
nodules, which have a transient meristem and are round in shape
(Newcombe, 1986, Newcombe, 1981, Newcombe et al., 1979, Turgeon and
Bauer, 1985).
The nod and nitrogen fixation (nif, fix) genes are mostly clustered on symbiotic
(Sym) plasmids in Rhizobium and Sinorhizobium (Martinez et al., 1990) but
are chromosomal in Azorhizobium (Goethals et al., 1989), Bradyrhizobium,
and M. loti (Chua et al., 1985, Pankhurst et al., 1983, Sullivan et al., 1995).
The nodulation of legumes is species and strain specific according to the Nod-
factors produced. The nodABC genes are found in all rhizobia and are known
as the common nod genes as they are interchangeable between species
(Kondorosi et al., 1984). The three proteins NodA, NodB and NodC are
responsible for synthesis of the LCO backbone of Nod factors. NodC protein
functions as a N-acetylglucosaminyltransferase, causing polymerisation of N-
acetylglucosamine units to form the backbone (Geremia et al., 1994, Spaink
et al., 1994). NodB is a chitooligosaccharide deacetylase that specifically
removes the acetyl group at the non-reducing end of N-acetylglucosamine
(John et al., 1993) and NodA transfers the acyl chain to the non-reducing end
(Atkinson et al., 1994, Röhrig et al., 1994).
8
The nodIJ genes are also considered to be common nod genes as they are
found in many rhizobial species, including R. leguminosarum bv. viciae, bv.
trifolii, R. etli and S. meliloti. NodI and NodJ are involved in transport of Nod
factors and are thought to be an ATP-binding cassette (ABC) transporter
(Cardenas et al., 1996, Evans and Downie, 1986, Fernandez-Lopez et al.,
1996, McKay and Djordjevic, 1993, Spaink et al., 1995, Vàzquez et al., 1993).
The organisation of the nod genes of R. leguminosarum bv. viciae is shown in
Figure 1.1. The organisation of nod genes differs between different species,
although nodDABCIJ are often clustered into one organisational unit.
Figure 1.1 The nod Genes of R. leguminosarum bv. viciae
Adapted from van Rhijn and Vanderleyden, 1995.
The arrows indicate the direction in which the nod genes are transcribed. The
common nod genes are dark blue and host-specific genes are purple. The asterisks
indicate the position of nod boxes, which NodD binds to.
Host-specific nod-factors (hsn) are subtly different in each strain and are also
responsible for host specificity. For example, the main factor that determines
host specificity in R. leguminosarum bv. viciae and bv. trifolii is NodE (Spaink
et al., 1991, Spaink et al., 1989). In S. meliloti, nodH and nodPQ are
responsible for specifying nodulation of alfalfa (Cervantes et al., 1989,
nodO nodT nodN nodM nodL nodE nodF nodD nodA nodB nodC nodI nodJ
* ***
9
Faucher et al., 1989, Roche et al., 1991, Schwedock and Long, 1989). In B.
japonicum, nodVW, which are unique to this species, are required for
nodulation of Macroptilium and Vigna species (Göttfert et al., 1990).
1.2.2 Nitrogen Fixation by Bacteroids
Inside bacteroids most nod genes are no longer expressed (Schlaman et al.,
1991), possibly because large quantities of Nod factors can elicit plant
defence reactions (Savouré et al., 1997) and bacteroids express nitrogen
fixing genes (nif and fix). These genes are responsible for the reduction of
atmospheric nitrogen to ammonium. The nif genes are structurally
homologous to those of Klebsiella pneumoniae, a free-living bacterium in
which nitrogen fixation has been extensively studied (Arnold et al., 1988). The
fix genes are essential for nitrogen fixation, but do not have homologues in K.
pneumoniae. These genes are usually organised into distinct clusters in
rhizobia and have been studied most in S. meliloti and B. japonicum.
The two-component enzyme complex responsible for the process of nitrogen
fixation is nitrogenase, which is encoded for by nifHDK. The α and β subunits
of Component I (MoFe protein) are encoded for by nifD and nifK respectively.
Component II (Fe protein) is encoded for by nifH. This enzyme is large and
slow acting and may account for up to 30% of bacteroid cell protein (Haaker
and Klugkist, 1987). The nifB, nifE and nifN genes are involved in the
biosynthesis of a co-factor of component I (Dean et al., 1993). The nifH, nifM,
10
nifQ and nifV genes are also required for synthesis and maturation of the
active enzyme complex (Filler et al., 1986, Howard et al., 1986, Imperial et al.,
1984, Kennedy et al., 1986, Robinson et al., 1987).
High oxygen levels inactivate nitrogenase (Adams and Chelm, 1988, Hill,
1988) and expression of nifA is also dependent on low oxygen concentration
(Ditta et al., 1987). NifA is a positive regulator of nif and fix genes and also
activates other genes that are not involved in nitrogen fixation, but the
functions are not known (Nienaber et al., 2000). The fixL and fixJ gene
products sense and transduce the low oxygen signal. FixJ then activates
transcription of nifA and fixK (Cebolla and Palomares, 1994). FixK is a
positive regulator of fixNOPQ, which encode the high affinity membrane-
bound cytochrome oxidase that supports bacteroid respiration in the micro-
aerobic conditions of the nodule (Preisig et al., 1993). Several other fix
genes, fixABCX and fixGHIS are also required for nitrogen fixation to occur in
rhizobia, as mutants in these genes abolish the process (reviewed in Fisher,
1994).
There are also plant genes that are expressed specifically in root tissue as a
result of the interaction with rhizobia. The plant-derived products are called
nodulins. One nodulin is leghaemoglobin, which is transcribed in nodules
(Fuller et al., 1983). The pigment in leghaemoglobin is responsible for
nodules appearing pink. Leghaemoglobin maintains a high oxygen flux, but at
a concentration about 104 to 105 times lower than in aerobic cultures, so that
11
nitrogen fixation is not inactivated and bacteroid respiration can still occur
(Appleby, 1984, Hunt and Layzell, 1993).
The process of nitrogen fixation is highly energetic. The minimum
stoichiometric requirements are eight electrons and 16 molecules of ATP
hydrolysed per N2 molecule fixed. Therefore, in addition to providing an
environment free from oxidative stress, the plant also provides carbon and
water so that the bacteroids can generate ATP and reductant for nitrogen
fixation. In return, the bacteroids channel fixed nitrogen to the plant. Until
recently, it was generally accepted that ammonium was the only product
channelled to the plant. However, evidence has emerged that bacteroids
provide alanine, in addition to ammonium. Ammonium is converted to alanine
by the enzyme alanine dehyrogenase (AldA) (Allaway et al., 2000, Kretovich
et al., 1986, Rosendahl et al., 1992, Waters et al., 1998, reviewed in Poole
and Allaway, 2000). As the fixed nitrogen is supplied to the host plant, rather
than being incorporated into compounds in the bacteroid, nitrogen fixation in
the nodule is not generally accompanied by growth of bacteroids.
1.2.3 Carbon Metabolism by the Bacteroid
Free-living rhizobia are capable of utilising a wide range of carbon compounds
for growth. However, the principal carbon compounds utilised by mature
bacteroids are C4-dicarboxylic acids, such as malate, succinate and fumarate,
which are provided by the host plant. Mutants defective in C4-dicarboxylic
12
acid uptake do not fix nitrogen, although they are able to form nodules (Arwas
et al., 1986, Arwas et al., 1985, El-Din, 1992, Engelke et al., 1987, Finan et
al., 1988, Finan et al., 1983, Humbeck and Werner, 1989, Lafontaine et al.,
1989b, Ronson et al., 1981). The C4-dicarboxylic acids are metabolised via
the tricarboxylic acid cycle (TCA) and subsequently via malic enzyme and
pyruvate dehydrogenase (Copeland et al., 1989, McKay et al., 1988).
Sucrose is the major carbohydrate translocated in legumes from shoots to
nodules (Antoniw and Sprent, 1978, Kouchi and Yoneyama, 1984, Reibach
and Streeter, 1983). Other carbon compounds present in nodules in addition
to organic acids include glucose, fructose, maltose, trehalose and cyclitols
(Kouchi and Yoneyama, 1984, Lafontaine et al., 1989a, Streeter, 1987, see
Section 1.3.3.1). However, sugars are catabolised at very low levels in
soybean and pea bacteroids (Glenn and Dilworth, 1981, Salminen and
Streeter, 1987). Furthermore, pea and clover bacteroids with mutations in
sugar catabolic enzymes are unaltered in symbiotic performance (Glenn et al.,
1984, Ronson and Primrose, 1979).
As free-living cultures of rhizobia, including R. leguminosarum bv. viciae,
utilise sugars, this indicates that there are fundamental differences in transport
and metabolism in bacteroids. One reason why C4-dicarboxylic acids are the
principal energy source of bacteroids may be because they are more readily
transported across the PBM than other carbon compounds such as sugars
(McKay et al., 1985, Saroso et al., 1986, Udvardi et al., 1988).
13
Carbon compounds in nodules that are not catabolised may perform other
roles. Such compounds include D-pinitol and proline (Ford, 1984, Keller and
Ludlow, 1993, Straub et al., 1996), which might function as osmoprotectants
in plants in times of drought stress (Section1.3.4.2).
14
1.2 Soil Carbon
In order for nodulation to occur, rhizobia must first be able to grow and survive
in the rhizosphere. Organic carbon, in the form of carbohydrates and organic
acids, provides energy for growth and survival for non-photosynthesising
micro-organisms. Carbohydrates comprise approximately 10% of soluble soil
organic carbon content (Cheshire, 1985). Most is in the form of
polysaccharides, with small amounts of monosaccharides and
oligosaccharides also present (Cheshire, 1979). The origin of these
compounds is dead plant tissue, root exudates and micro-organisms
(Cheshire, 1985). Composition of root exudates varies with biotic factors such
as species, age and nutrient status and abiotic factors such as temperature,
soil structure, pesticides and water (Rovira, 1965).
Leakage of carbon compounds from plant roots varies with both the species
and growth stage. Smith (1970) found that mature trees release different
amounts and types of compounds respective to seedlings of the same
species. It is worth noting that seedlings are the focus of most studies. The
exudates may also differ in composition and patterns of availability along a
root according to the age of the roots. For example, Jaeger et al. (1999)
found that tryptophan was released from older sections and sucrose from
younger sections of grass (Avena barbata).
The amount of exudate, root cap and mucilage available to micro-organisms
has been estimated at between 3 to 15% of the dry weight of the root
15
(Campbell and Greaves, 1990). Most reviewers conclude that around 20% of
fixed photosynthate is lost to the rhizosphere, a process known as
rhizodeposition (Campbell and Greaves, 1990, Metting, 1985, Sparling, 1985).
Much of this loss is thought to be through passive leakage and from damaged
regions of the plant (Klein et al., 1990, Whipps, 1990). The actual amount
available to micro-organisms is unknown as plants may reclaim some of the
exuded carbon by active uptake against a concentration gradient (Jones and
Darrah, 1996).
1.3 myo-Inositol
One of the many soluble sugar compounds that Rhizobium can utilise as a
sole carbon source is the cyclitol myo-inositol. This compound is abundant in
soil and the rhizosphere (c.f. Section 1.5). It has been shown that in soil
solution, Rhizobium numbers increase concomitantly with the disappearance
of myo-inositol (Wood and Stanway, 2000). Raggio et al. (1959) found that
supplementation of the growth medium of common bean (P. vulgaris) with
mesoinositol (myo-inositol) resulted in an increase in both the percentage of
nodulated roots and nodules per root containing R. leguminosarum bv.
phaseoli. The growth medium of the plants contained sucrose, so the
increase may not have simply been due to myo-inositol being utilised as a
carbon source. These data imply that myo-inositol may be involved in the
growth and survival of Rhizobium in soil and the rhizosphere.
16
myo-Inositol is a sugar alcohol with a six carbon ring structure. There are nine
stereoisomeric forms of inositol (L-chiro, D-chiro, myo, neo, scyllo, muco, cis,
epi, allo) but the myo- form appears to be by far the most common (Figure
1.2). The other forms most commonly encountered in nature are D-chiro, L-
chiro and scyllo-inositol (Loewus, 1990).
Figure 1.2 The Structural Formula of myo-Inositol
1.3.1 Synthesis of myo-Inositol
myo-Inositol de novo synthesis occurs by conversion from glucose-6-
phosphate in two steps, which are catalysed by L-myo-inositol 1-phosphate
synthase and inositol monophosphatase (Loewus, 1990). Control of synthesis
is through feedback inhibition of the synthase (Nelson et al., 1998) (Figure
1.3). The pathway was originally elucidated in yeast, but is believed to be the
same for all organisms (Loewus, 1990).
OH
H
OH OH
OH
OH
OH
HO
H
H
H
H
17
Figure 1.3 Synthesis of myo-Inositol
glucose-6-phosphate
L-myo-inositol 1-phosphate synthase
myo-inositol-1-phosphate
inositol monophosphatase
myo-inositol
1.3.2 myo-Inositol Utilisation by Bacteria
A pathway detailing the degradation of myo-inositol via 2-keto-myo-inositol
and D-2,3-diketo-4-deoxy epi-inositol, has been elucidated in the soil-dwelling
bacterium, Klebsiella aerogenes (Aerobacter aerogenes) (Anderson and
Magasanik, 1971). The final products were proposed to be dihydroxyacetone
phosphate, acetyl coenzyme A (CoA) and carbon dioxide (CO2).
It was postulated that the catabolic pathway is similar for R. leguminosarum
bv. viciae, as the first two enzymes in the pathway, myo-inositol
dehydrogenase and 2-keto-myo-inositol dehydratase have been shown to be
induced in the presence of myo-inositol (Poole et al., 1994). As a result of
findings in this project, additional steps have been added to the pathway. In
these steps, dihydroxyacetone phosphate forms pyruvate, which then
combines with pyruvate to form acetolactate (Figure 1.4).
18
Figure 1.4 Proposed Pathway of myo-Inositol Degradation in R.
leguminosarum bv. viciae
myo-inositol
myo-inositol dehydrogenase
2-keto-myo-inositol
2-keto-inositol dehydratase
D-2,3-diketo-4-deoxy-epi-inositol
*
2-deoxy-5-keto-D-gluconic acid
DKH kinase
diketohydroxy 6-phosphate
DKHP aldolase
malonic semialdehyde dihydroxyacetone phosphate
acetyl CoA + CO2
pyruvate
acetolactate synthase
acetolactate + CO2
The proposed pathway of myo-inositol catabolism is adapted from that of K.
aerogenes, B. subtilis and work in this thesis. Putative functions of Iol proteins from
B. subtilis are shown.
* The pathway of conversion of D-2,3-diketo-4-deoxy-epi-inositol to 2-deoxy-5-keto-
D-gluconic acid has not been characterised. Conversion may occur spontaneously,
or through the action of a hydratase.
MSA oxidative decarboxylase
NAD
NADH + H+
NAD
NADH + H+
H2O
H2O
CoA
ATP
ADP
TCA cycle
ADP
ATP
NAD
NADH + H+
IolA
IolD
IolC
IolG
IolJ
19
Pseudomonas species can also utilise myo-inositol as the sole carbon source
(Deshusses and Reber, 1972), as can Bacillus subtilis. An operon of
catabolic genes for the degradation of myo-inositol has been identified in B.
subtilis (Fujita and Fujita, 1983, Yoshida et al., 1994) (Figure1.5). The
proposed pathway of degradation corresponds to that of K. aerogenes
(Yoshida et al., 1997).
Figure 1.5 iol Operon of B. subtilis
Adapted from Yoshida et al., 1997.
The iol operon of B. subtilis is an 11.5kb region that contains 10 genes iolA-J
which are believed to be transcribed from the iol promoter. All 10 iol genes
are involved in myo-inositol catabolism, as disruption of each one individually
eliminated the ability to degrade myo-inositol. The iolR and iolS genes are
thought to be co-transcribed from the iolRS promoter (Yoshida et al., 1997).
The proposed functions of the iol genes are presented in Table 1.2.
iolS iolR iolA iolB iolC iolD iolE iolF iolG iolH iolI iolJ
iol promoter iolRS promoter
terminator terminator
20
Table 1.2 The iol Genes of B. subtilis
gene homologous proteins from
Genbank database
expected function of gene
iolA methylmalonic semialdehyde
dehydrogenase
methylmalonic semialdehyde
oxidative decarboxylase
iolB no significant homology to known
proteins
no known function
iolC gluconokinase gluconokinase
iolD acetolactate synthase no known function
iolE MocC protein of S. meliloti no known function
iolF proline/betaine or dicarboxylic acid
transporter
myo-inositol transporter
iolG MocA protein of S. meliloti myo-inositol 2-dehydrogenase
iolH no significant homology to known
proteins
no known function
iolI no significant homology to known
proteins
no known function
iolJ aldolase aldolase
iolR glucitol operon repressor operon repressor
iolS MocA protein of Agrobacterium
tumefaciens
no known function
Compiled from the results of Yoshida et al., (1997).
B. subtilis is the only bacterium to date that has been shown to possess an
operon of myo-inositol degrading genes. The genes responsible for the
degradation of myo-inositol may not be organised into an operon in rhizobial
species and these genes may be found both in the chromosome and on
plasmids. In S. meliloti, a gene encoding myo-inositol dehydrogenase was
identified that was not part of an operon of myo-inositol utilising genes
21
(Galbraith et al., 1998). Curing R. leguminosarum bv. trifolii of plasmids
resulted in the loss of the ability to utilise myo-inositol (Baldani et al., 1992).
1.3.2.1 myo-Inositol Uptake by Bacteria
myo-Inositol enters the cell through dedicated transport systems in K.
aerogenes. The system is bi-directional and can also transport scyllo-inositol,
which is not metabolised (Deshusses and Reber, 1977b). Attempts to identify
an active binding protein for myo- or scyllo-inositol were unsuccessful
(Deshussses and Reber, 1977a). In contrast, Reber et al. (1977) reported
that there are two uptake systems in Pseudomonas putida, a high affinity
system involving a periplasmic binding protein and a different, low affinity
system.
Mutation of a myo-inositol transport transport system in Pseudomonas sp.
JD34 caused very slow growth when myo-inositol was the sole carbon source
(Frey et al., 1983). The transport genes were mapped to a chromosomal
11.5kb region, but were not sequenced (Gauchat-Feiss et al., 1985). In
another Pseudomonas species myo-inositol was transported by a high affinity
transport system involving a 30kD myo-inositol-binding protein (Deshusses
and Belet, 1984). The protein was purified and sequencing of the N-terminal
segment revealed that it highly resembles the galactose binding protein of E.
coli (Mahoney et al., 1981).
22
1.3.3 myo-Inositol in the Environment
1.3.3.1 Plant myo-Inositol
myo-Inositol is ubiquitous in the environment. It has been shown to
accumulate in plant leaves exposed to air pollution, possibly due to membrane
disintegration (Bücker and Guderian, 1994). Analysis of several foodstuffs
such as oats (Avena sativa L.), soybean and barley (Hordeum vulgare L.)
revealed free inositol (Norris and Darbre, 1956).
Sulochana (1962) reported the presence of inositol in soil and root exudates
of cotton, along with many other compounds. Wood and Stanway, (2000)
identified inositol in soybean exudates, although it was not the most abundant
compound. However, a study of sugars in rice straw (Oryza sativa L.) and
straw compost by high resolution gas chromatography revealed many sugars,
predominantly xylose, arabinose and glucose, but no myo-inositol (Sugahara
et al., 1992). Sánchez-Mata et al. (1998) carried out high performance liquid
chromatography (HPLC) analysis of legume carbon compounds and found
that ciceritol, an inositol digalactoside was the principal sugar in chickpeas
(Cicer arietinum L.). It was also present in lentils (Lens sculenta L.).
Sucrose is a major constituent of nodules, along with other sugars including
glucose and fructose (Antoniw and Sprent, 1978, Kouchi and Yoneyama,
1984, Reibach and Streeter, 1983). However, there are several other carbon
23
compounds found in nodules and plant roots, including myo-inositol and other
cyclitols. myo-Inositol was the most abundant compound found in bacteroids
of B. japonicum on soybean (Skøt and Egsgaard, 1984, Streeter, 1987).
Streeter and Salminen (1985) reported that myo-inositol had been found in
nodules formed on several different legumes including soybean, clover, pea,
alfalfa and common bean. D-pinitol (1-D-4-O-methyl-myo-inositol, D-chiro-
inositol, oonitol and O-methyl-scyllo-inositol were also present in some
nodules, with the largest amounts of cyclitols found in nodules on roots of
soybean and clover. Only trace amounts of myo-inositol were found in
nodules formed on pea and in contrast to other legumes, common bean only
contains trace amounts of myo-inositol and no other cyclitols (Lafontaine et
al., 1989a, Streeter and Salminen, 1985).
Davis and Nordin (1983) reported that D-pinitol was the most abundant
compound in clover. D-pinitol, oonitol and chiro-inositol were also reported in
soybean cytosol and bacteroids of B. japonicum (Kouchi and Yoneyama,
1986). Kouchi and Yoneyama (1984) found D-pinitol, chiro-inositol, sequoyitol
and myo-inositol in the organs of plants, with high turnover in leaves, but low
turnover in nodules. D-pinitol was again the major cyclitol. Streeter and
Salminen (1985) reported that D-pinitol was present at a greater concentration
than sucrose in nodules formed on lupin (Lupinus angustifolius). Aminated
derivatives of myo-inositol called rhizopines have also been reported in
nodules containing S. meliloti (Murphy et al., 1987, see Section 1.3.6).
24
Despite the prevalence of cyclitols in plants and nodules, it is believed that
cyclitols are metabolically inert. When labelled 13CO2 was supplied to plants,
the labelled carbon was not readily incorporated into the cyclitols, suggesting
a low turnover, particularly in nodules (Davis and Nordin 1983, Kouchi and
Yoneyama, 1986). Studies on the first two enzymes in the catabolic pathway
of myo-inositol showed that they were not expressed in the bacteroid (Poole
et al., 1994, this work Section 5.2.10).
Clearly, myo-Inositol and derivatives are abundant in plant tissues and
nodules, but as they are unlikely to be used as carbon sources, they must
have other roles that have not yet been elucidated. Galbraith et al. (1998)
hypothesised that bacteroids may synthesise cyclitols in nodules to act as
osmoprotectants, as the bacteroids are subjected to high osmotic pressure.
The role of myo-inositol derivatives as osmoprotectants is discussed in
Section 1.3.4.2.
25
1.3.3.2 Soil myo-Inositol
Soil studies have primarily focused on inositol phosphates rather than free
inositol, due to the importance of these compounds in phosphorus supply to
plants. The results of the studies that have been carried out are conflicting,
partly due to differences in methodology (e.g., extraction and analysis) and to
the different composition of soils. Soil is an extremely complex and dynamic
environment, where compounds may be complexed into unavailable forms. A
variety of factors such as pH, presence or absence of plants, farming
strategies and microbial activity all contribute to soil composition (Wood,
1995).
Yoshida (1940) isolated crystals of pure inositol, as well as myo-inositol
hexaphosphate and other inositol phosphates, from three different samples of
Hawaiian soil. The inositol was obtained by acid hydrolysis of soil organic
phosphorus, extraction of which from soil was by solubility in acid and alkali.
The author postulated that the inositol was probably not present in the free
form, but as inositol monophosphate and that the separation technique may
have been responsible for releasing free inositol.
Lynch et al. (1958) studied the composition of four soils with different organic
matter content and discovered several carbon compounds, including inositol,
also by using acid hydrolysis to extract compounds. McKercher and
Anderson (1968) found small quantities of inositol in addition to other
unidentified carbohydrates following acid hydrolysis of three Canadian soils.
26
However, others employing similar techniques to isolate organic phosphorus
reported that they were unable to isolate pure inositol from soil, only myo-
inositol hexaphosphate (Bower 1945, Wrenshall and Dyer, 1940). Similarly,
no myo-inositol was found in soils derived from volcanic ash (andosols),
where the most abundant sugars were mannose, fucose and ribose (Yoshida
and Kumada, 1979).
Inositol was found in both the soils and exudates of cotton strains Neurospora
crassa and N. sitophila (Sulochana, 1962). HPLC analysis of soil solutions
extracted from two soils, showed that myo-inositol is the most common
soluble carbon compound present (Wood and Stanway, 2000). This method
does not involve any chemical changes to the soil and hence, it could be
argued that it is a more accurate measure of soil composition.
These studies indicate that myo-inositol is likely to be present in soils, but not
necessarily in the free form and when it is found in the free form, its presence
may be overlooked, due to the techniques used.
27
1.3.4 myo-Inositol Derivatives
myo-Inositol is not only used as a carbon source by bacteria. It is an essential
component of several biochemical pathways found in all organisms. myo-
Inositol acts as a co-factor in the formation of galactinol from UDP-galactose
(Frydman and Neufeld, 1963). This is the substrate for raffinose sugars,
which are believed to be important in plants for stress tolerance and
carbohydrate transport (Loewus and Dickinson, 1982).
A myo-inositol oxidation pathway has been identified in plants. myo-Inositol
forms D-glucuronate (although D-glucuronate can also be made from UDP-
glucose) (Loewus and Loewus, 1983), which then undergoes metabolism to
be incorporated into cell wall polysaccharides, pentose phosphate cycle
intermediates and hexonic and hexaric acids (Loewus, 1990). One product of
D-glucuronate, D-galacturonate forms oligosaccharides that act as signals to
activate plant defence mechanisms (Ryan, 1987). Therefore, myo-inositol is
an essential carbon source for cell wall synthesis in growing plant tissues
(Sasaki and Nagahashi, 1990).
In the following sections, the roles of various derivatives of myo-inositol are
discussed.
28
1.3.4.1 Auxins
Auxins are plant hormones that are required for lateral root formation
(Wightman et al., 1980). By conjugating myo-inositol to auxins, they are
temporarily inactivated, which allows long-distance transport of these
compounds from shoots to roots (Cohen and Bandurski, 1982). Auxin
accumulates in the inner cortical cells of alfalfa after inoculation by S. meliloti
(Hirsch et al., 1997). These are the cells that eventually become nodule
primordia (Yang et al., 1994). The high auxin levels are thought to be
necessary to stimulate the cell divisions that lead to nodule primordia
development. Nod-factors (lipo-chitin-oligosaccharides) produced by rhizobia
cause inhibition of auxin transport, which leads to an accumulation of auxins
at the site of nodule initiation (Mathesius et al., 1998). This is mediated by
flavonoids, which are also known to inhibit auxin transport (Jacobs and
Rubery, 1988).
1.3.4.2 Methyl Esters of myo-Inositol
Another important pathway involves monomethylation or dimethylation of
myo-inositol. It has been proposed that methyl esters of myo-inositol may
play a role as a preformed store of myo-inositol that can be utilised when
metabolic demands arise (Loewus, 1990). myo-Inositol and methylated
derivatives are also implicated in high salinity tolerance by some animals and
higher plants (Keller and Ludlow, 1994, Nelson et al., 1998).
29
For example, D-pinitol (1-D-4-O-methyl-myo-inositol) is a derivative of myo-
inositol that may protect proteins and membranes from dehydration and
denaturation in the tropical legume, pigeonpea (Cajanus cajan) and other
legumes (Keller and Ludlow, 1994). D-pinitol and the amino acid proline are
the main solutes to accumulate in drought-stressed pigeonpea leaves (Ford,
1984). Carbon flux is diverted from starch and sucrose accumulation into
polyols in response to low water potential caused by drought and salt stress
(Keller and Ludlow, 1994). D-pinitol and its precursor ononitol are thought to
lower the cytoplasmic osmotic potential and balance sodium accumulation in
the plant vacuole, in a similar mechanism to the role of myo-inositol and other
solutes in the renal cells of vertebrates (Burg, 1994).
Nelson et al. (1998) postulated that in the ice plant Mesembryanthemum
crystallinum, photosynthesis could simultaneously control root growth via
myo-inositol supply and osmotic stress protection via ononitol and D-pinitol
availability in the roots. myo-Inositol in the phloem may serve to signal
photosynthesis capacity to the roots, sustain membrane biosynthesis and act
as a facilitator of long term sodium transport, again important for osmotic
regulation (Nelson et al., 1998).
30
1.3.4.3 Di-myo-inositol 1,1’-phosphate
Di-myo-inositol 1,1’-phosphate (DIP) has been shown in the thermophilic
bacteria Pyrococcus woesei (Scholz et al., 1992) and Methanococcus igneus
to exert a thermo-stabilising effect on proteins within the cell. The cellular
concentration of this small molecular mass compound increases
comcomitantly with temperature increase (Ciulla et al., 1994). However, DIP
is synthesised directly from glucose-6-phosphate rather than from myo-inositol
(Scholz et al., 1998).
1.3.4.4 Phosphatidylinositol
myo-Inositol is a constituent of membrane lipids, such as phosphatidylinositol
(cardiolipin), which comprises 20% of the lipids of the inner mitochondrial
membrane in animals, as well as a large proportion of bacterial membrane
lipids. This compound is also important for signalling in eukaryotes.
Phosphatidylinositol is modified to produce the membrane lipid
phosphatidylinositol-4,5-bisphosphate. This is hydrolysed by the enzyme
phospholipase C to release inositol-1,4,5-triphosphate. Inositol-1,4,5-
triphosphate then combines with a receptor that activates a calcium pump or
transporter. Calcium then activates a variety of enzymes. Inositol-1,4,5-
triphosphate is hydrolysed and then converted back to phosphatidylinositol.
Similar pathways occur in plants and mammals (Salisbury and Ross, 1992).
31
Another role of phosphatidylinositol is in ice nucleation. Ice nucleating agents
limit supercooling and are found in lichens, higher plants, insects, molluscs
and bacteria (Duman et al., 1991). Kozloff et al., (1991) showed that P.
syringae is able to degrade myo-inositol hexyaphosphate when grown under
inorganic phosphorus limitation. This provides myo-inositol for production of a
membrane protein containing phosphatidylinositol that enables the bacterium
to cause ice nucleation. However, the bacterium was unable to utilise pure
free myo-inositol as a component of the protein (Blondeaux and Cochet,
1994). P. syringae, Erwinia herbicola and E. coli all contain
phosphatidylinositol and phosphatidylinositol synthase. The latter increases in
amount in the presence of an ice nucleation gene (Duman et al., 1991).
1.3.4.5 myo-Inositol Hexaphosphate
myo-Inositol is abundant in animals and higher plants as myo-inositol
hexaphosphate (phytate, or as the calcium-magnesium salt, phytin)
(Lehninger, 1975, Loewus et al., 1990) (Figure 1.6). Phosphorus is often one
of the major limiting factors for plant growth, so both plants and micro-
organisms have evolved systems for acquiring and safeguarding it.
Conjugating phosphorus to myo-inositol acts as a phosphorus store
(Cosgrove, 1980). The majority of inositol phosphates are the myo- isomer,
then the scyllo- isomer and very small quantities of dl- and neo- isomers
(Cosgrove, 1963, McKercher and Anderson, 1968, Omotoso and Wild, 1970).
32
Figure 1.6 Structure of myo-Inositol Hexaphosphate
Phytate may account for 50-80% of total phosphorus in seeds, including
cereal grains, oilseeds and legumes (Al-Asheh and Duvnjak, 1995, Cilliers
and van Niekerk, 1986, Knowles and Watkins, 1932, McCance and
Widdowsen, 1935, Raboy, 1990, Reddy et al., 1989, Reddy et al., 1982).
Phytate comprises approximately 1% of the dry weight of wheat bran
(Lasztity, 1991) and mature lily pollen (Baldi et al., 1988).
Phytate is also abundant in soil, presumably due to exudation from plants.
Studies have shown that phytate accounts for approximately 33% of organic
phosphorus in soil (Anderson, 1956, Yoshida, 1940, Bower, 1945) and up to
50% of inorganic phosphate (Anderson, 1980, Dalal, 1978). Phytate has also
been reported in both freshwater and saltwater sediments (De Groot and
Golterman, 1993, Suzumura and Kamatani, 1995).
Phytate can chelate various metals and bind proteins, diminishing the
bioavailability of proteins and nutritionally important minerals (Liu et al., 1998).
Phytate may be particularly important as a siderophore, due to its high affinity
PO
H
PO PO
PO
OH
OH
PO
H
PO
H
H
33
for iron. It is believed to be universal in eukaryotic tissues, where it may bind
to iron without forming harmful hydroxyl radicals (Hawkins et al., 1993). A
similar role has also been identified in prokaryotes as phytate can allow P.
aeruginosa to grow in iron-limiting conditions (Smith et al., 1994). Other
inositol phosphates may also have similar roles, as myo-inositol 1,2,3-
trisphosphate was shown to be able to act as a siderophore in P. aeruginosa.
The activity of iron uptake was similar to that of phytate (Spiers et al., 1996).
1.3.4.5.1 Phytase
The enzyme phytase catalyses the release of phosphate from phytate,
yielding yield inositol triphosphate, inositol monophosphate, phosphoric acid
and inositol (Anderson, 1915). Phytases have been found in plants, bacteria
and fungi. In the former two, they are usually intracellular, but fungal and
yeast phytases are generally extracellular (Howson and Davis, 1983).
Phytase activity in seeds increases during germination, releasing phosphate
(Reddy et al., 1989). Phytase may also be active in soil, as phytate
derivatives have been found alongside phytate (Bower 1945, Yoshida, 1940).
Soluble organic phosphorus that could be assimilated by maize was obtained
in the root medium of maize plants supplemented with phytate and phytase
(Findenegg and Nelemans, 1993). Pseudomonas species can synthesise
phytase and utilise phytate as a source of inorganic phosphate when it is
lacking in the culture medium (Cosgrove, 1970). Phytase production was
enhanced by inorganic phosphorus-limiting conditions (Shieh and Ware,
34
1968). Recently, several ruminal anaerobic bacteria have been found to have
high phytase activity, which may release phosphorus for utilisation by their
host animals (Yanke et al., 1998).
Of 200 randomly selected soil bacterial isolates, none could utilise phytate as
a sole carbon and phosphorus source (Richardson and Hadobas, 1997),
although they could utilise phytate as a phosphorus source when additional
carbon was supplied. Of a further 238 isolates obtained, two fluorescent
Pseudomonas strains CCAR53 and CCAR59, were able to utilise phytate as a
sole carbon source, depending on the pH of the medium. Therefore, phytate
release from plants and its subsequent breakdown by phytase may account
for the presence of free myo-inositol in soil.
1.3.6 Rhizopines
Rhizopines are compounds produced exclusively in legume root nodules,
which can be utilised by free-living rhizobia as both a carbon and nitrogen
source. They are similar in structure to opines, of which many different forms
have been identified in Agrobacterium species, which are Gram-negative, soil-
dwelling bacteria (Tempé and Schell, 1977). The first rhizopine was
discovered in alfalfa nodules containing S. meliloti strain L5-30 by Tempé and
Petit (1983) and is called L-3-O-methyl-scyllo-inosamine (3-O-MSI) (Figure
1.5). Subsequently, a derivative called scyllo-inosamine (SI) was discovered
in nodules containing S. meliloti strain Rm220-3 (Saint et al., 1993) (Figure
35
1.7). Both are aminated derivatives of myo-inositol, although by definition,
rhizopines do not need to be inositol derived (Murphy and Saint, 1992).
Figure 1.7 Structure of Rhizopines
3-O-MSI SI
The ability to synthesise and catabolise rhizopines appears to be an
infrequent trait amongst species of Rhizobium, although such species are
widespread in the environment (Wexler and Murphy, 1995). One study found
that of over 300 strains of Rhizobium, approximately 10% of S. meliloti strains
and 14% of R. leguminosarum bv. viciae strains possessed the genes to both
synthesise and catabolise rhizopines. No R. leguminosarum bv phaseoli or R.
leguminosarum bv. trifolii tested had the genes (Murphy et al., 1995).
Rossbach et al. (1995) found that strains of Agrobacterium, Bradyrhizobium,
Azorhizobium, Azospirillum, Escherichia and Pseudomonas species were
incapable of utilising rhizopines and did not contain known rhizopine catabolic
genes (see below).
OH
H
OH H
OH
OH
H2N
HO
H
H
H
H OH
H
OH H
OH
OH
H2N
CH3O
H
H
H
H
36
1.3.6.1 Rhizopine Synthetic and Catabolic Genes of S. meliloti
S. meliloti strains possess genes encoding both the synthesis of rhizopines
(mos) and catabolism (moc). These genes are closely linked and located on
the Sym plasmid in S. meliloti (Murphy et al., 1987). The expression of mos
genes in S. meliloti is directly controlled by the symbiotic nitrogen fixation
regulatory system (Murphy et al., 1988), so mos genes are expressed only
when Rhizobium has differentiated into a bacteroid and is expressing nif
genes. The moc genes are not regulated in the same way and are only
expressed in free-living rhizobia (Saint et al., 1993). Despite the link to
symbiosis, rhizopines are not essential for symbiosis, as mutants are still able
to nodulate and fix nitrogen (Murphy et al., 1988).
The mos locus contains four genes in S. meliloti strain L5-30, mosABC and
orf1. MosA is involved in a methylation step to form 3-O-MSI from SI (Rao et
al., 1995). The mosA gene is not present in S. meliloti strain Rm220-3, which
produces SI (Murphy et al., 1993). MosB may have a regulatory role and
MosC may be involved in export of rhizopines or their precursors. A fourth
gene, orf1, does not code for a known protein and is not required for rhizopine
synthesis (Murphy et al., 1993).
The moc locus contains six open reading frames (ORFs), of which four genes
are essential for rhizopine catabolism in S. meliloti (Rossbach et al., 1994).
MocB is believed to be a periplasmic binding protein, probably involved in the
transport of rhizopines into the cell. MocR is thought to be a regulatory
37
protein with a DNA-binding motif. MocA is thought to be a dehydrogenase
and MocC has no known function but is believed to be involved in inositol
catabolism (Rossbach et al., 1994). The latter two genes resemble genes in
the B. subtilis ino operon (Yoshida et al., 1997). The moc operon of R.
leguminosarum bv. viciae contains nine ORFs, of which six are essential for
degradation of 3-O-MSI (mocCABRDE) and two (mocCA) for degradation of
SI (Bahar et al., 1998).
Additional genes involved in myo-inositol catabolism are also required for
degradation of rhizopines. An inositol dehydrogenase mutant of S. meliloti
which could not grow on myo-inositol as the sole carbon source, was also
unable to catabolise the rhizopine L-3-O-MSI (Galbraith et al., 1998). The
same results were reported through a collaboration that used the myo-inositol
catabolic mutants of R. leguminosarum biovar viciae used in this project
(RU360 and RU361). The wild type strain 3841 does not possess the genes
to synthesise or catabolise rhizopines but can utilise myo-inositol as the sole
carbon source. After a plasmid containing rhizopine catabolic genes was
introduced into 3841 and the mutants RU360 and RU361, 3841 gained the
ability to utilise rhizopines as the sole carbon and nitrogen source, but the
mutants did not (Bahar et al., 1998).
McSpadden Gardener and deBruijn (1998) isolated 21 soil isolates that could
utilise SI as the sole carbon and nitrogen source. Hybridisation tests carried
out using rhizopine catabolic genes as probes were negative, indicating that
these strains do not use the known catabolic genes. Although 16S ribosomal
38
DNA analysis revealed that there were five distinct genera of bacteria, only
four Arthrobacter and two Sinorhizobium isolates passed a standard rhizopine
catabolism test and the Sinorhizobium isolates were unable to nodulate
alfalfa. These data indicate that there may be alternative genes for rhizopine
catabolism that are as yet unidentified.
1.3.6.2 Agrobacterium Opines
The Agrobacterium Opine Concept outlines a novel role for these compounds.
It was postulated that pathogenic agrobacteria growing in tumours on host
plants induce the production of selective growth substrates for other bacteria
capable of inciting infection that they do not utilise themselves (Guyon et al.,
1980). This process of aiding genetic siblings is known as kin selection. A
concept analogous to the opine concept, the Rhizopine Concept, was
postulated by Murphy and Saint, (1992). It was suggested that bacteroids
specifically produce and release nutrients into the rhizosphere to selectively
aid the growth and survival of free-living rhizobia in infection threads and the
rhizosphere. Strains that could utilise such compounds may thus have a
competitive advantage over non-utilising strains. However, the selective
advantage proposed by the concept would only apply if the environment were
limited in carbon, as there are many other carbon compounds that rhizobia
can utilise.
Another role for opines in A. tumefaciens involves a hierarchical cascade
where opine availability regulates quorum sensing, which in turn regulates
39
transfer of the Ti plasmid between bacteria and plants (Piper et al., 1999).
This plasmid contains the genes for synthesising and catabolising opines.
Quorum sensing (auto induction) is a mechanism by which bacteria regulate
the expression of certain genes in response to cell density. Bacteria gauge
the size of the population by sensing small, diffusible signal molecules known
as auto inducers that are produced by the bacteria. At a threshold
concentration that corresponds with cell density, these molecules regulate
transcription of certain genes, the products of which are assumed to be of
benefit to the bacteria in the particular conditions. The autoinducers are
usually N-acylated derivatives of L-homoserine lactone (acyl-HSL). It is
postulated that regulation of quorum sensing by opines informs the Ti plasmid
that nutritional conditions are conducive to conjugation Piper et al., 1999).
This regulation may also serve to increase the copy number of the plasmid so
that there is an increase in components of the opine catabolic system, which
may be advantageous when nutritional resources are limited (Li and Farrand,
2000).
The evolutionary factors involved in rhizopine synthesis and catabolism,
especially in relation to distribution of the genes, are not yet fully understood
(Wexler et al., 1996). Rhizosphere colonisation studies using rhizopine
positive and negative strains have found that rhizopine positive strains have a
competitive advantage, but the exact role of rhizopines is not known. (Gordon
et al., 1996, Heinrich et al., 1999) (c.f. Section 1.4).
40
1.4 Rhizosphere Growth and Competition for Nodulation
There has been extensive study of the genes involved in nodulation and
nitrogen fixation by Rhizobium. However, relatively little is known about the
traits that enable Rhizobium to grow and colonise the rhizosphere and
successfully nodulate plants in the presence of other strains. The ability for
one strain to dominate nodulation is termed competitiveness. Rhizobia
chosen for their improved nodulation of legumes in vitro have frequently been
out-competed by indigenous bacteria in situ due to unknown factors (Graham,
1981, Robleto et al., 1998).
There are two aspects of competition that are considered here, the ability to
grow and survive in the rhizosphere and the ability to occupy nodules. To
identify genes that are important for competition, many studies have
compared a wild type with a defined mutant by co-inoculating an equal ratio
into the rhizosphere and observing differences in phenotypic behaviour.
Genes of particular interest are ones where mutants appear to grow in the
rhizosphere and nodulate plants as successfully as the wild type when
inoculated alone, but which are disadvantaged when co-inoculated.
1.4.1 Competition for Rhizosphere Colonisation
Studies on Pseudomonas have revealed several factors important for
bacterial rhizosphere colonisation. These include motility (de Weger et al.,
41
1987), production of the O-antigen of lipopolysaccharide (de Weger et al.,
1989), amino acids (Simons et al., 1997) and vitamin B1 (Simons et al., 1996).
Recently, a study identified a P. fluorescens mutant in a two-component
regulatory system which, when inoculated singly onto potato cuttings,
colonised as effectively as the wild type. However, when co-inoculated in
equal numbers of 107 colony forming units, the mutant was impaired by 300-
1000-fold. The mutant was not distinguishable from the wild type with respect
to in vitro growth rate and factors known to be important for colonisation, so
the authors concluded that the two-component system must respond to some
unknown factor that stimulates a trait crucial for colonisation (Dekkers et al.,
1998).
Roberts et al. (1999) postulated that the quantities of carbon sources found in
seed exudates and the ability of micro-organisms to utilise them are important
in spermosphere colonisation. They studied a mutant of Enterobacter cloacae
in pfkA, which encodes a phosphofructokinase. The mutant was severely
deficient in its ability to colonise the spermosphere of several plant species.
However, the mutant was only limited in the spermosphere of plants that
released limited amounts of carbon.
Water-soluble vitamins may be important in competition for colonisation of the
rhizosphere. Colonisation of alfalfa plant roots by S. meliloti can be limited by
availability of biotin, thiamine and riboflavin (Streit et al., 1996). Alfalfa roots
release biotin (Rovira and Harris, 1961), which is a co-factor for carboxylases.
42
Biotin auxotrophs created by transposon mutagenesis competed poorly for
root colonisation when co-inoculated with the wild type into the rhizosphere,
although they grew when inoculated alone in very low numbers (fewer than
100 cells). The results indicated that both synthesis and uptake of biotin from
the rhizosphere were important for successful competition in the rhizosphere
(Streit et al., 1996).
The ability to utilise carbon compounds in the rhizosphere and the role in
competition has also been studied, particularly in relation to plasmid encoded
catabolic genes. Plasmids are important in determining competitive ability in
Rhizobium species as it has been shown that when cured of plasmids,
competitive ability was severely impaired. (Brom et al., 1992, Hynes, 1990,
Moënne-Loccoz and Weaver, 1995). However, the mechanisms are unknown
as the plasmids contain many genes.
1.4.2 Competition for Nodulation
Oresnik et al., (1998) showed that some catabolic loci present on plasmids
are important for competition for nodulation by R. leguminosarum bv. trifolii. A
mutant unable to utilise rhamnose occupied less than 10% of nodules when
co-inoculated with the wild type onto clover. However, sorbitol and adonitol
mutants were not impaired. The mutant was able to colonise the rhizosphere,
nodulate clover, fix nitrogen at rates similar to the wild type and neither in vitro
nor in vivo growth was slowed by the mutation. Rhamnose catabolic genes
43
were inducible by clover root extracts, indicating that this compound is present
in the rhizosphere and may be important in nodulation.
Rhamnose is a component of plant cell walls in the polysaccharide
rhamnogalacturonan (McNeil et al., 1984). Oresnik et al. (1998) postulated
that as plant cell walls are continuous with infection threads,
rhamnogalacturonan subunits or degradation products might be present in
infection threads. The rhamnose mutant strain may then grow more slowly in
the infection thread. Depending on when the mechanism that results in
infection threads being aborted occurs, proportionately more infection threads
formed by the mutant may be aborted, resulting in the wild type occupying the
majority of nodules.
A mutant of R. etli in the rosR gene grew in the rhizosphere and nodulated
bean as normal when inoculated alone. When co-inoculated with the wild
type, a 17,000-fold excess of the mutant was required to achieve equal nodule
occupancy with the wild type, although a high initial inoculum of 108 cells was
used. The mutant had altered cell surface characteristics (Araujo et al.,
1994). There is also evidence that overexpression of rosR might enhance
competitiveness (Bittinger et al., 1997). RosR is a negative transcriptional
regulator of at least 42 loci and a positive regulator of at least one. The
negatively regulated genes have homology with genes involved in
polysaccharide and carbohydrate metabolism, synthesis and transport of
opines and genes that may be involved in survival in the rhizosphere.
Mutations in these genes did not affect nodulation competitiveness. However,
44
when two rosR mutants were mutated in loci that restored the normal cell
surface characteristics, they became were more competitive than the rosR
mutant. Therefore, regulation by rosR is important for competition in some
cases (Bittinger and Handelsman, 2000).
Milcamps et al. (1998) reported that three strains of S. meliloti mutated in
genes induced by carbon deprivation were not at a competitive disadvantage
for nodule occupancy. One strain that was mutated in a putative sugar
transport system occupied more nodules than the wild type when co-
inoculated onto alfalfa. As in other studies, an equal ratio of wildtype to
carbon-deprivation induced mutants of S. meliloti were inoculated onto alfalfa.
The mutants were able to nodulate and had equal nitrogenase activity to the
wild type.
A region on the large plasmid of S. meliloti was identified as containing genes
that are responsible for nodulation efficiency and competitive ability. These
genes were designated nodulation formation efficiency genes (nfe) (Sanjuan
and Olivares, 1989). The expression of these genes was found to be
dependent on the NifA/NtrA regulatory system (Sanjuan and Olivares, 1991).
Mutation in this region caused a delay in nodule formation and a reduction in
nodulation competitiveness when co-inoculated with the wild type, although
again, the mechanism for this is unknown.
Chun and Stacey (1994) identified a gene from B. japonicum termed nfeC that
caused a significant delay in nodulation of soybean and was at a competitive
45
disadvantage for nodulation when co-inoculated with the wild type. The
mutant exhibited a 6-day delay in nodule formation and occupied less than
12% of nodules when co-inoculated with the wild type. The gene was linked
to the nod gene cluster and had two promoters, one that was expressed in
bacteroids and the other in free-living rhizobia. The mutant was able to fix
nitrogen effectively. However, the function of the gene was unknown.
1.4.2.1 Utilisation of Secondary Metabolites
Rhizobia may gain an advantage over other micro-organisms because of their
ability to utilise novel nutritional compounds such as rhizopines and plant
secondary metabolites, which include calystegins, betaines and homoserine.
Plants may deliberately release these compounds in order to promote
beneficial associations such as nodulation. For example, homoserine which is
produced by peas, has been shown to be a selective substrate for R.
leguminosarum bv. viciae (van Egeraat, 1975).
Calystegins are derivatives of tropane that can be utilised by S. meliloti as the
sole carbon and nitrogen source, although they are only released by non-
legumes (Tepfer et al., 1988). Catabolic genes (cac) are encoded on a non-
symbiotic plasmid and when inoculated alone, both cac+ and cac- strains of S.
meliloti were able to colonise the rhizosphere of calystegin positive and
negative plants. When co-inoculated in the presence of calystegin-producing
plants, the cac+ strain reached much higher population levels. There was no
advantage in the rhizosphere of calystegin-negative plants (Guntli et al.,
46
1999). However, this study was carried out with strains cured of the plasmid
containing the cac genes and so it is possible that there were other factors on
the plasmid that were responsible for the competitive advantage.
An opine producing strain of P. putida was more competitive in the
rhizosphere of mannopine producing tobacco plants (Wilson et al., 1995).
Similarly, Gordon et al. (1996) showed that the rhizopine-producing strain S.
meliloti L5-30 had a competitive advantage for nodulation of lucerne
(Medicago sativa) in soil when co-inoculated with a mutant strain, even though
when inoculated alone, the mutant had a similar rate of growth and nodulation
to the wild type. The mutant occupied less than 30% of nodules. This
competitive advantage remained four years after inoculation, even though
there had been turnover of nodules in that time (Heinrich et al., 1999). In
Chapter 4 of this work, evidence is presented that the ability to utilise myo-
inositol as a carbon source is also important for competition for nodulation.
In contrast, Bosworth et al. (1994) and Scupham et al. (1996) found that
disruption of the ability to utilise myo-inositol was beneficial to host plants,
although the mechanism for this is unknown. Bosworth et al. (1994) created
recombinant strains of S. meliloti by insertion of combinations of an
interposon, a modified nifA regulatory gene and the dctABD C4-dicarboxylic
acid transport genes into an inositol locus. The strains could no longer grow
when myo-inositol was the sole carbon source. The mutations were in myo-
inositol dehydrogenase (Galbraith et al., 1998). Interruption of the inositol site
by the interposon alone or by the interposon plus nifA/dctABD sometimes
47
caused increased grain yields of alfalfa in field trials measured over four years
but this was dependent on conditions in the fields. There was either no
increase or a decrease in yield when the inositol locus was interrupted by an
interposon plus nifA, indicating that the inositol locus might have a role in
nodulation competition (Bosworth et al., 1994, Scupham et al., 1996).
Catabolism of the betaine stachydrine (aka N,N-dimethylproline or proline
betaine) may contribute to root colonisation. Stachydrine is a quaternary
amine found in Medicago species that is a catabolite, an osmoprotectant in
times of osmotic stress (when catabolism is reduced) and an inducer of nod
genes. Although stachydrine catabolic genes are found on the Sym plasmid,
they are not under the NifA/NtrA regulatory system and are therefore not
essential for nitrogen fixation. Stachydrine mutants took three to four days
longer to form nodules than the wild type on alfalfa, although the mutants
colonised roots and formed nodules as effectively as the wild type when
inoculated alone (Goldmann et al., 1994).
Two other S. meliloti mutants impaired in the ability to utilise stachydrine as
the sole carbon and nitrogen source were able to colonise roots as effectively
as the wild type when inoculated separately. However, when co-inoculated
with the wild type, the mutants were at a serious competitive disadvantage.
Root colonisation was severely reduced and the final population count of the
mutant was half that of the wild type (Phillips et al., 1998). One of the
mutations was in the gene encoding proline dehydrogenase (putA).
Stachydrine is catabolised via proline, which is why putA mutants cannot
48
utilise this compound. Trigonelline and stachydrine released from alfalfa
exudates induce expression of NodD2 protein in S. meliloti (Phillips et al.
1992).
Jimenez-Surdo, et al., (1995) previously reported that a putA mutant of S.
meliloti was unable to utilise ornithine or proline as the sole carbon source.
The mutant was impaired in nodulation efficiency and competitiveness on
alfalfa roots, although nitrogen fixation occurred as normal. These results
were confirmed by Jimenez-Surdo, et al. (1997) who found that putA gene
expression was induced by root exudates from alfalfa, during root invasion
and nodule formation, but expression was not induced in differentiated
bacteroids. Therefore, secondary metabolites do not seem to be used by
bacteroids in nodules as carbon sources. However, the competitive
disadvantage of the mutant may be related to the role of proline as an
osmoprotectant (Straub et al., 1994), or stachydrine as an osmoprotectant or
inducer of nod gene expression, rather than simply because it cannot be
utilised as a carbon source.
1.4.3 Improving Nodulation Competitiveness
A potential method for improving nodulation competitiveness inolves utilisation
of antirhizobials such as trifolitoxin, produced by R. leguminosarum bv. trifolii
T24. The tfx genes, which encode the production and resistance of trifolitoxin
are present in several rhizosphere bacteria. When the tfx genes were
inserted into R. leguminosarum bv. trifolii and co-inoculated onto clover plants
49
with the wild type, the trifolitoxin-producing strain occupied over 91% of
nodules. The non-producing wild type occupied 41% (the numbers add up to
more than 100% because many nodules contained both strains). When
inoculated alone, both strains formed the same number of nodules and the
rate of nitrogen fixation was the same (Triplett, 1990).
A strain of R. etli which had the tfx genes introduced into it was also more
competitive than a non-producing strain in non-sterile soil (Robleto et al.,
1998), despite the fact that trifolitoxin is rapidly biodegraded in such conditions
(Bosworth et al., 1993). The trifolitoxin-producing strain occupied at least 20%
more nodules, even when it constituted only 5% of the inoculum. There was
no difference in the grain yield of common bean, indicating that there were no
adverse effects on plant productivity (Robleto et al., 1998). Similarly, Goel et
al. (1999) found that a bacteriocin producing strain Rhizobium VF10 occupied
significantly more nodules on green gram (Vigna radiata) than a bacteriocin
sensitive strain when co-inoculated.
Clearly, there are many genes that are important for successful competition
for rhizosphere colonisation and nodulation to occur, many of which have yet
to be identified. It is extremely interesting that knocking out just one gene can
cause loss of competitive ability, particularly when the function of the gene is
unknown. The work of Scupham et al. (1996) highlights the fact that when
engineering strains to improve competitive ability, it is important that sites of
gene insertion are not themselves important for competitive ability.
50
1.5 Research Objectives
myo-Inositol and its derivatives are ubiquitous throughout nature and several
micro-organisms are able to utilise myo-inositol as the sole carbon source.
Therefore, myo-inositol may be important in the growth and survival of
Rhizobium, either directly as a carbon source, in combination with other
compounds such as phosphorus (inositol phosphates) and nitrogen
(rhizopines), or through another as yet unidentified function.
The objectives of this project are as follows.
1. Identify and characterise the genes involved in myo-inositol utilisation in R.
leguminosarum bv. viciae. Ascertain whether the genes are arranged in a
regulon, an operon, or are located separately throughout the genome or
plasmids. Identify promoters that are induced by myo-inositol to discover
additional genes that are regulated by myo-inositol. Determine whether
there is a specific transport system for myo-inositol.
2. Investigate catabolic regulation in free-living rhizobia and in bacteroids.
Determine if myo-inositol is the preferred carbon source and whether myo-
inositol inhibits catabolism of other sugars or vice versa. Investigate
whether regulation of catabolism of myo-inositol is the same in free-living
rhizobia and in bacteroids.
51
3. Ascertain the importance of the ability to utilise myo-inositol in the
rhizosphere by studying growth in the rhizosphere of the mutants
compared with the wild type. Compare the relative growth rates of the
strains on different carbon sources in vitro, to determine whether the
mutations are specific to myo-inositol catabolic ability.
4. Nodulation ability and competition for nodulation. Discover whether the
myo-inositol mutants nodulate plants and if they do so at the same rate as
the wild type. Compare the rate at which the mutants and wild type fix
nitrogen. Determine whether the plants benefit from inoculation with the
mutants to the same extent as when inoculated with the wild type.
Investigate the mutants’ competitive ability to occupy nodules when co-
inoculated with the wild type.
52
Chapter 2 – Materials and Methods
53
2.1 Bacterial Strains and Plasmids
bacterial strain,
plasmid or
bacteriophage
relevant characteristics source or
reference
R. leguminosarum bv. viciae 3841 Streptomycin and trimethoprim resistant
derivative of strain 300 bv. viciae
Johnston and
Beringer (1975)
RU360 Strain 3841::Tn5-lacZ unable to grow when
myo-inositol is the sole carbon source.
Poole et al.
(1994)
RU361 Strain 3841::Tn5-lacZ unable to grow when
myo-inositol is the sole carbon source.
Poole et al.
(1994)
RU307 Strain 3841::Tn5-lacZ impaired in growth when
myo-inositol is the sole carbon source.
P.S. Poole
S. meliloti RMB7101 Strain RCR2011::Ω unable to grow when myo-
inositol is the sole carbon source. Streptomycin
and spectinomycin resistant.
E.W. Triplett
E. coli
DH5α Strain made competent for transformation,
supE44, ∆lacU169 (φ80 lacZ∆M15), hsdR17,
recA1, endA1, gyrA96, thi-1, relA1.
Hanahan (1983)
XL1- and
XL2-Blue Super and ultracompetent Epicurian coli® cells
for transformation, recA1, endA1.
Stratagene Ltd
803 Host strain for pRK2013 and pIJ1687, met-, gal- Wood (1966)
bacteriophage
RL38 General transducing phage of R.
leguminosarum
Buchanan-
Wollaston (1979)
plasmid
pRK2013 Self transmissible helper plasmid. Kanamycin
resistant.
Figurski and
Helinski (1979)
54
pBluescript
SK-
Phagemid; f1- origin of replication; ColE1
replicon; SK polylinker; 2.96kb; standard
cloning vector. Ampicillin resistant.
Stratagene Ltd
pIJ1687 nodC:phoA fusion. J.A. Downie
Economou
(1990)
pCR2.1
TOPO
TA vector for PCR product cloning. Kanamycin
and ampicillin resistant.
Invitrogen
pOT1 Promoter probe vector, containing gfpuv gene.
Gentamycin resistant.
Schofield (1999)
pRU426 12.4kb EcoRI fragment containing Tn5 from
RU307 in pACYC184.
P.S. Poole
pRU3078,
pRU3079
pLAFR1 (Friedman et al., 1982) cosmids in E.
coli strain 803 containing strain 3841 genomic
DNA that complements RU360.
Poole et al.
(1994)
pRU3111 pLAFR1 (Friedman et al., 1982) cosmid in E.
coli strain 803 containing strain 3841 genomic
DNA that complements RU361.
this project
pRU438 11.5kb SalI fragment containing Tn5-lacZ from
RU361 in pBluescript SK-.
this project
pRU439 0.24kb BamHI fragment containing part of
IS50L of Tn5-lacZ from pRU438 in pBluescript
SK-.
this project
pRU445 3.2kb NotI fragment from pRU438 in
pBluescript SK-.
this project
pRU472 9kb SstI fragment containing 6kb of Tn5-lacZ
from RU360 in pBluescript SK-.
this project
pRU476 1.2kb NotI fragment containing IS50R of Tn5-
lacZ from pRU438 in pBluescript SK-.
this project
pRU482 Adjacent 7.5kb + 2.5kb PstI fragments from
pRU3111 in pBluescript SK-.
this project
pRU542 7.5kb SalI fragment from pRU3111 in
pBluescript SK-.
this project
55
pRU544 0.2kb EcoRI fragment from pRU3111 in
pBluescript SK-.
this project
pRU555 6kb SalI fragment from pRU482 in pBluescript
SK-.
this project
pRU556 7.5kb PstI fragment, different to pRU482 from
pRU3111 in pBluescript SK-.
this project
pRU560 5kb NotI fragment from pRU482 in pBluescript
SK-.
this project
pRU683 0.7kb PstI-SpeI PCR product from pRU3078 in
pCR2.1 TOPO.
this project
pRU702 2.5kb PstI-SpeI PCR product from pRU3078 in
pCR2.1 TOPO.
this project
pRU703 0.7kb PstI-SpeI fragment from pRU683 in
pOT1.
this project
pRU705 2.5kb XbaI-SpeI fragment from pRU702 in
pOT1.
this project
pRU706 As pRU705, but opposite orientation. this project
pRU707 4kb PmeI-SpeI PCR product from pRU3078 in
pCR2.1 TOPO.
this project
pRU713 4kb PmeI-SpeI fragment from pRU707 in
pOT1.
this project
56
2.2 Culture Conditions
Rhizobia were grown at 26-28°C either on tryptone yeast medium (TY)
(Beringer, 1974) or on acid minimal salts (AMS) medium derived from that of
Brown and Dilworth (1975). The changes to AMS were; phosphate (0.5mM),
MgSO4 (2mM) and buffering by MOPS (20mM) pH 7.0. All carbon and
nitrogen sources added to AMS were used at 10mM, unless otherwise stated.
Antibiotic concentrations (in µg ml-1) were gentamycin 20, kanamycin 40,
spectinomycin 100, streptomycin 500, tetracycline 2 (in AMS) and 5 (in TY).
Liquid cultures were incubated in a rotary shaker at 200-250 rpm.
All Escherichia coli strains DH5α, 803, XL1-blue and XL2-blue were grown in
Luria-Bertani (LB) liquid or solid medium at 37°C (Sambrook et al., 1989).
Antibiotic concentrations (in µg ml-1) were ampicillin 50, gentamycin 10,
kanamycin 20 and tetracycline 10.
2.2.1 Calculation of Mean Generation Time
Growth curves were plotted using the logarithm of cell density at OD600nm.
The mean generation time (g) was calculated using the equation g = 0.693/k.
The growth rate constant (k) is equal to the slope of the graph x 2.303 (Stanier
et al., 1988).
57
2.3 DNA and Genetic Manipulations
Plasmid and cosmid DNA isolation was performed using the Flexiprep method
(Pharmacia) according to the manufacturer's instructions. Rhizobium
chromosomal DNA was isolated using DNA Isolator (Genosys) according to
the manufacturer's instructions. Extracted DNA was dissolved in TE buffer
(Sambrook et al., 1989). Restriction enzymes digests, ligations and sub-
clonings were performed according to Sambrook et al., (1989). Transductions
were performed with the bacteriophage RL38 as described by Buchanan-
Wollaston (1979). Transductants were selected for on TY agar containing
kanamycin (80µg ml-1).
DNA was analysed by gel electrophoresis in 0.8% agarose with TAE buffer
(Sambrook et al., 1989). Visualisation of DNA was by staining of gels with
ethidium bromide at a concentration of 10µg ml-1 (10mg ml-1 stock). Loading
buffer allowed the movement of the DNA to be observed on the gel
(Sambrook et al., 1989). DNA fragments cut directly out of agarose gels for
subcloning of plasmids were extracted using Qiaex II (Qiagen) according to
the manufacturer’s instructions.
Southern analysis was carried out on DNA digested with different restriction
enzymes and separated on 0.8% agarose gels. DNA was transferred to a
positively charged , Hybond N+, nylon membrane (Amersham). The DNA
was hybridised with a fluorescent labelled probe and the signal detected with
58
the CDP-star detection kit (Amersham) according to the manufacturer's
instructions.
Triparental matings were carried out using the transfer functions of the helper
plasmid pK2013 (Figurski and Helinski, 1979) in E. coli strain 803.
Transformations were carried out using heat shock at 42°C, according to
Sambrook et al., (1989) with E. coli strain DH5α, or with Stratagene E. coli
strains XL1-blue and XL2-blue according to the manufacturer's instructions.
2.3.1 PCR Amplification
Polymerase chain reaction (PCR) amplification of cosmid pRU3078 DNA was
carried out using the primers detailed in table 2.1. All the primers were
synthesised by Genosys. There was an initial denaturation for 5 minutes at
94°C, 1.5 minutes at 58°C, then 30 cycles of 1.5 minutes at 72°C and a final
10 minutes at 72°C. This final stage ensured an overhang A was added, for
cloning into pCR2.1 TOPO (Invitrogen) according to the manufacturer’s
instructions. The reaction was carried out in a volume of 50µl, which
contained 5µl 10x polymerase buffer, 1.5mM MgCl2, 0.8mM dNTPs, 50pmol
primers, 1% DMSO and 3 units of Bio-X-act DNA polymerase (Bioline).
59
2.3.2 DNA Sequencing
Most DNA sequencing was carried out with a Pharmacia ALF automated DNA
sequencer. The enzyme used was ThermoSequenase. Universal M13
forward and reverse primers were used for sequencing of DNA in the plasmid
Bluescript SK-, LacZB20 or IS50R primers were used for sequencing DNA
adjacent to Tn5-lacZ. Custom Cy5-labelled primers were made by Pharmacia
(Table 2.1). Other sequencing was carried out by MWG-Biotech Ltd, using a
Li-Cor machine. Primers were designed by MWG-Biotech Ltd from a 100-
200bp region supplied to the company. The company did not give details of
the actual primers used.
60
Table 2.1 PCR Amplification and Custom DNA Sequencing Primers
primer Sequence Details p164 ggaagcctatacccgtgttc Downstream of iolA,
5’-3’ direction. p173 ccttgagatttggcattggtg Upstream of iolB, 3’-5’
direction. p174 gcacaagctctggccggc Downstream of iolD,
5’-3’ direction. p175 gctcgacatctcctattccc Sequencing further
downstream from p164, 5’-3’ direction.
p176 gccgaccaggaagcatacga Upstream of iolA, 3’-5’ direction.
p182 gggcggctcgtttcctcg Sequencing further upstream from p176, 3’-5’ direction.
p184 gctcaagctcaacatcatcg Sequencing further downstream from p174, 5’-3’ direction.
p213 ttttttttctgcagcgagctgatttcctgcttcg Upstream of iolD, 5’-3’ direction with PstI added.
p214 aaaaaaaaactagtcaggaagagaacggggata In iolD, 3’-5’ direction with SpeI added.
p228 ttttttttactagtgcggaggagcggtgccg At the end of iolD, 3’-5’ direction with SpeI added.
p216 aaaaaaaaactagtttcgcgctgtcagattattt At the end of iolB, 3’-5’ direction with SpeI added.
p230 ttttttttgtttaaaccgagctgatttcctgcttcg Upstream of iolD, 5’-3’ direction with PmeI added.
61
2.4 Analysis of Sequence Data
DNA sequence data was analysed using the Gapped BlastX algorithm with
default parameters to search the SwissProt and Genbank/EMBL sequence
databases. This was carried out via the National Centre for Biotechnology
Information (NCBI) molecular biology server home page
(www.ncbi.nlm.nih.gov). BlastX translated the DNA sequence into all six
possible reading frames and a list was compiled of proteins most closely
related to the query sequence. The statistical significance of the alignment
score for each pairwise comparison was evaluated by comparing it to the
mean score obtained from comparison of each sequence to random
permutations of the other (Altschul et al., 1997). Other sequence analysis
was carried out using software accessed via the EXPASY molecular biology
server (www.expasy.ch) or using Genetics Computer Group (gcg) software,
through the Human Genome Mapping Project (HGMP) website
(www.hgmp.mrc.ac.uk).
2.5 Statistical Analysis of Data
Data were subjected to Analysis of Variance using Genstat 3.2 software.
62
2.6 Transport Assay
For R. leguminosarum bv. viciae strains, cells were prepared and transport
assays performed as previously described (Poole et al., 1985). The total
substrate concentration was 25µM. Samples of 0.1ml were taken at intervals
of 1 minute for up to 5 minutes, Millipore filtered and counted. For competition
assays, a 5-fold excess (125µM) of a non-labelled substrate was added 5
seconds prior to addition of the substrate to be assayed. The specific
activities of labelled substrates in the assays were; D-[U-14C]glucose (310
MBq mmol-1) and D-[U-14C]myo-inositol (310 MBq mmol-1).
2.7 Enzyme Assays
2.7.1 myo-Inositol Enzyme Assay
Supernatant from 400ml cultures of R. leguminosarum bv. viciae were
obtained by centrifugation at 4000rpm for 30 minutes, then washed in 40mM
N-2-hydroxyethylpiperazine-N’-ethanesulfonic acid (HEPES). pH7.0 and re-
centrifuged (Poole et al., 1994). Cells were then resuspended in 10ml 40mM
HEPES, pH7.0, containing 2mM dithriothreitol (DTT). Bacteroids of 3841
were isolated under argon within an AtmosBag (Aldrich) with argon-purged
63
isolation buffer (Bergersen and Turner, 1990) and by using a Percoll density
gradient method (Reibach et al., 1981) by D. Allaway in the laboratory.
myo-Inositol dehydrogenase and 2-keto-myo-inositol dehydratase were
assayed by procedures modified from Berman and Magasanik (1966). The
myo-inositol dehydrogenase assay contained in 1ml; NH4Cl, 50mM; NAD+,
0.4mM; sodium carbonate, pH10, 50mM; and myo-inositol, 100mM. The 2-
keto-myo-inositol dehydratase assay contained in 1ml; Tris-HCl, pH8.5,
50mM; and 2-keto-myo-inositol, 1mM.
The myo-inositol dehydrogenase assay was followed at 340nm. Activity was
calculated using the molar extinction coefficient of NADH at 340nm, which is
6220. The 2-keto-myo-inositol dehydratase assay was followed at 260nm.
Activity was calculated using the molar extinction coefficient at 260nm, which
is 6000. The protein concentration was determined according to the method
of Lowry et al. (1951) using bovine serum albumin as standard.
2.7.2 β-Galactosidase Assay
β-galactosidase assays were conducted according to the method of Miller
(1972) except that chloroform permeabilisation was replaced by 5 minutes
incubation of cells in 0.5mg ml-1 lysozyme, followed by 15 minutes incubation
in 1mM EDTA. Cells were lysed by addition of 0.001% sodium dodecyl
sulfate (SDS) (Poole et al., 1994).
64
2.7.3 Alkaline Phosphatase Assays
Cultures of R. leguminosarum bv. viciae containing a nodC-phoA reporter
plasmid pIJ1687 (Economou, 1990) were grown in 10ml AMS with appropriate
carbon sources, in the presence of hesperetin 1µM to induce expression of
the nod gene. Controls were grown in the absence of hesperetin. Cells were
harvested and the assay carried out according to the method of Brickman and
Beckwith (1975). Cells were assayed for alkaline phosphatase activity with p-
nitrophenylphosphate (Sigma). The reaction was stopped with 1M K2HPO4.
The samples were cleared by centrifugation for two minutes and the optical
density read at 420nm.
2.8 GFP-UV Assay
GFP-UV has an excitation maximum of 395nm and an emission maximum of
509nm (Clontech). GFP-UV expression was measured in cultures grown
overnight in AMS plus appropriate nutritional factors. 200µl was aliquoted into
a 96-well microtitre plate (Iwaki) and the assay performed using a
Biolumin960 plate reader (Molecular Dynamics). Cell density was measured
at OD630nm and fluorescence of GFP-UV at an excitation of 405/10nm and
emission of 505/10nm. Specific fluorescence was calculated using the
equation A-X/B-Y. A is the fluorescence of the sample, X is fluorescence of
the blank, B is the OD630nm of the sample and Y is the OD630nm of the blank.
Colonies grown on AMA plus appropriate nutritional factors were observed on
65
an UV transilluminator (UVP model TL33E), fitted with 420nm bulbs and a
long wave emission filter.
Bacteria harvested from the rhizosphere and bacteroids in nodules were
assessed for GFP-UV expression under a Nikon Optiphot epifluorescence
microscope, magnification 1000x using a GFP filter set 11003 (Chroma
Technology). The filter consisted of a band pass exciter of 425nm with a
40nm width, a long pass emitter of 475nm and a dichroic long pass of 460nm
(Figure 2.1). The filter set is non-specific which means that only material
expressing GFP-UV will appear green and all auto-fluorescent material will
appear yellow.
Figure 2.1 GFP-UV Filter Set
Wavelength
excitation fluorescence
400 450 500 550
100
T
0
66
2.9 Plant Assays
2.9.1 Seed Sterilisation
Seeds of Pisum sativum cv. Feltham First (common pea) (Sutton seeds) and
Vicia sativa (common vetch) (Chiltern seeds) were surface sterilised by
immersion in 100% ethanol for 5 minutes, followed by immersion in 3%
sodium hypochlorite for 10 minutes, rinsing after each treatment with sterile
distilled water three times. Sterilised seeds were germinated on wetted sterile
filter paper in petri dishes for up to seven days in the dark at 26°C (M. Wood,
pers. comm.). The germination rate was approximately 90%. Seeds
contaminated by fungi after surface sterilisation numbered fewer than 1%,
whereas more than 50% of unsterilised seeds were contaminated after
germination.
2.9.2 Nodulation Competition
P. sativum seedlings were added to sterile 250ml glass conical flasks
containing 250ml sterile vermiculite. V. sativa seedlings were added to glass
boiling tubes containing 50ml sterile vermiculite or 0.2% water agar containing
nitrogen-free rooting solution. Only seedlings with an established root and
shoot that were not visibly contaminated were planted. The vermiculite was
then wetted with sterile nitrogen-free rooting solution. This contained 1mM
67
CaCl2.H2O, 100µM KCl, 800µM MgSO4.7H2O, 10µM FeEDTA, 35µM H3BO3,
9µM MnCl2.4H2O, 0.8µM ZnCl2, 0.5µM NaMoO4.2H2O, 0.3µM CuSO4.5H2O.
7.2mM KH2PO4 and 7.2mM Na2HPO4 were autoclaved separately and added
just prior to use, to prevent precipitation. There was no exogenous carbon or
nitrogen in the system (Poole et al., 1994).
At three to five days after planting, the seeds were inoculated with 1ml of a
103, 104, 105, or 106 cfu ml-1 bacterial culture of 3841, RU360, RU361 or
RU307. The bacterial number was calculated by cell density at OD600nm. An
OD600 of 1 was taken to be 109 cfu ml-1. The cultures were washed twice in
AMS to ensure removal of all nutritional compounds and antibiotics. When a
mutant and wildtype were co-inoculated, the mutant was inoculated first to
avoid absorption effects. Controls were inoculated with sterile AMS. Aliquots
of the bacterial cultures were plated onto TY agar with appropriate antibiotics
to confirm that an OD600 of 1 was actually 109 cfu ml-1.
P. sativum plants were incubated in a growth cabinet with a 16 hour
photoperiod at 25°C. The temperature was lowered to 16°C for the dark
period. Relative humidity was 94%. V. sativa seeds were incubated at 22°C
in a growth room illuminated by a Philips Sont-Agro grow light, with a 16 hour
photoperiod. Plants were harvested four to six weeks post-inoculation.
68
2.9.3 Nodule Harvesting
Nodules were removed from roots and surfaced sterilised by immersion in
sodium hypochlorite for 10 minutes, followed by three rinses in sterile distilled
water (Poole et al., 1994). Nodules were crushed and plated onto TY agar,
with appropriate combinations of streptomycin, kanamycin and tetracycline to
distinguish between the wildtype and mutant strains. Sample colonies were
subsequently streaked onto AMA with appropriate nutritional factors, to
confirm the phenotype of the colonies.
2.9.4 Acetylene Reduction
Acetylene reduction was carried out on whole P. sativum plants four weeks
post-inoculation according to the method described by Trinick et al., (1976).
2.9.5 Plant Dry Weight
P. sativum and V. sativa plants with the root systems removed were dried for
48 hours in an oven at 55°C. The plants were then weighed.
69
2.9.6 Nodule Mass
All nodules were removed from each P. sativum plant and weighed to give
total wet nodule mass.
2.9.7 Rhizosphere Growth
P. sativum and V. sativa seedlings were planted in 20ml glass Universal
bottles containing sterile vermiculite and 12ml nitrogen-free rooting solution
(c.f. section 2.9.2). The seeds were inoculated on the same day as planting
with 1ml of a 103 or 106 cfu ml-1 bacterial culture of 3841, RU360, RU361 or
RU307. Harvesting occurred every one to two days post-inoculation, for up to
two weeks. The contents of the Universal, minus the plant shoot, were
macerated with a pestle and mortar with 10ml nitrogen-free rooting solution
added. The mixture was filtered through sterile muslin and centrifuged for 2
minutes at 4000rpm, to remove debris. The supernatant was then plated onto
TY agar with appropriate antibiotics to determine colony numbers for each
strain (Schofield, 1999).
CHAPTER 3 - MYO-INOSITOL CATABOLIC MUTANTS OF RHIZOBIUM
LEGUMINOSARUM BIOVAR VICIAE ........................................................... 70
3.1 Introduction ............................................................................................ 71
3.2.1 Growth Characteristics of the myo-Inositol Mutants.................................... 72
3.2.2 Cosmids Carrying myo-Inositol Catabolic Genes........................................ 76
70
3.2.3 Southern Hybridisation................................................................................ 79
3.2.4 Transduction of RU307 ............................................................................... 85
3.2.5 Sequencing of myo-Inositol Catabolic Genes ............................................. 86
3.2.5.1 RU360 .................................................................................................. 86
3.2.5.1.1 Complementation of the iol region of RU360 ...................................... 102
3.2.5.2 RU361 ................................................................................................ 104
3.2.3.3 RU307 ................................................................................................ 119
3.2.5 Uptake of Glucose and myo-Inositol ......................................................... 125
3.3 Discussion............................................................................................ 133
70
Chapter 3 - myo-Inositol Catabolic Mutants of
Rhizobium leguminosarum biovar viciae
71
3.1 Introduction
In order to identify the genes involved in myo-inositol utilisation by R.
leguminosarum bv. viciae, three mutant strains of R. leguminosarum bv. viciae
strain 3841 were characterised. Strain 3841 is capable of growth when myo-
inositol is the sole carbon source. Following introduction of a plasmid
containing rhizopine degradation genes, 3841 grew on rhizopines as the sole
carbon and nitrogen source (Bahar et al., 1998).
Strains RU360 and RU361 are Tn5-lacZ mutants that cannot grow on myo-
inositol as the sole carbon source (Poole et al., 1994). They also cannot grow
when rhizopines are the sole carbon source following introduction of a plasmid
containing rhizopine degradation genes (Bahar et al., 1998). Strain RU360
was previously shown to be unable to induce expression of the first two
enzymes in the proposed myo-inositol degradation pathway. However, the
strain retains the ability to nodulate V. sativa and reduces acetylene at the
same rate as the wildtype (Poole et al., 1994).
Strain RU307 is a Tn5 mutant that was isolated by its ability to grow in the
presence of glutamic acid gamma hydrazide, a compound toxic to the wild
type strain 3841 (P.S. Poole, pers. comm.). During this project, this strain was
found to be severely impaired in the ability to utilise myo-inositol as the sole
carbon source. Sequencing data indicated that RU307 might be mutated in
an uptake system for myo-inositol, so the ability of the mutants and RU307 to
transport myo-inositol into the cell was studied.
72
3.2 Results
3.2.1 Growth Characteristics of the myo-Inositol Mutants
The mutants RU360 and RU361 were identified by their inability to grow when
myo-inositol was the sole carbon source (Poole et al., 1994). Strain RU307
was identified as being severely impaired in the ability to grow when myo-
inositol was the sole carbon source. In order to characterise the catabolic
pathway for myo-inositol in R. leguminosarum bv. viciae, the mutants and
3841 were tested for the ability to grow in AMS on different carbon sources.
These were 10mM myo-inositol, 10mM glucose, 10mM glucose plus 10mM
myo-inositol, 20mM pyruvate, 20mM pyruvate plus 10mM myo-inositol.
Growth of the strains on each carbon source was measured at OD600nm. The
mean generation times for each strain in each carbon source were calculated
over a period of at least six hours during linear growth. The mean generation
times were similar for the mutants and 3841 in all combinations of carbon
sources tested other than myo-inositol (Table 3.1). Strains RU360 and
RU361 could not grow when myo-inositol was the sole carbon source. Strain
RU307 grew on myo-inositol, but the mean generation time was almost four
times higher than that for 3841 grown on myo-inositol. Growth by all strains
was slightly slower on pyruvate than on glucose. The mean generation times
were also similar when the strains were grown in glucose or pyruvate plus
myo-inositol. This indicates that the presence of myo-inositol in the medium
did not inhibit growth on other carbon compounds.
73
Two cosmids (pRU3078 and pRU3079) were previously isolated that
complemented RU360, restoring the ability to utilise myo-inositol (Poole et al.,
1994). A cosmid (pRU3111) was isolated during this project that
complemented RU361 (c.f. Section 3.2.2). These complemented strains,
RU360/pRU3078, RU360/pRU3079 and RU361/pRU3111 were also tested for
growth on 10mM glucose, 10mM glucose plus 10mM myo-inositol and 10mM
myo-inositol (Table 3.1).
The mean generation times for the complemented strains were similar to the
mutants and 3841 on glucose and glucose plus myo-inositol. When grown on
myo-inositol as the sole carbon source, the cosmids restored the ability to
grow as well as 3841.
74
Table 3.1 Mean Generation Times for the Mutants and 3841
strain myo-inositol
(hrs)
glucose (hrs) glucose +
myo-inositol
(hrs)
pyruvate (hrs) pyruvate +
myo-inositol
(hrs)
3841 3.41 3.52 3.57 4.39 4.12
RU307 13.36 3.67 3.85 4.05 4.1
RU360 * 3.41 3.37 4.45 3.91
RU361 * 3.58 3.6 4.16 3.93
RU360/
pRU3078
3.78 4.15 3.79 N/T N/T
RU360/
pRU3079
3.4 3.85 3.56 N/T N/T
RU361/
pRU3111
3.28 3.58 3.54 N/T N/T
Each value represents the average of three cultures, which was then
used to determine the mean generation time.
* = too low to be measured.
N/T = not tested.
75
Strains RU360, RU361 and RU307 were also able to grow on AMS and AMA
containing 10mM of the sugar polyols mannitol, sorbitol and glycerol, with no
apparent deficiency compared with 3841. The mutant strains and 3841 were
also tested for their ability to utilise myo-inositol hexaphosphate (phytate) as
the sole carbon source. Phytate is abundant in the rhizosphere and some
micro-organisms produce enzymes that can cleave phytic acid, to release the
phosphorus stored in the compound (c.f. Section 1.6.3). It was postulated that
Rhizobium might be able to utilise the myo-inositol in phytate for growth,
especially if phosphorus were limiting. However, there was no growth by any
of the strains in the presence or absence of phosphate when phytate was the
sole carbon source. It was not tested whether phytate could be used as a
phosphorus source in the presence of other carbon sources. Therefore,
phytate is unlikely to be a source of energy in the rhizosphere for R.
leguminosarum bv. viciae.
The data presented above indicate that there is a specific pathway for myo-
inositol utilisation in R. leguminosarum bv. viciae, mutation of which does not
affect growth on other carbon sources. There was no detectable effect on
growth on pyruvate or glucose caused by the presence of the transposon
insertion or by the presence of complementing cosmids. The presence of
myo-inositol did not inhibit growth of the mutants on pyruvate or glucose.
76
3.2.2 Cosmids Carrying myo-Inositol Catabolic Genes
In order to characterise the region surrounding the myo-inositol mutations and
to try to identify other genes involved in myo-inositol utilisation, a R.
leguminosarum bv. viciae cosmid library was used to isolate cosmids that
could complement the mutants, restoring the ability to utilise myo-inositol.
The cosmids pRU3078 and pRU3079 were previously isolated from this
library for their ability to restore to RU360 the ability to utilise myo-inositol
(Poole et al., 1994).
The cosmid library was conjugated into RU361 and colonies spread on AMA
containing 10mM myo-inositol as the sole carbon source. Two colonies were
obtained that grew on myo-inositol and were tetracycline resistant, indicating
the presence of a cosmid. Restriction enzyme analysis showed that both
colonies contained the same cosmid, which was designated pRU3111.
Attempts to isolate a cosmid that complemented RU307 for the ability to utilise
myo-inositol were unsuccessful. The same library was also used to try to
complement a S. meliloti strain RMB7101, a myo-inositol dehydrogenase (idh)
mutant. Again, this was unsuccessful.
Each cosmid was conjugated into each of the mutants to determine whether
they could cross-complement. The cosmids pRU3078 and pRU3079 were
unable to complement RU361 and pRU3111 was unable to complement
RU360. None of the cosmids complemented RU307. The profiles of the
77
three cosmids digested with five restriction enzymes, EcoRI, NotI, PstI, SalI
and SstI are shown in Figure 3.1.
Cosmids pRU3078 and pRU3079 contain five EcoRI bands of the same size,
two NotI bands, two PstI bands, two SalI bands and three SstI bands. Cosmid
pRU3111 has two EcoRI bands of the same size as pRU3078 and pRU3079,
two NotI bands, one SalI band, one SstI band and no PstI bands.
The results suggest that pRU3078 and pRU3079 contain fragments of the
same DNA although they are not identical, as each cosmid contains additional
fragments. They probably contain overlapping parts of the chromosome.
Cosmid pRU3111 does not appear to contain the same DNA as there are only
a few bands of the same size as those in pRU3078 and pRU3079.
78
Figure 3.1 Restriction Enzyme Digests of Cosmids pRU3078, pRU3079
and pRU3111.
0.8% agarose gel showing DNA bands stained with ethidium bromide visualised on a
UV transilluminator.
1 = pRU3078 EcoRI, 2 = pRU3079 EcoRI, 3 = pRU3111 EcoRI, 4 = pRU3078 NotI, 5
= pRU3079 NotI, 6 = pRU3111 NotI, 7 = 1kb ladder 8 = pRU3078 PstI, 9 = pRU3079
PstI, 10 = pRU3111 PstI, 11 = pRU3078 SalI, 12 = pRU3079 SalI, 13 = pRU3111
SalI, 14 = pRU3078 SstI, 15 = pRU3079 SstI, 16 = pRU3111 SstI.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
←3kb
←5kb
←1.6kb
79
3.2.3 Southern Hybridisation
In order to determine whether each complementing cosmid contained the
DNA interrupted by the transposon, or whether they contained genes that
suppressed the mutation, Southern hybridisation studies were carried out.
The cosmids pRU3078, pRU3079 and pRU3111 were probed with DNA
cloned from RU360 (pRU472, c.f. Section 3.2.5.1) and RU361 (pRU476, c.f.
Section 3.2.5.2). These clones both contain part of Tn5-lacZ and flanking
downstream DNA. A 1kb DNA ladder was used as a positive control in the
Southern blot, as both probes were cloned into Bluescript SK-, which contains
DNA that is homologous to DNA in the mass ladder. The cosmids were each
digested with five enzymes, EcoRI, NotI, PstI, SalI and SstI.
The results of the cosmids probed with pRU472 are shown in Figure 3.2.
Plasmid pRU472 DNA bound to cosmids pRU3078 and pRU3079 but not to
pRU3111. There are two EcoRI sites within pRU472. The probe bound to
two fragments in both pRU3078 and pRU3079, one of approximately 900bp
and one of approximately 3kb. There was also faint binding to a fragment
approximately 2.7kb in pRU3079. This corresponds with sequence data that
there should be binding to fragments of 902bp, at least 2115bp and at least
2593bp (c.f. Section 3.2.5.1). There should have been binding to another
fragment in pRU3078, but this may not have been observed if it was a similar
size to the 3kb fragment. There are no known NotI sites within the sequence
and the probe only bound to a large fragment at the top of the gel in both
pRU3078 and pRU3079.
80
There is one PstI site in the probe DNA sequence. The probe bound to a
fragment of approximately 4.5kb in both cosmids and to a large fragment at
the top of the gel in pRU3078. The sequence data indicate that there should
have been binding to fragments of at least 1938bp and 3668bp. There should
have been binding to another fragment in pRU3079, but this may not have
been observed if it was a similar size to the 4.5kb fragment (c.f. Section
3.5.2.1). There is no SalI site in pRU472 but there are two SalI sites within
the region sequenced, which encompass the probe DNA, giving a fragment of
4694bp, to which the probe bound to in both pRU3078 and pRU3079 (c.f.
Section 3.2.5.1). There is one SstI site at one end of pRU472 and one in the
region sequenced. The probe bound to one fragment of 3117bp in both
pRU3078 and pRU3079, which corresponds with the sequence data (c.f.
Section 3.2.5.1).
The data indicate that cosmids pRU3078 and pRU3079 contained the same
region of DNA that restores the ability to utilise myo-inositol to the mutants.
Cosmid pRU3079 contained additional DNA to pRU3078, but this was not
required for complementation of RU360.
81
Figure 3.2 Hybridisation of pRU3078, pRU3079 and pRU3111 to pRU472
Southern blot with each lane containing digested DNA as detailed below.
1 = 1kb ladder, 2 = pRU3078 EcoRI, 3 = pRU3079 EcoRI, 4 = pRU3111 EcoRI, 5 =
pRU3078 NotI, 6 = pRU3079 NotI, 7 = pRU3111 NotI, 8 = pRU3078 PstI, 9 =
pRU3079 PstI, 10 = pRU3111 PstI, 11 = pRU3078 SalI, 12 = pRU3079 SalI, 13 =
pRU3111 SalI, 14 = pRU3078 SstI, 15 = pRU3079 SstI, 16 = pRU3111 SstI.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
4.5kb→3kb→
0.9kb→
2kb→
0.5kb→
82
The results of the cosmids probed with pRU476 are shown in Figure 3.3.
Plasmid pRU476 DNA bound to pRU3111 DNA but not to pRU3078 or
pRU3079. Two EcoRI fragments of approximately 1.1kb and 2.2kb
hybridised. There was one EcoRI site within pRU476 and two within the
entire region sequenced. The binding corresponds with a fragment of 1143bp
and one larger than 1535bp (c.f. Section 3.2.5.2). A NotI fragment of
approximately 7.5kb hybridised. There is a NotI site at the end of pRU476.
The binding corresponds with a fragment larger than 771bp. There was also
binding to a fragment of approximately 3kb. This does not correspond with
known data and it is not known why this fragment bound (c.f. Section 3.2.5.2).
A PstI fragment of approximately 7.5kb hybridised. There were no PstI sites
within pRU476 or the entire region sequenced. Therefore, this fragment
should encompass the entire iolA gene and surrounding region.
A SalI fragment of approximately 7.5kb hybridised. There were no SalI sites
within pRU476 and two sites in the region sequenced, giving a fragment of
approximately 7.5kb (Figure 3.8). Based on the size of pRU438, which was
isolated as a SalI clone, binding was expected to a fragment that was
approximately 4kb in size. It was subsequently discovered by restriction
mapping that the upstream SalI site did not exist in the complementing cosmid
pRU3111 (c.f. Section 3.2.5.2). It is postulated that when RU361 was
digested with SalI, star activity occurred, followed by a forced ligation. This
could have caused the creation of a SalI site, which gave the fragment that
was then cloned into pBluescript SK-, resulting in pRU438. Alternatively, there
could have been a deletion between SalI sites. Only one colony was obtained
83
from the RU361 subcloning. This is probably because the true SalI fragment
containing Tn5-lacZ would have been too large to clone into pBluescript SK-.
One SstI fragment of approximately 4kb hybridised. There were no SstI sites
within pRU476 and one site downstream of iolA. This corresponds with
binding to a fragment encompassing the entire iolA gene at least 3167bp in
size (c.f Section 3.2.5.2).
The results indicate that cosmid pRU3111 contains the DNA that was
interrupted by Tn5-lacZ in RU361. The data also confirmed the results that
the two mutants RU360 and RU361 are mutated in different loci.
84
Figure 3.3 Hybridisation of pRU3078, pRU3079 and pRU3111 to pRU476
Southern blot with each lane containing digested DNA as detailed below.
1 = pRU3078 EcoRI, 2 = pRU3079 EcoRI, 3 = pRU3111 EcoRI, 4 = pRU3078 NotI, 5
= pRU3079 NotI, 6 = pRU3111 NotI, 7 = 1kb ladder 8 = pRU3078 PstI, 9 = pRU3079
PstI, 10 = pRU3111 PstI, 11 = pRU3078 SalI, 12 = pRU3079 SalI, 13 = pRU3111
SalI, 14 = pRU3078 SstI, 15 = pRU3079 SstI, 16 = pRU3111 SstI.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
7.5kb→
2.2kb→
1.1kb→
0.5kb→
4kb→
2kb→
3kb→
85
3.2.4 Transduction of RU307
Poole et al. (1994) carried out transduction of Tn5-lacZ from RU360 with the
bacteriophage RL38 back into 3841 and showed that the insertion of Tn5-lacZ
was tightly linked to the inability to grow on myo-inositol. Transduction of Tn5
from RU307 with RL38 had previously shown that the ability to grow on
glutamic acid gamma hydrazide was tightly linked to the transposon insertion
(P.S. Poole, pers. comm.). To ascertain whether the reduced growth
capability on myo-inositol was also due to the insertion of Tn5, the
transduction of Tn5 from RU307 into 3841 was repeated.
Colonies were selected for kanamycin resistance. Eleven transductants were
tested for growth on AMA with 10mM myo-inositol as the sole carbon source.
No growth was visible for any of the transductants until at least eight days
after they had been streaked out. All the transductants grew as normal on
glucose. This corresponds with the phenotype of RU307 and confirms that
the reduced ability to grow on myo-inositol is tightly linked to the transposon
insertion.
86
3.2.5 Sequencing of myo-Inositol Catabolic Genes
3.2.5.1 RU360
Part of Tn5-lacZ was cloned from RU360 as a 12378bp SstI fragment into
pBluescript SK- (pRU472), with 4030bp chromosomal DNA flanking the IS50R
(Figure 3.4). This enzyme digests in the lacZ part of Tn5-lacZ, so only DNA
downstream of the transposon was obtained. Attempts to obtain an intact
Tn5-lacZ using the enzyme SalI, which does not digest Tn5-lacZ were
unsuccessful, probably because the resulting fragment that would be obtained
would be too large to clone into pBluescript SK- efficiently.
87
Figure 3.4 Plasmid pRU472
pRU47211595 bp
am picillin res is tance
MCS
iolB
iolE
iolD
lacZ
f1 (-) origin
ColE1 origin
Tn5-lacZ
Sal I
Sst I (760)Sst I (9394)
Eco R I (702)
Eco R I (1824)
Eco R I (7437)
Eco R I (8335)
PstI (712)
PstI (2513)
PstI (3561)
PstI (6421)
PstI (7260)
SstI fragment from RU360 containing part of Tn5-lacZ and adjacent chromosomal
DNA cloned into pBluescript SK-.
88
The plasmid pRU472 was completely sequenced on both strands and three
open reading frames (ORFs) were identified. BlastX analysis of the deduced
amino acid sequences revealed homology with proteins encoded by genes in
the myo-inositol degradation operon (iol) of Bacillus subtilis (Figure 3.5). The
amino acid sequence of the ORF interrupted by the transposon had identity to
several bacterial acetolactate synthases. A 9bp repeat was created in RU360
by insertion of Tn5-lacZ (1775-1784bp). The sequence that was repeated is
highlighted in Figure 3.7. Highest identity was 41% (p-value of 10-127) with
IolD found in the myo-inositol catabolic iol operon of B. subtilis. The ORF was
designated iolD. The beginning of the gene was not contained on pRU472.
Downstream of iolD was an ORF whose deduced amino acid sequence had
homology with three proteins in the databases, MocC of R. leguminosarum
bv. viciae, MocC of S. meliloti and IolE of B. subtilis. These proteins have no
known function. The highest identity was 42% (p-value of 3 x 10-62) over the
entire amino acid sequence of MocC of R. leguminosarum bv. viciae. The
ORF was designated iolE and began 46bp downstream of the end of iolD.
Downstream of the iolE gene was an ORF with 62% identity (p-value of 5 x
10-58) with IolB of B. subtilis. This protein has no other homologues in the
databases and has no known function. The ORF was designated iolB and
began 246bp downstream of the end of iolE. The end of the gene was not
contained on pRU472. All three genes are predicted to be transcribed in the
same direction as the transposon but they are not organised in the same
order as in B. subtilis.
89
Figure 3.5 RU360 iol operon
iolD iolE iolB orf1
transposon insertion s i te
PstI (1938)
EcoR I (2115)
EcoR I (3013)
SalI (754)
SalI (5448)SstI (955) SstI (4072)
The diagram represents the entire region of DNA sequenced from pRU3078 and
encompasses the DNA from pRU472. The entire region sequenced was 5.606kb.
pRU472
1kb
SstI SstI
90
In order to obtain the full DNA sequence of the interrupted genes and to
identify any other genes in the region surrounding the myo-inositol locus,
attempts were made to subclone pRU3078 and pRU3079. No fragment large
enough to encompass the iol region and surrounding DNA was identified by
Southern hybridisation. Therefore, sequencing was carried out on cosmid
pRU3078 directly with primers designed to sequence upstream and
downstream of the region encompassed by pRU472. A region of 5606bp was
sequenced on both strands. This contained the entire sequence of the three
putative iol genes (EMBL Accession Number AJ276296) (Figure 3.5).
Putative Shine-Dalgarno sequences indicating ribosome binding sites (Shine
and Dalgarno, 1974) were identified for each putative gene (Figure 3.7).
A TTG start site for iolD corresponds with a TTG start of the closest
homologue, iolD of B. subtilis. A putative Shine-Dalgarno sequence was
identified upstream of the TTG start. However, a TTG start is less common
than ATG and it is not known whether the start of the putative iolD gene is the
same as the closest homologue. If the entire gene were considered as the
largest ORF starting at ATG, which also had a putative Shine-Dalgarno
sequence upstream, then the putative iolD gene would contain an additional
201bp upstream of the start of its closest homologue.
Other homologues in the Genbank and EMBL databases are larger than the
B. subtilis iolD and are of a similar size to the iolD gene identified here, with
ATG start sites. Testcode analysis was carried out on the sequence to
determine whether the sequence is likely to be coding (Figure 3.6). The
91
results indicate that the 201bp upstream of the TTG start site are likely to be
coding.
Further analysis is required to confirm the correct sequence, but in order to
ensure that the start of iolD is definitely present, the iolD gene was considered
to be the largest ORF, which starts with ATG. The iolD gene is therefore
1881bp, encoding a protein of 627 amino acids with a predicted molecular
weight of 66841.9 and an Isoelectric Point (PI) of 6.1. The smaller ORF would
be 1680bp, encoding a protein of 560 amino acids, with a predicted molecular
weight 59363.5 and a PI of 5.8.
The iolE gene is 939bp, encoding a predicted protein of 313 amino acids, with
a molecular weight of 344440.3 and a PI of 5.6. The iolB gene is 795bp,
encoding a predicted protein of 295 amino acids, with a molecular weight of
29157 and a PI of 5.8.
Further sequence was obtained for 1kb either side of the iol genes. A putative
ORF was identified that began 407bp downstream of iolB. Sequencing did
not extend far enough to give the end of the putative ORF. BlastX analysis of
the deduced amino acid sequence of the region surrounding the iol genes
revealed no homology with known sequences. The ORF was designated orf1
but testcode analysis indicates that this region is unlikely to be coding.
Sequencing of the region upstream of iolD did not reveal any homology with
sequences in the databases and no putative ORFs. The entire sequence,
92
with the deduced amino acid sequence for the four ORFs is presented in
Figure 3.7.
The sequence data obtained suggest that the putative genes iolD, iolE and
iolB comprise an operon that is a distinct locus to other genes involved in
myo-inositol utilisation.
93
Figure 3.6 Testcode Analysis of RU360
Testcode analysis of the iol region from pRU472 and pRU3078. Sequence that is in
the top portion of the graph is likely to be coding. Sequence in the middle portion is
ambiguous and sequence in the bottom portion is unlikely to be coding.
The ATG and TTG start sites of iolD are marked with arrows.
T = testcode.
iolD iolE iolB orf1
0 1000 2000 3000 4000 5000
bp
14
12
10
T 8
6
4
ATG TTG
94
Figure 3.7 Entire Sequence of pRU472 and Surrounding DNA
1 CGACTTGAAG ACGGAGCAGC AGGAGAAGCT GCGGACGCTG TTCGAGGCCG GCTGAACTTC TGCCTCGTCG TCCTCTTCGA CGCCTGCGAC AAGCTCCGGC 51 CCCGCAAGGT CGGCCGCGAG CTGCTGGTGG AGATCATCGC CGGCAAGAAC GGGCGTTCCA GCCGGCGCTC GACGACCACC TCTAGTAGCG GCCGTTCTTG 101 GGCCACTCAC CGACGACACG ATCGCGACCG CGCTGGAGGA ACTCTATGCG CCGGTGAGTG GCTGCTGTGC TAGCGCTGGC GCGACCTCCT TGAGATACGC 151 CTCGGCATCA AGCCGGATTG GTGGAAGCTG GAGCCGCACG GCATCCCGTG GAGCCGTAGT TCGGCCTAAC CACCTTCGAC CTCGGCGTGC CGTAGGGCAC 201 AAGCCTGGAA GAAGATCGAT GCGGTGATCG CCAAAAATGA TCCATGGTGC TTCGGACCTT CTTCTAGCTA CGCCACTAGC GGTTTTTACT AGGTACCACG 251 CGCGGTATCG TGCTGCTCGG GCTCGAGGCG CCCGCCGGCG AGCTGATTTC GCGCCATAGC ACGACGAGCC CGAGCTCCGC GGGCGGCCGC TCGACTAAAG 301 CTGCTTCGAG GCGACGCTGG CAGCTCCCTC CGTGAAGGGT TTCGCCGTCG GACGAAGCTC CGCTGCGACC GTCGAGGGAG GCACTTCCCA AAGCGGCAGC 351 GCCGGACGAT CTTTGCCGAT CCGGCGCGCG CCTGGCTTTC AGGCGAAATG CGGCCTGCTA GAAACGGCTA GGCCGCGCGC GGACCGAAAG TCCGCTTTAC 401 AACGACGAGG AAGCGATCGC CGATATGGCC GGCCGCTTCC AGCAACTGAC TTGCTGCTCC TTCGCTAGCG GCTATACCGG CCGGCGAAGG TCGTTGACTG 451 AGAGGCGTGG CTGAAGACGC GCGGACATCA GTAGACTTAG AATTAAGACC TCTCCGCACC GACTTCTGCG CGCCTGTAGT CATCTGAATC TTAATTCTGG 501 CCTCCCCAAC CCCTCCCCAC AAGGGGGGAC TTAACCCGGA GCACCGCCCT GGAGGGGTTG GGGAGGGGTG TTCCCCCCTG AATTGGGCCT CGTGGCGGGA 551 CACGCCATCA GCAAGCTTCC CTTTGGAGGA GACGTTGCGG CGAGAATGTA GTGCGGTAGT CGTTCGAAGG GAAACCTCCT CTGCAACGCC GCTCTTACAT SD 601 GAGAGCGCGG CAGGTCAGCC CTCCCCTTGT GGGGAGGGGT TGGAGAGGGG CTCTCGCGCC GTCCAGTCGG GAGGGGAACA CCCCTCCCCA ACCTCTCCCC iolD +3 M R K E R L R E D P M G K T I R 651 AAAATGCGAA AAGAAAGATT GCGGGAGGAC CCGATGGGCA AGACGATACG TTTTACGCTT TTCTTTCTAA CGCCCTCCTG GGCTACCCGT TCTGCTATGC +3 L T M A Q A V A H F L K V Q M T 701 GTTGACGATG GCGCAGGCCG TCGCACATTT CCTGAAAGTA CAGATGACGA CAACTGCTAC CGCGTCCGGC AGCGTGTAAA GGACTTTCAT GTCTACTGCT +3 I V D G K K V P I F G G V W A I F
95
751 TCGTCGACGG CAAGAAAGTG CCGATCTTCG GCGGTGTCTG GGCGATCTTC AGCAGCTGCC GTTCTTTCAC GGCTAGAAGC CGCCACAGAC CCGCTAGAAG SD +3 G H G N V A G I G E A L Y Q V R E 801 GGTCACGGCA ACGTCGCCGG TATCGGCGAG GCGCTCTATC AGGTGCGCGA CCAGTGCCGT TGCAGCGGCC ATAGCCGCTC CGCGAGATAG TCCACGCGCT putative ttg start +3 E L T T Y R A H N E Q G M A H A 851 GGAATTGACG ACCTATCGCG CCCACAACGA ACAGGGCATG GCGCATGCCG CCTTAACTGC TGGATAGCGC GGGTGTTGCT TGTCCCGTAC CGCGTACGGC +3 A I A Y A K A N F R T R F M A C T 901 CGATTGCCTA TGCCAAGGCG AATTTCCGCA CGCGCTTCAT GGCCTGCACG GCTAACGGAT ACGGTTCCGC TTAAAGGCGT GCGCGAAGTA CCGGACGTGC +3 S S I G P G A L N M V T A A G V A 951 AGCTCGATCG GTCCGGGCGC GCTGAACATG GTGACGGCCG CCGGCGTGGC TCGAGCTAGC CAGGCCCGCG CGACTTGTAC CACTGCCGGC GGCCGCACCG +3 H V N R I P V L F L P G D V F A 1001 GCATGTCAAT CGTATCCCCG TTCTCTTCCT GCCGGGCGAC GTCTTCGCCA CGTACAGTTA GCATAGGGGC AAGAGAAGGA CGGCCCGCTG CAGAAGCGGT +3 N R A P D P V L Q Q I E D S A T A 1051 ACCGCGCGCC GGATCCGGTG CTGCAACAGA TCGAGGATTC GGCGACGGCA TGGCGCGCGG CCTAGGCCAC GACGTTGTCT AGCTCCTAAG CCGCTGCCGT +3 S V S A N D A F R S V S R Y F D R 1101 TCGGTTTCGG CCAATGATGC CTTCCGTTCG GTTTCGCGCT ATTTCGACCG AGCCAAAGCC GGTTACTACG GAAGGCAAGC CAAAGCGCGA TAAAGCTGGC +3 I T R P E Q I I T A L K R A M Q 1151 CATCACCCGG CCCGAGCAGA TCATCACGGC GCTGAAGCGC GCCATGCAGG GTAGTGGGCC GGGCTCGTCT AGTAGTGCCG CGACTTCGCG CGGTACGTCC +3 V L T D P L D C G P V T L S L C Q 1201 TCCTGACCGA CCCGCTCGAT TGCGGCCCGG TGACATTGTC GCTCTGCCAG AGGACTGGCT GGGCGAGCTA ACGCCGGGCC ACTGTAACAG CGAGACGGTC +3 D V Q A E A Y D Y P E S L F A E K 1251 GACGTTCAGG CAGAAGCCTA TGATTATCCG GAAAGCCTGT TTGCGGAAAA CTGCAAGTCC GTCTTCGGAT ACTAATAGGC CTTTCGGACA AACGCCTTTT +3 V W T T R R P Q P D A D E L A N 1301 AGTCTGGACG ACCCGCCGGC CGCAGCCGGA TGCGGACGAG CTGGCGAATG TCAGACCTGC TGGGCGGCCG GCGTCGGCCT ACGCCTGCTC GACCGCTTAC +3 A I A L I K A S Q K P V I V A G G 1351 CCATCGCGCT GATCAAGGCA TCGCAGAAGC CGGTGATCGT TGCCGGCGGC GGTAGCGCGA CTAGTTCCGT AGCGTCTTCG GCCACTAGCA ACGGCCGCCG +3 G V L Y S Q A T K E L A A F A E A 1401 GGCGTGCTTT ATTCGCAGGC GACGAAGGAA CTTGCCGCCT TTGCCGAGGC CCGCACGAAA TAAGCGTCCG CTGCTTCCTT GAACGGCGGA AACGGCTCCG
96
+3 H G I P V V V S Q A G K S A I N 1451 CCACGGCATT CCGGTCGTCG TCAGCCAGGC CGGCAAGTCG GCGATCAACG GGTGCCGTAA GGCCAGCAGC AGTCGGTCCG GCCGTTCAGC CGCTAGTTGC +3 E T H P L A L G S V G V T G T S A 1501 AAACCCATCC GCTGGCGCTC GGCTCGGTCG GCGTCACCGG CACGTCGGCG TTTGGGTAGG CGACCGCGAG CCGAGCCAGC CGCAGTGGCC GTGCAGCCGC +3 A N A I A E E T D L V I A V G T R 1551 GCGAATGCGA TCGCCGAAGA GACGGACCTC GTCATCGCCG TCGGCACGCG CGCTTACGCT AGCGGCTTCT CTGCCTGGAG CAGTAGCGGC AGCCGTGCGC +3 C Q D F T T G S W A L F K N D S 1601 CTGCCAGGAT TTCACCACCG GCTCCTGGGC GCTGTTCAAG AACGACAGCC GACGGTCCTA AAGTGGTGGC CGAGGACCCG CGACAAGTTC TTGCTGTCGG +3 L K M I G L N I A A Y D A V K H D 1651 TGAAGATGAT CGGCCTCAAT ATCGCCGCCT ATGACGCGGT GAAGCACGAC ACTTCTACTA GCCGGAGTTA TAGCGGCGGA TACTGCGCCA CTTCGTGCTG +3 S H P L V A D A R E G L K A L S A 1701 AGCCATCCGC TGGTGGCGGA CGCCCGCGAA GGGCTGAAGG CGCTTTCGGC TCGGTAGGCG ACCACCGCCT GCGGGCGCTT CCCGACTTCC GCGAAAGCCG 9bp overlap by Tn5-lacZ in RU360 +3 G L S G W K A P A A L A E K A A 1751 AGGGCTTTCG GGCTGGAAGG CGCCGGCAGC ACTCGCCGAG AAGGCAGCGG TCCCGAAAGC CCGACCTTCC GCGGCCGTCG TGAGCGGCTC TTCCGTCGCC +3 A E K K I W M E A A A R A M A T T 1801 CGGAGAAAAA GATCTGGATG GAGGCTGCGG CCAGGGCGAT GGCGACGACC GCCTCTTTTT CTAGACCTAC CTCCGACGCC GGTCCCGCTA CCGCTGCTGG +3 N A A L P S D A Q V I G A V A R T 1851 AATGCCGCCC TGCCCTCCGA TGCGCAGGTG ATCGGCGCGG TGGCGCGCAC TTACGGCGGG ACGGGAGGCT ACGCGTCCAC TAGCCGCGCC ACCGCGCGTG +3 I G G E N T T V L C A A G G L P 1901 GATCGGCGGC GAGAATACGA CGGTTCTTTG CGCTGCAGGC GGCCTTCCCG CTAGCCGCCG CTCTTATGCT GCCAAGAAAC GCGACGTCCG CCGGAAGGGC +3 G E L H K L W P A T A P G S Y H M 1951 GCGAATTGCA CAAGCTCTGG CCGGCGACGG CTCCGGGCAG CTATCACATG CGCTTAACGT GTTCGAGACC GGCCGCTGCC GAGGCCCGTC GATAGTGTAC +3 E Y G F S C M G Y E I A G G L G A 2001 GAATACGGCT TTTCCTGCAT GGGCTACGAG ATCGCCGGCG GGCTCGGCGC CTTATGCCGA AAAGGACGTA CCCGATGCTC TAGCGGCCGC CCGAGCCGCG +3 K M A R P E R D V V V M V G D G 2051 CAAGATGGCG CGTCCCGAAC GGGACGTCGT CGTCATGGTC GGCGACGGTT GTTCTACCGC GCAGGGCTTG CCCTGCAGCA GCAGTACCAG CCGCTGCCAA +3 S Y M M M N S E L A T S V M L G L 2101 CCTACATGAT GATGAATTCC GAGCTTGCGA CCTCGGTCAT GCTCGGCCTC
97
GGATGTACTA CTACTTAAGG CTCGAACGCT GGAGCCAGTA CGAGCCGGAG +3 K L N I I V L D N R G Y G C I N R 2151 AAGCTCAACA TCATCGTGCT CGATAATCGC GGTTACGGCT GCATCAACCG TTCGAGTTGT AGTAGCACGA GCTATTAGCG CCAATGCCGA CGTAGTTGGC +3 L Q M G T G G A N F N N L L K D 2201 ATTGCAAATG GGAACCGGCG GCGCCAACTT CAACAATCTG CTCAAGGACT TAACGTTTAC CCTTGGCCGC CGCGGTTGAA GTTGTTAGAC GAGTTCCTGA +3 S Y H E V M P E I D F R A H A E S 2251 CCTATCACGA GGTGATGCCG GAGATCGATT TCCGCGCGCA TGCCGAAAGC GGATAGTGCT CCACTACGGC CTCTAGCTAA AGGCGCGCGT ACGGCTTTCG +3 M G A I A V K V A S I A E L E Q A 2301 ATGGGCGCCA TCGCCGTCAA GGTCGCCTCG ATCGCCGAGC TGGAACAGGC TACCCGCGGT AGCGGCAGTT CCAGCGGAGC TAGCGGCTCG ACCTTGTCCG +3 L A D S R K N D R T S V F V I D 2351 GCTCGCCGAT TCCAGGAAGA ACGACCGCAC GTCGGTCTTC GTCATCGACA CGAGCGGCTA AGGTCCTTCT TGCTGGCGTG CAGCCAGAAG CAGTAGCTGT +3 T D P L I T T E A G G H W W D V A 2401 CCGATCCGCT GATCACCACA GAAGCCGGCG GCCACTGGTG GGATGTCGCG GGCTAGGCGA CTAGTGGTGT CTTCGGCCGC CGGTGACCAC CCTACAGCGC +3 V P E V S S R S E V N R A H E A Y 2451 GTGCCTGAGG TCAGCTCGCG CAGTGAGGTC AACAGGGCGC ATGAGGCCTA CACGGACTCC AGTCGAGCGC GTCACTCCAG TTGTCCCGCG TACTCCGGAT +3 V K A R A A Q R V G 2501 TGTCAAAGCA CGTGCTGCCC AGCGCGTCGG CTGATCGATT GCTGTCATTT ACAGTTTCGT GCACGACGGG TCGCGCAGCC GACTAGCTAA CGACAGTAAA iolE +3 SD M L L R R M L 2551 TTCTGGAGGA GCGGCACCGC TCCTCCGCAA TGCTTTTAAG GAGAATGTTG AAGACCTCCT CGCCGTGGCG AGGAGGCGTT ACGAAAATTC CTCTTACAAC +3 M K A K L G M S P I A W W N D D L 2601 ATGAAGGCCA AACTCGGCAT GTCGCCCATC GCTTGGTGGA ACGACGACCT TACTTCCGGT TTGAGCCGTA CAGCGGGTAG CGAACCACCT TGCTGCTGGA +3 P E L S D D V S L E E S V R Q S 2651 TCCTGAACTC AGCGACGACG TGTCTCTCGA GGAATGCGTG CGGCAGTCCC AGGACTTGAG TCGCTGCTGC ACAGAGAGCT CCTTACGGAC GCCGTCAGGG +3 R S A G F T G M E Q G R R F P S N 2701 GAAGTGCTGG CTTTACCGGC ATGGAGCAAG GCCGCCGTTT CCCCAGCAAT CTTCACGACC GAAATGGCCG TACCTCGTTC CGGCGGCAAA GGGGTCGTTA +3 P E E M L P I L R A A D V T L C G 2751 CCCGAGGAGA TGCTGCCCAT CCTGCGCGCC GCCGACGTGA CGCTGTGCGG GGGCTCCTCT ACGACGGGTA GGACGCGCGG CGGCTGCACT GCGACACGCC +3 G W F S G T L V N E E L A A N K
98
2801 TGGCTGGTTC TCCGGCACGC TGGTCAATGA GGAGCTTGCC GCCAACAAGG ACCGACCAAG AGGCCGTGCG ACCAGTTACT CCTCGAACGG CGGTTGTTCC +3 D R I A P M I A L F K A V N A P C 2851 ACCGCATCGC GCCGATGATC GCGCTGTTCA AGGCGGTCAA TGCACCTTGC TGGCGTAGCG CGGCTACTAG CGCGACAAGT TCCGCCAGTT ACGTGGAACG +3 I V Y G E V G R S I Q G D R S K P 2901 ATCGTTTATG GCGAAGTCGG TCGCTCCATC CAGGGCGACC GCTCCAAGCC TAGCAAATAC CGCTTCAGCC AGCGAGGTAG GTCCCGCTGG CGAGGTTCGG +3 L A T K P R L S D D E M K A Y A 2951 GCTCGCGACC AAGCCGCGCC TTTCCGATGA CGAGATGAAG GCCTATGCGC CGAGCGCTGG TTCGGCGCGG AAAGGCTACT GCTCTACTTC CGGATACGCG +3 R R V T E F G E W C A E Q G M P L 3001 GCCGCGTCAC GGAATTCGGG GAATGGTGCG CCGAGCAAGG CATGCCGCTT CGGCGCAGTG CCTTAAGCCC CTTACCACGC GGCTCGTTCC GTACGGCGAA +3 S Y H H H M A A V V E T E P E L D 3051 TCCTATCACC ACCACATGGC GGCCGTGGTC GAGACCGAGC CGGAACTCGA AGGATAGTGG TGGTGTACCG CCGGCACCAG CTCTGGCTCG GCCTTGAGCT +3 A F M R H S G E G I P L L L D A 3101 CGCCTTCATG CGTCATTCGG GCGAAGGCAT CCCGCTGCTG CTCGATGCCG GCGGAAGTAC GCAGTAAGCC CGCTTCCGTA GGGCGACGAC GAGCTACGGC +3 G H L A F A G G D V L R A I G N H 3151 GCCATCTCGC CTTTGCCGGC GGCGACGTGC TGCGCGCCAT CGGAAACCAC CGGTAGAGCG GAAACGGCCG CCGCTGCACG ACGCGCGGTA GCCTTTGGTG +3 H A R I N H V H V K D I R K P V V 3201 CACGCCCGCA TCAACCATGT TCACGTCAAG GACATCCGCA AGCCTGTCGT GTGCGGGCGT AGTTGGTACA AGTGCAGTTC CTGTAGGCGT TCGGACAGCA +3 D G L D R S R Q S F L D A V A L 3251 GGATGGGCTG GACCGCAGCC GGCAGTCCTT CCTCGATGCG GTGGCGCTTG CCTACCCGAC CTGGCGTCGG CCGTCAGGAA GGAGCTACGC CACCGCGAAC +3 G A F T V P G D G S L D F G A I V 3301 GCGCCTTCAC GGTGCCGGGC GACGGCTCGC TCGATTTCGG CGCCATCGTC CGCGGAAGTG CCACGGCCCG CTGCCGAGCG AGCTAAAGCC GCGGTAGCAG +3 Q R L A D H G Y E G W F V V E A E 3351 CAGCGGCTTG CCGATCACGG CTATGAAGGC TGGTTTGTCG TCGAGGCCGA GTCGCCGAAC GGCTAGTGCC GATACTTCCG ACCAAACAGC AGCTCCGGCT +3 Q D P R K A P P Q K M A E I G H 3401 ACAGGATCCG CGCAAGGCCC CGCCGCAGAA AATGGCCGAG ATCGGCCACG TGTCCTAGGC GCGTTCCGGG GCGGCGTCTT TTACCGGCTC TAGCCGGTGC +3 A E L M R V M T A A G Y T V E T E 3451 CCGAATTGAT GCGCGTCATG ACGGCCGCGG GCTATACAGT GGAAACGGAA GGCTTAACTA CGCGCAGTAC TGCCGGCGCC CGATATGTCA CCTTTGCCTT +3 G F P K G
99
3501 GGCTTCCCCA AGGGATAGCG AGCACAAGAC CCCTCCCCAC AAGGGGGAGA CCGAAGGGGT TCCCTATCGC TCGTGTTCTG GGGAGGGGTG TTCCCCCTCT 3551 GGCTGATCAG GACAGCCGGA GCAAGACGCC CCCTCATCCG CCCTCCGGAC CCGACTAGTC CTGTCGGCCT CGTTCTGCGG GGGAGTAGGC GGGAGGCCTG 3601 ACCTTTGCCT GGGTTAAGCC ACCGGACTCA ACCCGTCCTT CGGACCCCCG TGGAAACGGA CCCAATTCGG TGGCCTGAGT TGGGCAGGAA GCCTGGGGGC 3651 CTGGGGAGAA GGGGAAAGAG GCGGCGCGGC ACATCCCTTC TCCCCTCGGG GACCCCTCTT CCCCTTTCTC CGCCGCGCCG TGTAGGGAAG AGGGGAGCCC 3701 GAGAAGGTGG CGGCAGCCGG ATGAGGGGGG GCCGCACACG CCAATTTACG CTCTTCCACC GCCGTCGGCC TACTCCCCCC CGGCGTGTGC GGTTAAATGC iolB +2 SD M P N L K V K P S G T H 3751 AGGAGAAACA CCAATGCCAA ATCTCAAGGT GAAACCATCG GGCACGCATG TCCTCTTTGT GGTTACGGTT TAGAGTTCCA CTTTGGTAGC CCGTGCGTAC +2 G R V T H V T P E N A G W T Y V G 3801 GCCGCGTCAC CCATGTCACC CCGGAAAACG CCGGCTGGAC CTATGTCGGC CGGCGCAGTG GGTACAGTGG GGCCTTTTGC GGCCGACCTG GATACAGCCG +2 F D L H R M K P G E T V S G E T G 3851 TTCGATCTGC ATCGGATGAA GCCGGGCGAG ACCGTTTCGG GAGAGACGGG AAGCTAGACG TAGCCTACTT CGGCCCGCTC TGGCAAAGCC CTCTCTGCCC +2 D R E V C L V W V T G K G K A S 3901 CGATCGCGAA GTCTGCCTCG TCTGGGTGAC CGGCAAGGGC AAGGCGTCTG GCTAGCGCTT CAGACGGAGC AGACCCACTG GCCGTTCCCG TTCCGCAGAC +2 A G T K D F G T L G G R M N P F E 3951 CCGGCACCAA GGATTTCGGC ACGCTCGGCG GCCGGATGAA CCCGTTCGAG GGCCGTGGTT CCTAAAGCCG TGCGAGCCGC CGGCCTACTT GGGCAAGCTC +2 G A P H A L Y I P M E S T W S V T 4001 GGCGCGCCGC ACGCGCTCTA CATCCCGATG GAATCGACAT GGTCGGTGAC CCGCGCGGCG TGCGCGAGAT GTAGGGCTAC CTTAGCTGTA CCAGCCACTG +2 A E T D L E L A V C S A P G G G 4051 GGCGGAGACC GATCTGGAGC TCGCGGTCTG CTCGGCACCC GGCGGCGGCA CCGCCTCTGG CTAGACCTCG AGCGCCAGAC GAGCCGTGGG CCGCCGCCGT +2 T Y Q A K E I P P G T H P Q V T R 4101 CCTATCAGGC CAAGGAAATC CCGCCCGGCA CGCATCCGCA GGTGACGCGC GGATAGTCCG GTTCCTTTAG GGCGGGCCGT GCGTAGGCGT CCACTGCGCG +2 G K G T N V R Y V N N I M P E D D 4151 GGCAAGGGCA CGAATGTGCG CTATGTCAAC AATATCATGC CGGAAGATGA CCGTTCCCGT GCTTACACGC GATACAGTTG TTATAGTACG GCCTTCTACT +2 S S A H S L L V V E V I T P G G 4201 CAGCTCGGCG CATTCGCTGC TGGTCGTCGA GGTGATCACG CCGGGTGGAC GTCGAGCCGC GTAAGCGACG ACCAGCAGCT CCACTAGTGC GGCCCACCTG
100
+2 H T S S Y P P H K H D Q D D L P N 4251 ACACCTCCTC CTATCCGCCG CACAAACACG ACCAGGACGA TCTGCCGAAC TGTGGAGGAG GATAGGCGGC GTGTTTGTGC TGGTCCTGCT AGACGGCTTG +2 E S F L E E T Y Y H R L N P P Q A 4301 GAGAGCTTCC TGGAAGAGAC CTATTACCAT CGCCTCAACC CGCCGCAGGC CTCTCGAAGG ACCTTCTCTG GATAATGGTA GCGGAGTTGG GCGGCGTCCG +2 F A F Q R V Y T D D R S L D E A 4351 GTTCGCTTTC CAGCGCGTCT ATACCGACGA TCGTTCGCTT GACGAGGCAA CAAGCGAAAG GTCGCGCAGA TATGGCTGCT AGCAAGCGAA CTGCTCCGTT +2 M A L E D G D V T L V P S Y H P C 4401 TGGCGCTCGA GGACGGCGAT GTGACGCTGG TGCCGAGCTA CCACCCCTGT ACCGCGAGCT CCTGCCGCTA CACTGCGACC ACGGCTCGAT GGTGGGGACA +2 A A C H G Y D L Y Y L N V M A G P 4451 GCCGCCTGCC ATGGCTACGA CCTCTATTAC CTCAACGTCA TGGCCGGGCC CGGCGGACGG TACCGATGCT GGAGATAATG GAGTTGCAGT ACCGGCCCGG +2 Q R I W K F H N A A E H E W L L 4501 GCAGCGGATC TGGAAATTCC ACAATGCCGC CGAGCACGAG TGGCTGCTGA CGTCGCCTAG ACCTTTAAGG TGTTACGGCG GCTCGTGCTC ACCGACGACT +2 K A 4551 AGGCGTAAGC GTCGCGTTCG ATCGCTAAAT AATCTGACAG CGCGAAGCTG TCCGCATTCG CAGCGCAAGC TAGCGATTTA TTAGACTGTC GCGCTTCGAC 4601 CAACTGATGG TTCACTGCCT CCATCCAAAC ACGGATGGAG GATCACCATG GTTGACTACC AAGTGACGGA GGTAGGTTTG TGCCTACCTC CTAGTGGTAC 4651 CACAGCCTGA ATTTCACGAT ACCGGCCCGA AATATTCGCT CTTCGCGTCG GTGTCGGACT TAAAGTGCTA TGGCCGGGCT TTATAAGCGA GAAGCGCAGC 4701 TACTCGACCC AGCCTGTTCC CTGCCGCAAT CACGGCGGCG CGCTGGCTGG ATGAGCTGGG TCGGACAAGG GACGGCGTTA GTGCCGCCGC GCGACCGACC 4751 CGCGCAAAAT AAACGAACGA CGCAACCTGA ATGCGTTGAT GGAGCTTTCC GCGCGTTTTA TTTGCTTGCT GCGTTGGACT TACGCAACTA CCTCGAAAGG 4801 GACGAACAGC TGAAGGATAT CGGCCTTTCC AGGGGGCAGA CGGAAAGCGA CTGCTTGTCG ACTTCCTATA GCCGGAAAGG TCCCCCGTCT GCCTTTCGCT 4851 TGTTCATGTC TACAGCCGTT ACTAAAGTGC TGTTATAAGT ACTGTCGGTT ACAAGTACAG ATGTCGGCAA TGATTTCACG ACAATATTCA TGACAGCCAA 4901 CTGGCCTGTC TGATACGGAA TATGCAATGT CCCAGTTCTC AACGGCACCC GACCGGACAG ACTATGCCTT ATACGTTACA GGGTCAAGAG TTGCCGTGGG orf1 +3 M V T I R D I V C N G E 4951 CTTCTGAAAA CGATATGGTC ACTATTCGTG ACATCGTCTG CAATGGCGAG GAAGACTTTT GCTATACCAG TGATAAGCAC TGTAGCAGAC GTTACCGCTC +3 C R H K S D E E C A H R T S L V Y 5001 TGCCGTCATA AGAGCGACGA GGAATGCGCC CACAGGACGA GCCTCGTCTA
101
ACGGCAGTAT TCTCGCTGCT CCTTACGCGG GTGTCCTGCT CGGAGCAGAT +3 P Y R G V F M R H V G R N D T V 5051 TCCCTATCGC GGCGTCTTCA TGCGGCATGT CGGCCGCAAT GATACGGTGG AGGGATAGCG CCGCAGAAGT ACGCCGTACA GCCGGCGTTA CTATGCCACC +3 A E A I R S C S S I P A R D T G S 5101 CGGAAGCAAT CAGGTCTTGT TCTTCAATAC CGGCCAGGGA TACCGGATCA GCCTTCGTTA GTCCAGAACA AGAAGTTATG GCCGGTCCCT ATGGCCTAGT +3 A T L S R V A T P A S I W R S T T 5151 GCCACCCTAT CGAGGGTGGC GACGCCTGCA TCGATCTGGC GATCGACGAC CGGTGGGATA GCTCCCACCG CTGCGGACGT AGCTAGACCG CTAGCTGCTG +3 P C S K S Y A E G A G A A R P A 5201 TCCATGCTCG AAGAGTTACG CCGAAGGAGC AGGCGCAGCC CGGCCCGCCC AGGTACGAGC TTCTCAATGC GGCTTCCTCG TCCGCGTCGG GCCGGGCGGG +3 L F L P P P A P A D R S A R A G L 5251 TTTTCCTTCC GCCGCCAGCG CCGGCGGATC GATCCGCGCG CGCAGGCCTG AAAAGGAAGG CGGCGGTCGC GGCCGCCTAG CTAGGCGCGC GCGTCCGGAC +3 V A L L R H G L S R N V A E T L E 5301 GTTGCGCTCC TGCGCCATGG TCTCAGCCGC AACGTCGCCG AAACACTGGA CAACGCGAGG ACGCGGTACC AGAGTCGGCG TTGCAGCGGC TTTGTGACCT +3 A E I L A L T L V R R S L G R A 5351 GGCGGAAATA TTGGCGCTGA CGCTCGTGCG CCGTTCGCTC GGGCGAGCGC CCGCCTTTAT AACCGCGACT GCGAGCACGC GGCAAGCGAG CCCGCTCGCG +3 H I G T A G G A P A P A G R N S S 5401 ACATCGGCAC GGCAGGCGGG GCGCCAGCGC CGGCCGGCAG AAACTCGTCG TGTAGCCGTG CCGTCCGCCC CGCGGTCGCG GCCGGCCGTC TTTGAGCAGC +3 T R G K S W L L F P P I L A R A L 5451 ACCCGGGGCA AAAGCTGGTT GCTTTTCCCT CCGATTCTCG CGCGGGCGCT TGGGCCCCGT TTTCGACCAA CGAAAAGGGA GGCTAAGAGC GCGCCCGCGA +3 G T T F R E N R H P K W G V S A 5501 TGGGACCACT TTCCGGGAAA ATCGCCACCC GAAGTGGGGC GTCTCGGCCG ACCCTGGTGA AAGGCCCTTT TAGCGGTGGG CTTCACCCCG CAGAGCCGGC +3 V L S H P G S F Q Q V R R R T P L 5551 TTTTATCTCA CCCAGGGTCT TTCCAGCAGG TCCGAAGGCG GACGCCACTT AAAATAGAGT GGGTCCCAGA AAGGTCGTCC AGGCTTCCGC CTGCGGTGAA +3 F T 5601 TTTACC AAATGG Letters in red indicate amino acids. For amino acid code, see Appendix
3.1. Start and stop codons for each ORF are bold and underlined.
Putative Shine-Dalgarno sequences (SD) are bold.
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3.2.5.1.1 Complementation of the iol region of RU360
In order to determine whether all three genes identified in RU360 are
necessary for myo-inositol catabolism, PCR amplification was carried out on
the iol region of pRU3078 (Figure 3.8). The fragments obtained were cloned
into pCR2.1 TOPO and then subcloned into pOT1, which is a broad host
range vector. Primers p213 and p228 were designed to amplify a 2308bp
fragment containing the entire iolD gene. Primer p228 was designed with a
SpeI site. This fragment was then cloned into the plasmid pOT1, as an XbaI-
SpeI fragment. The resulting plasmid was named pRU706.
Primers p230 and p228 were designed to give a 4343bp fragment containing
the entire three iol genes identified, ending 38bp beyond the end of iolB. The
restriction enzyme sites PmeI and SpeI were included in the primers p230 and
p228 respectively, to enable directional cloning of the fragment. The resulting
plasmid was named pRU713.
The plasmids pRU706 and pRU713 were conjugated into RU360. Six
colonies from each conjugation that were streptomycin, kanamycin and
gentamycin resistant were streaked onto AMA containing 10mM myo-inositol
as the sole carbon source. None of the colonies containing strain
RU360/pRU706 were able to grow on myo-inositol, indicating that the
presence of the entire iolD gene was not sufficient to restore the ability to
utilise myo-inositol to RU360. All six colonies containing RU360/pRU713
grew on myo-inositol as the sole carbon source, indicating that the iolD, iolE
103
and iolB genes enable complementation. This suggests that the transposon
insertion in iolD has a polar effect on the downstream genes and therefore
one or both of these genes is essential for myo-inositol catabolism. It is not
known whether the putative gene orf1 is required for myo-inositol utilisation,
as it may have its own promoter, but as the sequence is unlikely to be coding,
this region was not investigated further.
Figure 3.8 Subclones of RU360
io lD iolE iolB orf1
PstI (1938)
EcoR I (2115)
EcoR I (3013)SalI (754)SalI (5448)SstI (955)
SstI (4072)
The 5.606kb region sequenced from pRU3078 with arrows detailing the
sequence encompassed by the subclones.
1kb
pRU703
pRU706
pRU713
transposon insertion site
104
3.2.5.2 RU361
An intact Tn5-lacZ with flanking chromosomal DNA was cloned from RU361
as an approximately 12400bp SalI fragment into pBluescript SK- (pRU438)
(Figure 3.9). The clone contained 230bp of chromosomal DNA upstream of
the transposon and approximately 3800bp downstream. A subclone of
pRU438 was subsequently constructed in pBluescript SK- (pRU476) in order
to facilitate sequencing and for use in Southern hybridisation studies. Plasmid
pRU476 was a 1370bp NotI clone, consisting of 599bp of Tn5-lacZ DNA with
771bp adjacent chromosomal DNA.
Sequencing was carried out on both strands of 230bp of chromosomal DNA
upstream of Tn5-lacZ and on the 771bp downstream of Tn5-lacZ contained in
pRU476. A 9bp repeat was created in RU361 by insertion of Tn5-lacZ (1525-
1533bp). The sequence that was repeated is highlighted in Figure 3.12.
BlastX analysis of the deduced amino acid sequence of the DNA revealed an
ORF with homology to several methylmalonate semialdehyde
dehydrogenases, including IolA of the B. subtilis iol operon. Highest identity
was 68% with MmsA of Mycobacterium tuberculosis (p-value of 1 x 10-145).
The ORF was designated iolA and is predicted to be transcribed in the
opposite direction to the transposon. The end of the gene was not contained
on pRU438.
105
Sequencing was also carried out on 500bp of DNA from the SalI site
downstream of the transposon. The deduced amino acid sequence had no
homology with any known sequences.
Figure 3.9 Plasmid pRU438
pRU43815339 bp
Am picillinCol1E origin
iolA
iolA
lacZ
f1 (-) origin
Tn5-lacZ
Sal I (675)
Sal I (13053)
Sst I (2911)
Sst I (13138)
Eco R I (3975)
Eco R I (9740)
Eco R I (13080)
Not I (4574)
Not I (8654)
Not I (10017)
Not I (13117)
PstI (4664)
PstI (5712)
PstI (8572)
PstI (13090)
SalI fragment from RU361 containing intact Tn5-lacZ and flanking chromosomal DNA
cloned into pBluescript SK-.
The size of the plasmid is approximate, as the entire chromosomal insert DNA was
not sequenced.
106
The transposon clone pRU438 did not contain the entire iolA gene. As the
SalI fragment in pRU438 was smaller than the SalI fragment that bound in the
Southern hybridisation, it was decided to utilise the complementing cosmid to
obtain the entire gene sequence and to identify any adjacent genes. This was
in case pRU438 contained deletions of crucial sequence. The cosmid was
known to contain the correct sequence as it complemented RU361 (Figure
3.10). Attempts were made to clone a PstI fragment approximately 7.5kb in
size, as this was the size of the PstI fragment that bound to the probe in the
Southern blot and was expected to encompass the entire iolA gene.
A 10.2kb fragment was obtained, containing three PstI fragments, one of
approximately 7.5kb fragment, one of approximately 2.5kb and one of 213bp.
The 7.5kb fragment was cloned into pBluescript SK- and the resulting plasmid
designated pRU482. Attempts to clone the 7.5kb fragment alone by cutting
the fragment out of an agarose gel and cloning into pBluescript SK- failed. It
is postulated that a lethal gene product would be produced if the end of the
7.5kb fragment was not covered by a piece of DNA that would prevent
transcription from the cloning vector.
Sequencing using custom primers to the iolA gene revealed that pRU482
contained DNA identical to that interrupted by Tn5-lacZ in RU361. The entire
iolA gene was sequenced on both strands (EMBL Accession Number
AJ276297) and a Shine-Dalgarno sequence identified upstream of the start
site (Shine and Dalgarno, 1974). The iolA gene was 1494bp, encoding a
107
predicted protein of 498 amino acids, which has a molecular weight of 53528
and a PI of 5.8.
Downstream of iolA an ORF was identified whose deduced amino acid
sequence had identity to Aau3 of S.meliloti. The Aau3 protein is required for
growth on polyhydroxybutyrate (PHB) cycle intermediates. There was 74%
identity (p-value of 6 x 10-54) over the amino acid sequence. Other
homologues included Yhde of B. subtilis and YjeB of E. coli. None of the
other homologues had any known function. The ORF was designated aau3
and began 336bp downstream of the putative end of iolA. A putative Shine-
Dalgarno sequence was identified (Shine and Dalgarno, 1974). The aau3
gene is predicted to be transcribed in the same direction as iolA. Sequencing
was only carried out on one strand.
Sequencing was continued for 1206bp upstream of the iolA gene, using
custom primers. An ORF was identified beginning 405bp upstream of the
start of iolA. BlastX analysis of the deduced amino acid sequence showed
that the ORF had 35% identity (p-value of 9 x 10-12) over the first 173 amino
acids of 282 with a hypothetical gene from A. tumefaciens. The gene does
not have any known function. The next highest homology was 31% (p-value
of 3 x 10-9) with a hypothetical transcriptional activator gene, act, from P.
aeruginosa. The ORF was designated orf1 and is predicted to be transcribed
in the opposite direction to iolA. Sequencing did not extend far enough to give
the end of orf1. This was because when the sequencing was carried out,
108
there were no homologous genes registered in the databases, so it was not
considered necessary to sequence further.
Testcode analysis was carried out on the region sequenced in pRU482
(Figure 3.11). The results show that iolA and aau3 are likely to be coding, but
orf1 is unlikely to be coding. Based on sequencing results, it is postulated that
the iolA gene is not part of an operon of myo-inositol utilising genes. The
complete DNA and amino acid sequence of iolA along with flanking DNA is
presented in Figure 3.12.
109
Figure 3.10 Restriction Map of pRU3111
iolAaau3 dppF
gntK
dppC
dppB
orf1
NotI (3497)
HindII (2504) HindII (9716)
SalI (2502)
SalI (9714)
EcoR I (6702)
EcoR I (9002)
EcoR I (11202)PstI (6)
PstI (7512)PstI (7722)
PstI (10228)
PstI (17500)
Restriction map of part of approximately 17.5kb of pRU3111, based on restriction
mapping and sequence data. Putative genes are marked according to sequencing
results. The three PstI fragments of pRU482 are arranged in the proposed correct
orientation, with each fragment indicated in a different colour.
The orientation as cloned into pRU482 is shown below. The two smallest fragments
are flipped round with respect to the correct orientation.
pRU482
pRU438
pRU476
pRU556
pRU544
1kb
PstI PstI PstI PstI
PstI PstI PstI PstI
PstI PstI
SalI SalI
NotI NotI EcoRI EcoRI
pRU482
Tn5-lac insertion site
110
Figure 3.11 Testcode Analysis of RU361
Testcode analysis of the iol region from pRU438 and pRU482. Sequence that is in
the top portion of the graph is likely to be coding. Sequence in the middle portion is
ambiguous and sequence in the bottom portion is unlikely to be coding.
T = testcode.
aau3 iolA orf1
0 1000 2000 3000
bp
14
12
10
T
8
6
4
111
Figure 3.12 Sequence of iolA and Surrounding DNA
1 AGAAGTTCAT CCTGGTCTTT TGATCAGGCG GCGGGGCAAC GATCGCGGGT TCTTCAAGTA GGACCAGAAA ACTAGTCCGC CGCCCCGTTG CTAGCGCCCA -3 G P R K I L R R P L S R P 51 TTGCGATAGG CGGCTCGCCG GTGATTCCGA GAAGGAAGTT GATCTGCGGA AACGCTATCC GCCGAGCGGC CACTAAGGCT CTTCCTTCAA CTAGACGCCT -3 A I P P E G T I G L L F N I Q P 101 CGCGCTTTGA CGAGGTCGTC GATGGAATAT CGAAGTACCG CAAAGAAAGC GCGCGAAACT GCTCCAGCAG CTACCTTATA GCTTCATGGC GTTTCTTTCG -3 R A K V L D D I S Y R L V A F F A 151 GTTGAGCGCC TTGCGCAGCG CCGAATTCAA ACCGCAGCTG TCGACCAGGG CAACTCGCGG AACGCGTCGC GGCTTAAGTT TGGCGTCGAC AGCTGGTCCC -3 N L A K R L A S N L G C S D V L 201 GGCATTCGAC CTCGCCGTCG TCCTCGAAGC ATTCGGCCAT GGCGAAACTG CCGTAAGCTG GAGCGGCAGC AGGAGCTTCG TAAGCCGGTA CCGCTTTGAC -3 C E V E G D D E F C E A M A F S 251 TCTTCCGTCA CCCGAACGAC GTCGAAAAGA CTAATATCGG CAGCCGGTTT AGAAGGCAGT GGGCTTGCTG CAGCTTTTCT GATTATAGCC GTCGGCCAAA -3 D E T V R V V D F L S I D A A P K 301 GCCCAAGCGC ACGCCACCGT TGCGACCGCG CACGGTTTCG ACCAGACCCG CGGGTTCGCG TGCGGTGGCA ACGCTGGCGC GTGCCAAAGC TGGTCTGGGC -3 G L R V G G N R G R V T E V L G 351 CCTTGTTCAG CGGCTGAAGG ATCTTGAAAA GAAAGAGCTC GGAAACGCCA GGAACAAGTC GCCGACTTCC TAGAACTTTT CTTTCTCGAG CCTTTGCGGT -3 K N L P Q L I K F L F L E S V G 401 TAGGCCCTGG CGATTTCCGG AATCCGGCTC AAGTGCCCGT CGTTGGACAG ATCCGGGACC GCTAAAGGCC TTAGGCCGAG TTCACGGGCA GCAACCTGTC -3 Y A R A I E P I R S L H G D N S L 451 CACAATACAT AACTGCGAMC CGCATAGTTC GCTCTGCTTC GTCAACCGCA GTGTTATGTA TTGACGCTKG GCGTATCAAG CGAGACGAAG CAGTTGGCGT -3 V I C L Q S G C L E S Q K T L R M aau3 501 TGCCATTCTC CTGCACTCGT CTGTTCAGAC CBVTVTAGGC GGTTTGCSTA ACGGTAAGAG GACGTGAGCA GACAAGTCTG GVBABATCCG CCAAACGSAT SD 551 GTTTTGAACA ATTCCCAAAA TATGAAATTC GCATTCAGCT TATGAGGCCA CAAAACTTGT TAAGGGTTTT ATACTTTAAG CGTAAGTCGA ATACTCCGGT 601 TTTGCCGCGA GGTCAATCCC GCTCCAGGAT GCTGATKTAS YCGAGGATGG AAACGGCGCT CCAGTTAGGG CGAGGTCCTA CGACTAMATS RGCTCCTACC
112
651 CTCGCCTGGG CTCGGGCATG CCTTGCACAT CAGTTTGACC TCGGCACTGG GAGCGGACCC GAGCCCGTAC GGAACGTGTA GTCAAACTGG AGCCGTGACC 701 GGGAATAGGA GATGTCGAGC AGACCGCGCC CTTGTTTCAG GACATCCTCG CCCTTATCCT CTACAGCTCG TCTGGCGCGG GAACAAAGTC CTGTAGGAGC 751 ACGCCGGACC GATATCGCCG GCAATCAGCA TGACGAGGAA AGATTCGGGA TGCGGCCTGG CTATAGCGGC CGTTAGTCGT ACTGCTCCTT TCTAAGCCCT 801 GCCAAAAGGC TCCCCAATTA CAGATTTCAG ATTGGAATGT CGTGAAGATC CGGTTTTCCG AGGGGTTAAT GTCTAAAGTC TAACCTTACA GCACTTCTAG 851 ACTTCATCGT CGGCATGACG AATTCAGCGC CGCTCTTGAT GCCCGAGGGC TGAAGTAGCA GCCGTACTGC TTAAGTCGCG GCGAGAACTA CGGGCTCCCG -1 K M T P M V F E A G S K I G S P 901 CAGCGGGCCG TGACGGTCTT GGTCTTCGTC CAGAACTTGA TCGAATCCGT GTCGCCCGGC ACTGCCAGAA CCAGAAGCAG GTCTTGAACT AGCTTAGGCA -1 W R A T V T K T K T W F K I S D T 951 GCCGTGCTGG TTGAGGTCGC CGAAGCTCGA GGCCTTCCAG CCGCCGAAGG CGGCACGACC AACTCCAGCG GCTTCGAGCT CCGGAAGGTC GGCGGCTTCC -1 G H Q N L D G F S S A K W G G F 1001 AGTGGTAGGC GAGCGGAACC GGGATCGGGA CGTTGATGCC GATCATGCCG TCACCATCCG CTCGCCTTGG CCCTAGCCCT GCAACTACGG CTAGTACGGC -1 H Y A L P V P I P V N I G I M G 1051 ATATTGATGC GTGAGGCAAA ATCGCGGGCG GCATCGCCGT CACGCGTGAA TATAACTACG CACTCCGTTT TAGCGCCCGC CGTAGCGGCA GTGCGCACTT -1 I N I R S A F D R A A D G D R T F 1101 GATGGCGACG CCGTTGCCAT ATTCGTGCTT CATCGGCAGC GACAGCGCCT CTACCGCTGC GGCAACGGTA TAAGCACGAA GTAGCCGTCG CTGTCGCGGA -1 I A V G N G Y E H K M P L S L A 1151 CCTCGTAGTT CTGGGCACGA ACGACGGAGA GGACAGGTCC GAAGATCTCG GGAGCATCAA GACCCGTGCT TGCTGCCTCT CCTGTCCAGG CTTCTAGAGC -1 E Y N Q A R V V S L V P G F I E 1201 GTCTTATAGA TATCCATGTC AGGCGTGACG TGATCGAACA GGCAACCGCC CAGAATATCT ATAGGTACAG TCCGCACTGC ACTAGCTTGT CCGTTGGCGG -1 T K Y I D M D P T V H D F L C G G 1251 GACGAAGTAG CCGTCTTCAT AGCCCTGGAG TTTGAAATCG CGGCCGTCGA CTGCTTCATC GGCAGAAGTA TCGGGACCTC AAACTTTAGC GCCGGCAGCT -1 V F Y G D E Y G Q L K F D R G D 1301 CGACGAGCTT GGCGCCTTCC TCGATGCCGC GGTCGATCAG GCCGCGAACA GCTGCTCGAA CCGCGGAAGG AGCTACGGCG CCAGCTAGTC CGGCGCTTGT -1 V L K A G E E I G R D I L G R V 1351 CGGGTATAGG CTTCCTTGGT AACGAGCGGG CCCATGTCGG CCTTGTCGTC GCCCATATCC GAAGGAACCA TTGCTCGCCC GGGTACAGCC GGAACAGCAG -1 R T Y A E K T V L P G M D A K D D
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1401 GGTATAGGGT CCGATGCGCA GGGATTCGAT CTTCGGCGTC AGCTTTTCGA CCATATCCCA GGCTACGCGT CCCTAAGCTA GAAGCCGCAG TCGAAAAGCT -1 T Y P G I R L S E I K P T L K E 1451 CGAGGCGGTT GGCAGTCTCC TCGCCGACCG GAACGGCAAC AGAGATCGCC GCTCCGCCAA CCGTCAGAGG AGCGGCTGGC CTTGCCGTTG TCTCTAGCGG -1 L R N A T E E G V P V A V S I A 9bp overlap by Tn5-lacZ in RU361 1501 ATGCAGCGCT CGCCAGCCGA ACCGTAGCCT GCGCCCATCA GCGCGTTGAC TACGTCGCGA GCGGTCGGCT TGGCATCGGA CGCGGGTAGT CGCGCAACTG -1 M C R E G A S G Y G A G M L A N V 1551 GGCCTGATCC AGGTCAGCAT CCGGCATGAT GATCATATGG TTCTTGGCGC CCGGACTAGG TCCAGTCGTA GGCCGTACTA CTAGTATACC AAGAACCGCG -1 A Q D L D A D P M I I M H N K A 1601 CGCCGAAGCA CTGGGCGCGT TTGCCGTTCA TCGCCGCGGT GCCATAGACG GCGGCTTCGT GACCCGCGCA AACGGCAAGT AGCGGCGCCA CGGTATCTGC -1 G F C Q A R K G N M A A T G Y V 1651 TAGCGGGCGA TCGGCGTCGA GCCGACGAAG GAGACGGCGC CGATATCGGG ATCGCCCGCT AGCCGCAGCT CGGCTGCTTC CTCTGCCGCG GCTATAGCCC -1 Y R A I P T S G V F S V A G I D P 1701 ATCGGTGAGG ATCGCATCGA CGGCACCCTT ATCGCCATTG ACGACGTTGA TAGCCACTCC TAGCGTAGCT GCCGTGGGAA TAGCGGTAAC TGCTGCAACT -1 D T L I A D V A G K D G N V V N 1751 GGATGCCGGC CGGCAAACCG GCCTCGATCA TCAGTTCGGC GAGACGGATC CCTACGGCCG GCCGTTTGGC CGGAGCTAGT AGTCAAGCCG CTCTGCCTAG -1 I G A P L G A E I M L E A L R I 1801 GGCAGGGAGG GATCGCGCTC GGAGGGCTTC AGGATAAAGG CGTTGCCGCA CCGTCCCTCC CTAGCGCGAG CCTCCCGAAG TCCTATTTCC GCAACGGCGT -1 P L S P D R E S P K L I F A N G C 1851 GGCGATCGCC GGCGCAAACA TCCACATCGG GATCATGCCC GGGAAATTGA CCGCTAGCGG CCGCGTTTGT AGGTGTAGCC CTAGTACGGG CCCTTTAACT -1 A I A P A F M W M P I M G P F N 1901 AGGGCGTAAT GCCGGCGCCG ATGCCGACCG GCTGGCGGAT CGAATACATG TCCCGCATTA CGGCCGCGGC TACGGCTGGC CGACCGCCTA GCTTATGTAC -1 P T I G A G I G V P Q R I S Y M 1951 TCGATCGCCG GCCCGGCGCC TTCGGTAAAC TCGCCCTTGG CGAGATGGGG AGCTAGCGGC CGGGCCGCGG AAGCCATTTG AGCGGGAACC GCTCTACCCC -1 D I A P G A G E T F E G K A L H P 2001 AATGCCGCAG ACGAATTCAC AGACTTCGAG GCCACGGATG ACGTCGCCCT TTACGGCGTC TGCTTAAGTG TCTGAAGCTC CGGTGCCTAC TGCAGCGGGA -1 I G C V F E C V E L G R I V D G 2051 TGGCATCCTC GATCGTCTTG CCGTGCTCCT TGGAGAGGAT TTCGGCAAGC ACCGTAGGAG CTAGCAGAAC GGCACGAGGA ACCTCTCCTA AAGCCGTTCG -1 A D E I T K G H E K S L I E A L
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2101 TCATCCATAT GCTTGTTCAG GAGTTCGACG AATTTGAAGA AGACGCGGGC AGTAGGTATA CGAACAAGTC CTCAAGCTGC TTAAACTTCT TCTGCGCCCG -1 E D M H K N L L E V F K F F V R A 2151 GCGGCGCTGC GGATTGGTGG CGGCCCATTT CGGCTGCGCA GCCTTGGCGT CGCCGCGACG CCTAACCACC GCCGGGTAAA GCCGACGCGT CGGAACCGCA -1 R R Q P N T A A W K P Q A A K A 2201 TTTCAACCGC GGCGCGCAAT TCCTCGACGC TTGCGAGCGC GACCGTCGCC AAAGTTGGCG CCGCGCGTTA AGGAGCTGCG AACGCTCGCG CTGGCAGCGG -1 E V A A R L E E V S A L A V T A 2251 TGGACTTCGC CGGTTGCCGG ATTGTAGACG TTGCTTACGC GGCCGCTGGT ACCTGAAGCG GCCAACGGCC TAACATCTGC AACGAATGCG CCGGCGACCA -1 Q V E G T A P N Y V N S V R G S T 2301 GCCGGCAACC TGTTTGCCGC CGATGAAATG ACCGATCTCA CGCATGGGTG CGGCCGTTGG ACAAACGGCG GCTACTTTAC TGGCTAGAGT GCGTACCCAC -1 G A V Q K G G I F H G I E R M iolA 2351 TTCCTCCTGT TTTTGGATGG CCGACAATCG CACTTCAATT TGCACAAATC AAGGAGGACA AAAACCTACC GGCTGTTAGC GTGAAGTTAA ACGTGTTTAG 2401 AATGCGCTGA TATAAGCAAC CGTTGTGCGC AAATTATAGC TCTATCGTTG TTACGCGACT ATATTCGTTG GCAACACGCG TTTAATATCG AGATAGCAAC 2451 CATCAGGCGC TGCCGCTGCT TTCAAACTTG GCACGGCACG CCAACAAGTC GTAGTCCGCG ACGGCGACGA AAGTTTGAAC CGTGCCGTGC GGTTGTTCAG 2501 AAACGCCGAC AAGTCAAACC CTGGGGGAGG TCAAACCGAT GAGCCGCAAA TTTGCGGCTG TTCAGTTTGG GACCCCCTCC AGTTTGGCTA CTCGGCGTTT 2551 CCCGTTGTCA GAATGGCGGA GCTGGAGATC GATCCGGACA CGCTTGAAAC GGGCAACAGT CTTACCGCCT CGACCTCTAG CTAGGCCTGT GCGAACTTTG 2601 CTATCGCGCA TTGCTCACGG AAGAAATCGA AGCATCCCTA GCGCTGGAAG GATAGCGCGT AACGAGTGCC TTCTTTAGCT TCGTAGGGAT CGCGACCTTC 2651 ACGGCGTCCT TTCCCTGAGC GCTGTTTCCA TCAGGGACAA CCCAAACCGG TGCCGCAGGA AAGGGACTCG CGACAAAGGT AGTCCCTGTT GGGTTTGGCC 2701 ATCCGTATCC TTGAGGTCTA CGCCGACCAG GAAGCATACG AGGCCCATCT TAGGCATAGG AACTCCAGAT GCGGCTGGTC CTTCGTATGC TCCGGGTAGA 2751 GCGAACACCG CACTTCCTTA AGTACAAAAA CCAGACGGCG CACATGGTCA CGCTTGTGGC GTGAAGGAAT TCATGTTTTT GGTCTGCCGC GTGTACCAGT 2801 CATCGCTGAC GCTTATCGAG GTCGATCCGA TCGCAATGCG TGCCAAGCCA GTAGCGACTG CGAATAGCTC CAGCTAGGCT AGCGTTACGC ACGGTTCGGT orf1 +1 M N W D D V R I F L A V A R T G Q 2851 TGAACTGGGA CGATGTCCGA ATTTTCCTCG CTGTCGCCCG CACCGGACAA ACTTGACCCT GCTACAGGCT TAAAAGGAGC GACAGCGGGC GTGGCCTGTT
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+1 I L A A S K R L G P Q S R H A L 2901 ATCCTTGCCG CCTCGAAGCG GCTGGGGCCT CAATCACGCC ACGCTCTCGC TAGGAACGGC GGAGCTTCGC CGACCCCGGA GTTAGTGCGG TGCGAGAGCG +1 P A D F A R R G A E D A A F H P 2951 GCCGGCTGAC TTCGCTCGAA GAGGCGCTGA AGACGCGGCT TTTCATCCGC CGGCCGACTG AAGCGAGCTT CTCCGCGACT TCTGCGCCGA AAAGTAGGCG +1 P H E W L R A D G R R E V F L A S 3001 CGCACGAATG GCTGCGAGCT GACGGCCGAA GAGAAGTGTT CCTGGCATCC GCGTGCTTAC CGACGCTCGA CTGCCGGCTT CTCTTCACAA GGACCGTAGG +1 A E R W K R K C S Q H R A A S A 3051 GCAGAGCGAT GGAAACGGAA ATGCTCGCAG CACAGGGCAG CCTCGGCACA CGTCTCGCTA CCTTTGCCTT TACGAGCGTC GTGTCCCGTC GGAGCCGTGT +1 R Y G N C R D C A R R R A R R L 3101 CAGATACGGC AATTGCCGGG ACTGTGCGCG TCGGCGCGCC CGACGGCTTC GTCTATGCCG TTAACGGCCC TGACACGCGC AGCCGCGCGG GCTGCCGAAG +1 R C F L S C A R M G R L I E R H P 3151 GGTGTTTCCT TTCTTGCGCG CGCATGGGCA GGTTGATCGA GCGCCATCCG CCACAAAGGA AAGAACGCGC GCGTACCCGT CCAACTAGCT CGCGGTAGGC +1 E L K I Q L V P V P R S F S L S 3201 GAACTGAAGA TCCAGCTCGT GCCGGTTCCG CGCTCTTTCT CGCTCTCGCT CTTGACTTCT AGGTCGAGCA CGGCCAAGGC GCGAGAAAGA GCGAGAGCGA +1 R E A D I A I T L Q R P D Q G R 3251 GCGCGAGGCC GATATCGCCA TCACGCTCCA GCGACCGGAC CAAGGGCGGC CGCGCTCCGG CTATAGCGGT AGTGCGAGGT CGCTGGCCTG GTTCCCGCCG +1 L V S S K L R I T R S G L Y A S 3301 TCGTTTCCTC GAAGCTGAGG ATTACACGC TCCGGCCTTT ATGCCTCG AGCAAAGGAG CTTCGACTCC TAATGTGCG AGGCCGGAAA TACGGAGC
Letters in red indicate amino acids. For amino acid code, see Appendix 3.1. Start
and stop codons for each ORF are bold and underlined. Putative SD sequences are
bold.
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Sequencing of the end of the pRU482 insert DNA using a primer from the
vector revealed that approximately 4kb upstream of the start of iolA, there
were two partial ORFs. BlastX analysis of the deduced amino acid sequence
of one ORF revealed homology to gluconokinases. Highest identity was with
the E. coli thermosensitive gluconokinase 2 (GTNK). There was 54% identity
(p-value of 6.2 x 10-13) from the start of the protein to amino acid 63 of 187.
The ORF was designated gtnK.
Ending 20bp upstream of the start of gtnK, the other partial ORF had identity
with the ATP binding protein component of dipeptide or oligosaccharide ABC
transporter systems (Dpp). Highest identity was 67% with DppF of E. coli (p-
value of 1.0 x 10-20). The identity was from amino acid 281 to 233, the end of
the protein.
When the other end of the 10kb fragment of pRU482 was sequenced, the
deduced amino acid sequence of the 213bp PstI fragment had identity to the
middle part of DppF of E. coli. There was 66% identity (p-value of 1.0 x 10-19).
The sequence identity was from amino acid 209 to 279 of 334. Based on the
sequence homology, it was postulated that the true position of this fragment
should be at the other end of the 10kb fragment, as the amino acid sequence
followed on immediately to the above sequence. The 213bp fragment was not
naturally linked to the adjacent 2.5kb PstI fragment because the homology to
DppF ended abruptly at the next PstI site. The deduced amino acid sequence
of the DNA from the next PstI site had 75% identity (p-value of 4 x 10-3)
homology over the first 133 amino acids of 334 to DppB of E.coli. Sequencing
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of another PstI fragment detailed below contained sequence that was identical
to this sequence, indicating that the 2.5kb fragment is also not naturally in that
position. When subcloning non-specific fragments, it is possible that
fragments will re-ligate to each other in the wrong order, as the ends are
identical. Based on restriction mapping of pRU3111 and sequencing of
another subclone, pRU544, it is postulated that the 2.5kb fragment was also
created unnaturally and that this PstI fragment should have been much
bigger.
Two further fragments that were subcloned from pRU3111 yielded partial
ORFs whose deduced amino acid sequence had homology with other DPP
proteins. A second 7.5kb PstI fragment was cloned into pBluescript SK-. The
resulting plasmid was designated pRU556. Sequencing of 500bp of each end
of the insert in pRU556 revealed that this was not the same fragment as that
in pRU482. At one end of the fragment the deduced amino acid sequence
revealed a gene with 66% identity (6 x 10-24) from amino acid 115 to 270 of
339 to DppB of E. coli. At the other end of the insert there was no homology
with known sequences.
A 2kb EcoRI fragment was also cloned from pRU3111 into pBluescript SK-.
The resulting fragment was designated pRU544. Sequencing of
approximately 500bp of each end of the insert revealed DNA whose deduced
amino acid sequence had homology to DppB and at the other end with DppC.
There was 51% identity (p-value of 3.5 x 10-28) over the first 110 amino acids
of DppB of E. coli. There was 67% identity (p-value of 1.2 x 10-48) over the
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final 149 amino acids (152 to 300) of DppC of E. coli. Restriction mapping
and sequence homologies to DppB indicate that this EcoRI fragment overlaps
the PstI fragment of pRU556 (Figure 3.8).
Restriction mapping of pRU556 and pRU544, along with homology of the
deduced amino acid sequences with known genes meant that it was possible
to postulate the relative positions of pRU556, pRU544 and pRU482. A
restriction map of part of pRU3111 with putative gene positions marked is
shown in Figure 3.10.
Apart from iolA, none of the putative genes identified had any homology with
ones involved in myo-inositol utilisation. For this reason, these subclones
were not investigated further during this project.
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3.2.3.3 RU307
An intact Tn5 was cloned from RU307 as an approximately 7900bp EcoRI
fragment, with approximately 2100bp of chromosomal DNA flanking both ends
of the transposon into the cloning vector pACYC184 by co-workers in the
laboratory (Figure 3.13). The resulting plasmid was designated pRU426.
The upstream region of Tn5 consisted of 700bp of chromosomal DNA, which
was sequenced on both strands. Downstream of Tn5 were approximately
1400bp of chromosomal DNA. Sequencing was carried out for approximately
500bp from either end of this region of chromosomal DNA. A region of
approximately 400bp in the middle was not sequenced. A 9bp overlap was
created in RU307 by insertion of Tn5 (691-699bp). The sequence that was
repeated is highlighted in Figure 3.14.
BlastX analysis of the deduced amino acid sequence revealed that the
transposon interrupted an ORF with identity to the ATP binding component of
ABC transport systems involved in the uptake of D-galactose and methyl-
galactoside (Mgl). The highest homology was 54% identity over the entire
sequence of MglA of E. coli (p-value of 2 x 10-71), with a gap of approximately
400bp in the middle of the gene as the entire region was not sequenced. The
gene encoded by the ORF was designated intA (myo-inositol transport). A
putative Shine-Dalgarno sequence was identified (Shine and Dalgarno, 1974).
There was no stop codon present on pRU426, so it is postulated that the
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putative gene is at least 200bp longer than its homologues. The gene is
predicted to be transcribed in the same direction as Tn5.
Ending 5bp upstream of the putative start of the intA gene was an ORF whose
deduced amino acid sequence had 75% identity (p-value of 1 x 10-38) over the
final 100 amino acids of 309 of MocB of S. meliloti and R. leguminosarum bv.
viciae. This gene encodes a periplasmic binding protein that binds to
rhizopines and is thought to be part of a rhizopine transport system. The gene
encoded by the ORF was designated intB and is predicted to be transcribed in
the same direction as intA. There was also high homology with D-galactose
binding proteins. This was also the case for the myo-inositol binding protein
of a Pseudomonas species (Deshusses and Belet, 1984). Plasmid pRU426
did not contain the beginning of the gene. The sequence of pRU426 is
presented in Figure 3.14
On the basis of the sequence data, it was postulated that RU307 is mutated in
an operon of genes encoding an ABC transport system that might be
responsible for uptake of myo-inositol or a derivative. This led to the
discovery during this project that RU307 was severely impaired in the ability to
utilise myo-inositol as the sole carbon source (c.f. Section 3.2.1).
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Figure 3.13 pRU426
pRU42612145 bp
tetracycline res is tance
intBintA
intA
p15A origin
Tn5
Sst I
Eco R I (2)
Eco R I (7902)
Not I (1294)
Not I (5920)
Sal I (3384)
Sal I (10047)
PstI (1384)
PstI (2432)
PstI (3355)
PstI (5838)
PstI (7303)
Approximately 7900bp EcoRI fragment from RU307 containing intact Tn5 and
flanking chromosomal DNA cloned into pACYC184.
The size of the plasmid is approximate, as the entire chromosomal insert DNA was
not sequenced.
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Figure 3.14 Sequence of pRU426
intB +1 E F D A V I S N N D E M A I G A I 1 GAATTCGACG CCGTGATCTC CAACAACGAC GAAATGGCGA TCGGCGCCAT CTTAAGCTGC GGCACTAGAG GTTGTTGCTG CTTTACCGCT AGCCGCGGTA +1 Q A L K A A G K D M T K V V V G 51 CCAAGCGCTG AAGGCGGCCG GCAAGGATAT GACCAAGGTC GTCGTCGGCG GGTACGCGAC TTCCGCCGGC CGTTCCTATA CTGGTTCCAG CAGCAGCCGC +1 G V D A T Q D A L A A M Q A G D L 101 GTGTCGATGC CACGCAGGAC GCGCTTGCCG CCATGCAGGC GGGCGATCTC CACAGCTACG GTGCGTCCTG CGCGAACGGC GGTACGTCCG CCCGCTAGAG +1 D V T V F Q D A A G Q G K G S L 151 GACGTCACGG TGTTCCAGGA TGCCGCCGGC CAGGGCAAGG GCTCTCTCGA CTGCAGTGCC ACAAGGTCCT ACGGCGGCCG GTCCCGTTCC CGAGAGAGCT +1 A A L K L A K G E K I E K K V Y 201 TGCAGCGCTC AAGCTCGCTA AAGGCGAGAA GATCGAAAAG AAGGTCTACA ACGTCGCGAG TTCGAGCGAT TTCCGCTCTT CTAGCTTTTC TTCCAGATGT +1 I P F Q L V T P A N V K D F V T K 251 TTCCCTTCCA GCTCGTCACG CCTGCCAACG TCAAGGACTT CGTCACCAAG AAGGGAAGGT CGAGCAGTGC GGACGGTTGC AGTTCCTGAA GCAGTGGTTC intA +3 M P H R M R A L P I L F R +1 N SD 301 AACTAAGGCG GATGCCACAC AGGATGCGGG CTTTGCCSAT CCTTTTTCGC TTGATTCCGC CTACGGTGTG TCCTACGCCC GAAACGGSTA GGAAAAAGCG +3 L P V S G G D D M A V S P T T M A 351 TTACCCGTAT CCGGAGGAGA TGATATGGCC GTCAGCCCGA CAACCATGGC AATGGGCATA GGCCTCCTCT ACTATACCGG CAGTCGGGCT GTTGGTACCG +3 A V R A S G A V P N A E Y L L S 401 CGCCGTGCGC GCGAGCGGCG CAGTCCCGAA TGCGGAATAT CTCTTGAGCG GCGGCACGCG CGCTCGCCGC GTCAGGGCTT ACGCCTTATA GAGAACTCGC +3 A E G V R K E F P G V V A L D D V 451 CCGAGGGTGT CCGCAAGGAA TTTCCTGGTG TCGTCGCACT CGACGATGTG GGCTCCCACA GGCGTTCCTT AAAGGACCAC AGCAGCGTGA GCTGCTACAC +3 Q F R L K R A S V H A L M G E N G 501 CAGTTCCGGC TAAAGCGCGC CTCCGTGCAT GCGCTGATGG GCGAAAACGG GTCAAGGCCG ATTTCGCGCG GAGGCACGTA CGCGACTACC CGCTTTTGCC +3 A G K S T L M K I L A G I Y T P 551 CGCCGGCAAA TCGACATTGA TGAAGATCCT CGCCGGCATC TATACGCCGG GCGGCCGTTT AGCTGTAACT ACTTCTAGGA GCGGCCGTAG ATATGCGGCC
123
+3 D K G D I R L K G I E I Q L K S P 601 ACAAGGGCGA TATTCGCCTG AAAGGGATCG AGATCCAGCT GAAATCTCCG TGTTCCCGCT ATAAGCGGAC TTTCCCTAGC TCTAGGTCGA CTTTAGAGGC 9bp overlap by Tn5 in RU307 +3 L D A L E N G I A M I H Q E L N L 651 CTCGACGCAC TCGAGAATGG GATTGCCATG ATCCATCAGG AGCTGAACCT GAGCTGCGTG AGCTCTTACC CTAACGGTAC TAGGTAGTCC TCGACTTGGA +3 M P F M T V A E N I W I R R E P 701 GATGCCGTTC ATGACGGTTG CCGAAAATAT CTGGATTCGC CGCGAACCGA CTACGGCAAG TACTGCCAAC GGCTTTTATA GACCTAAGCG GCGCTTGGCT +3 K N R L G F I D H G V M H R M T E 751 AGAACCGCCT CGGTTTCATC GATCACGGCG TGATGCACCG CATGACCGAG TCTTGGCGGA GCCAAAGTAG CTAGTGCCGC ACTACGTGGC GTACTGGCTC +3 E L F T R L N I A I D P D I E V R 801 GAATTGTTTA CTCGGCTCAA TATCGCAATC GATCCTGATA TCGAGGTCCG CTTAACAAAT GAGCCGAGTT ATAGCGTTAG CTAGGACTAT AGCTCCAGGC +3 F L S V A N R Q M V E I A K A V 851 ATTCCTCTCG GTCGCCAACC GGCAGATGGT CGAGATCGCC AAGGCGGTTT TAAGGAGAGC CAGCGGTTGG CCGTCTACCA GCTCTAGCGG TTCCGCCAAA +3 S Y N S D V L I M D E P T S A L T 901 CCTATAATTC CGATGTGCTG ATCATGGACG AGCCGACTTC GGCGCTGACC GGATATTAAG GCTACACGAC TAGTACCTGC TCGGCTGAAG CCGCGACTGG +3 E R E V E H L F R I I R D L K A Q 951 GAGCGCGAGG TCGAGCATCT TTTCCGTATC ATCCGCGACC TCAAGGCCCA CTCGCGCTCC AGCTCGTAGA AAAGGCATAG TAGGCGCTGG AGTTCCGGGT +3 G I G I V Y I T H K M N E L F E 1001 GGGTATCGGC ATCGTCTACA TCACCCACAA GATGAACGAG CTTTTCGAGA CCCATAGCCG TAGCAGATGT AGTGGGTGTT CTACTTGCTC GAAAAGCTCT +3 I A D E F S V F R D G R Y I G T H 1051 TCGCCGACGA GTTCTCCGTC TTCCGTGACG GGCGATATAT CGGCACGCAT AGCGGCTGCT CAAGAGGCAG AAGGCACTGC CCGCTATATA GCCGTGCGTA +3 A S T D V T R D D I I R M M V G R 1101 GCCTCGACCG ACGTCACCCG CGACGACATC ATCCGCATGA TGGTCGGGCG CGGAGCTGGC TGCAGTGGGC GCTGCTGTAG TAGGCGTACT ACCAGCCCGC +3 E I T Q M F P K E E G A D R R G 1151 CGARATCACC CARATGTTTC CCAAGGAAGA AGGTGCCGAT CGGCGAGGTC GCTYTAGTGG GTYTACAAAG GGTTCCTTCT TCCACGGCTA GCCGCTCCAG +3 H A F R Q G F L S Q R R L Q K V S 1201 ATGCTTTCCG TCAAGGATTT TTGTCTCAAC GGCGTCTTCA AAAAGTYTCC TACGAAAGGC AGTTCCTAAA AACAGAGTTG CCGCAGAAGT TTTTCARAGG
124
Unsequenced region of approximately 400bp. +3 G P P E A P A K K G K K S A R E N 1551 GGCCCCGTCG AGGCACCTGC GAAGAATGGC AAAAAAAGTG CGCGTGAAAA CCGCGGCAGC TCCGTGGACG CTTCTTACCG TTTTTTTCAC GCGCACTTTT +3 A N L Y E R V E N L S G G N Q Q 1601 CGCCAATCTT TACGAGCGGG TGGAAAATCT TTCGGGCGGC AATCAGCARA GCGGTTAGAA ATGCTCGCCC ACCTTTTAGA AAGCCCGCCG TTAGTCGTYT +3 K V L I G R W L L T N P R I L I L 1651 AGGTGCTGAT CGGGCGCTGG CTGCTCACCA ATCCGCGCAT CCTCATCCTC TCCACGACTA GCCCGCGACC GACGAGTGGT TAGGCGCGTA GGAGTAGGAG +3 D E P T R G I D V G A K A E I H R 1701 GACGAGCCGA CGCGCGGCAT CGATGTCGGC GCCAAGGCGG AAATCCACCG CTGCTCGGCT GCGCGCCGTA GCTACAGCCG CGGTTCCGCC TTTAGGTGGC +3 L V T E M A R D G V A V V M I S 1751 GTTGGTCACG GAGATGGCGC GAGACGGTGT GGCGGTGGTG ATGATCTCGT CAACCAGTGC CTCTACCGCG CTCTGCCACA CCGCCACCAC TACTAGAGCA +3 S E M P E V L G M S D R I M V M H 1801 CCGAGATGCC TGAGGTTCTC GGGATGAGCG ACCGCATCAT GGTCATGCAC GGCTCTACGG ACTCCAAGAG CCCTACTCGC TGGCGTAGTA CCAGTACGTG +3 E G R V T G F L N R D E A T Q I K 1851 GAGGGACGCG TGACCGGTTT CCTCAATCGC GACGAAGCAA CGCAGATCAA CTCCCTGCGC ACTGGCCAAA GGAGTTAGCG CTGCTTCGTT GCGTCTAGTT +3 V M E L V R S D A L T A I A Q G 1901 GGTGATGGAG CTGGTGCGCA GTGATGCGTT GACGGCGATC GCCCAAGGGA CCACTACCTC GACCACGCGT CACTACGCAA CTGCCGCTAG CGGGTTCCCT +3 R F D M T T K A A E G A A P L A T 1951 GGTTTGACAT GACTACCAAG GCAGCAGAGG GCGCGGCTCC GCTCGCAACC CCAAACTGTA CTGATGGTTC CGTCGTCTCC CGCGCCGAGG CGAGCGTTGG +3 R Q R R R R I P T E L S I F L V L 2001 CGGCAAAGGC GGCGGCGCAT ACCGACGGAG CTCAGCATTT TCCTCGTGCT GCCGTTTCCG CCGCCGCGTA TGGCTGCCTC GAGTCGTAAA AGGAGCACGA +3 V G I A L I Y E V L G W M F I G 2051 CGTCGGCATC GCGCTCATCT ACGAAGTGCT TGGCTGGATG TTCATCGGCC GCAGCCGTAG CGCGAGTAGA TGCTTCACGA ACCGACCTAC AAGTAGCCGG +3 Q S F L M N 2101 AAAGCTTCCT CATGAATTC TTTCGAAGGA GTACTTAAG Letters in red indicate amino acids. For amino acid code, see Appendix
3.1. Start and stop codons for each ORF are bold and underlined.
Putative SD sequences are bold.
125
3.2.5 Uptake of Glucose and myo-Inositol
Results presented above show that RU307 was severely impaired in the
ability to utilise myo-inositol as the sole carbon source and that this phenotype
was linked to the transposon insertion. The DNA sequence of the region
interrupted by the transposon had identity with components of ABC sugar
transport systems. The deduced amino acid sequence of a partial ORF
immediately upstream of the mutation resembled MocB of the rhizopine
catabolic system and D-galactose binding proteins. A myo-inositol uptake
mutant of Pseudomonas sp. JD34 also only grew very slowly on myo-inositol
(Frey et al., 1983). The myo-inositol binding protein of another Pseudomonas
species also resembled D-galactose binding proteins (Deshusses and Belet,
1984). Therefore, it was hypothesised that RU307 is mutated in an uptake
system for myo-inositol.
Previously, Poole et al. (1994), measured uptake of myo-inositol in 3841,
RU360 and RU361, using myo-[2-3H]-inositol. The authors reported that there
was constitutive uptake of radio-labelled myo-inositol by the strains but uptake
was at a lower rate than glucose uptake. It was reported that in K. aerogenes,
active transport of myo-[2-3H]-inositol is complicated by the presence of a
dehydrogenase that attacks the substrate at C-2, causing loss of the label
(Deshusses and Reber, 1972).
Therefore, 14C-labelled myo-inositol was used to measure uptake in 3841 and
in the mutants RU360, RU361 and RU307 (Figure 3.15). The strains were
126
grown on 20mM pyruvate or 20mM pyruvate plus 10mM myo-inositol as the
sole carbon sources. Uptake was also measured for 3841 and RU307 grown
on 10mM myo-inositol alone. All uptake experiments were also carried out
with 14C-labelled glucose, to determine whether the mutants were affected in
their ability to transport glucose (Figure 3.16).
The uptake data were subjected to Analysis of Variance (Appendix 3.2).
There was no significant difference in the rates of uptake of myo-inositol by
each strain grown on 20mM pyruvate, RU360 and RU307 grown on pyruvate
plus myo-inositol and RU307 grown on myo-inositol (P>0.1) (Figure 3.15).
When grown on 20mM pyruvate and 10mM myo-inositol, there was a 2.5-fold
increase in the rate of uptake of myo-inositol by 3841 and a 1.7-fold increase
by RU361 compared with the rate of uptake when grown on pyruvate.
Although the increase in uptake by RU361 was significantly higher than
RU361 grown on pyruvate alone, (P<0.05), it was still significantly lower than
the rate of uptake by 3841 (P<0.05). There was a 7-fold increase in uptake by
3841 when grown on 10mM myo-inositol as the sole carbon source. These
data indicate that there is an inducible system for the transport of myo-inositol
in R. leguminosarum biovar viciae that is expressed in 3841 but not in the
mutants.
Uptake of glucose occurred in all strains when grown either on 20mM
pyruvate, 20mM pyruvate with 10mM myo-inositol, or 10mM myo-inositol as
the sole carbon source (Figure 3.15). The data were subjected to Analysis of
127
Variance (Appendix 3.3). There was no significant difference in the rate of
uptake of glucose by the strains on different carbon sources (P>0.1). These
data indicate that the mutants were not impaired in the ability to transport
glucose into the cell.
128
Figure 3.15 Uptake of 14C-labelled myo-inositol
Cells were grown in AMS containing either 20mM pyruvate (P), 10mM myo-inositol (I)
or 20mM pyruvate plus 10mM myo-inositol (PI).
Mean uptake of myo-inositol per minute over four minutes. Mean results of three
experiments with standard deviation values.
129
Figure 3.16 Uptake of Glucose
Cells were grown in AMS containing either 20mM pyruvate (P), 10mM myo-inositol (I)
or 20mM pyruvate plus 10mM myo-inositol (PI).
P = 20mM pyruvate, I = 10mM myo-inositol.
Mean uptake of glucose over four minutes. Mean results of three experiments with
standard deviation values.
130
The deduced amino acid sequence of the putative intA gene of RU307 has
high homology to MglA, a component of the galactose and methyl-galactoside
uptake system. Therefore, the uptake system might not be specific to one
compound. To determine the specificity of the transport system, the uptake of
myo-inositol by 3841 was measured after addition of a 5-fold excess of
different carbon compounds (Figure 3.17). The uptake data were subjected to
Analysis of Variance (Appendix 3.4). Addition of a 5-fold excess of unlabelled
myo-inositol significantly inhibited uptake of radio-labelled myo-inositol
(P<0.05) but none of the other carbon compounds inhibited myo-inositol
uptake. There was no significant difference in the rate of uptake of myo-
inositol when different carbon compounds were added (P>0.1).
The inhibition experiment was also carried out using radio-labelled glucose.
The uptake system for glucose is well defined in bacteria, so no inhibition was
expected. Unlabelled carbon compounds were added in 5-fold excess prior to
addition of the radio-labelled carbon (Figure 3.18). The uptake data were
subjected to Analysis of Variance (Appendix 3.5). Addition of a 5-fold excess
of unlabelled myo-inositol significantly inhibited uptake of radio-labelled myo-
inositol (P<0.05) but none of the other carbon compounds inhibited myo-
inositol uptake. There was no significant difference in the rate of uptake of
myo-inositol when different carbon compounds were added (P>0.1).
The results indicate that the inducible transport system for myo-inositol uptake
in R. leguminosarum bv. viciae is myo-inositol-specific.
131
Figure 3.17 Uptake of myo-Inositol When Excess Carbon Compounds
are Added
Cells were grown in AMS containing 10mM myo-inositol.
Mean uptake by 3841 of radio-labelled myo-inositol over four minutes when 5-fold
excess carbon compound added. Mean results of three experiments with standard
deviation values.
132
Figure 3.18 Uptake of Glucose When Excess Carbon Compounds are
Added
Cells were grown in AMS containing 10mM myo-inositol.
Mean uptake of glucose over four minutes when 5-fold excess of carbon
compound added. Mean results of three experiments with standard deviation
values.
133
3.3 Discussion
There is a specific pathway of myo-inositol catabolism in R. leguminosarum
bv. viciae, as the mutants RU360, RU361 and RU307 were all able to grow at
similar rates to 3841 on several difference carbon sources, but RU360 and
RU361 were unable to grow in vitro when myo-inositol was the sole carbon
source. Strain RU307 was able to grow on myo-inositol but was severely
limited, as growth was at a quarter of the rate of 3841 on myo-inositol. The
data also show that the presence of a transposon did not affect growth on
other carbon sources. myo-Inositol did not inhibit growth of the mutants when
present in the growth medium in addition to pyruvate or glucose, indicating
that there are no toxic products produced by partial breakdown of myo-inositol
by the mutants.
When complemented by cosmids, RU360/pRU3078, RU360/pRU3079 and
RU361/pRU3111 grew at the same rates on glucose or myo-inositol as 3841.
These data indicate that the ability to utilise myo-inositol was completely
restored and also that the presence of a cosmid did not inhibit growth. The
inability of the mutants and 3841 to grow when phytic acid was the sole
carbon source, either in the presence or absence of phosphate, indicates that
this compound is not a likely energy source in the rhizosphere for R.
leguminosarum bv. viciae.
Sequencing of the genes interrupted by Tn5-lacZ in the mutants RU360 and
RU361 showed that the pathway for myo-inositol utilisation in R.
134
leguminosarum bv. viciae is not organised in a single operon, as occurs in B.
subtilis (Yoshida et al., 1997). There must be at least one other locus of myo-
inositol degradation genes, as only putative genes involved in the final stages
of myo-inositol catabolism were discovered. These genes may not be
encoded on the chromosome, but on plasmids as occurs in R. leguminosarum
bv. trifolii (Baldani et al., 1992) and with rhizopine catabolism genes of S.
meliloti (Murphy et al., 1987) (c.f. Section 1). This could be ascertained by
curing 3841 of plasmids and determining whether the ability to grow on myo-
inositol as the sole carbon source is retained.
The discovery that the interrupted gene in RU360 may encode a protein that
has high homology to acetolactate synthases, including IolD of B. subtilis, led
to the hypothesis that the catabolic pathway as elucidated for K. aerogenes is
not complete. Acetolactate synthase is required for the synthesis of
acetolactate from pyruvate and acetyl CoA. This compound is the precursor
of the amino acids valine, leucine and isoleucine. In B. subtilis, the product of
the iolD gene is also essential for myo-inositol breakdown (Yoshida et al.,
1997). Therefore, it is proposed that the catabolic pathway requires an extra
step (Figure 3.17).
135
Figure 3.17 Proposed Pathway of myo-Inositol Catabolism in R.
leguminosarum bv. viciae
myo-inositol
myo-inositol dehydrogenase
2-keto-myo-inositol
2-keto-inositol dehydratase
D-2,3-diketo-4-deoxy-epi-inositol
*
2-deoxy-5-keto-D-gluconic acid
DKH kinase
diketohydroxy 6-phosphate
DKHP aldolase
malonic semialdehyde dihydroxyacetone phosphate
acetyl CoA + CO2
pyruvate
acetolactate synthase
acetolactate + CO2
Putative functions of Iol proteins from B. subtilis are shown.
* The pathway of conversion of D-2,3-diketo-4-deoxy-epi-inositol to 2-deoxy-5-keto-
D-gluconic acid has not been characterised. Conversion may occur spontaneously,
or through the action of a hydratase.
MSA oxidative decarboxylase
NAD
NADH +
NAD
NADH + H+
H2O
H2O
CoA
ATP
ADP
ADP
ATP
NAD
NADH +
TCA cycle
IolA
IolD
IolJ
IolC
iolG
136
E. coli and S. typhimurium have three acetolactate synthases that are all
under different modes of regulation, but the results of one enzyme activity
study suggested that R. leguminosarum bv. viciae has only one copy
(Royuela et al., 1998). If iolD encodes an acetolactate synthase that is
essential for myo-inositol catabolism, then there must be more than one gene
encoding acetolactate synthase in R. leguminosarum bv. viciae, as RU360
would not be able to grow on any carbon source without supplementation in
the medium of valine and isoleucine.
Other acetolactate synthase genes must be under regulatory systems that are
not induced by myo-inositol, otherwise they would be expected to compensate
for the mutation in RU360. To test whether iolD is essential for myo-inositol
catabolism in R. leguminosarum bv. viciae, just the iolE and iolB genes could
be cloned and tested for complementation, or point mutation could be carried
out on iolD, to eliminate any polar effect on downstream genes. It was shown
that iolD of B. subtilis is essential (Yoshida et al., 1997) and due to time
constraints this was not considered a priority in this project. To test whether
iolD is a 1680bp or 1881bp gene, PCR amplification of the two ORFs could be
carried out in order to produce two different size clones, which could then be
assayed for acetolactate synthase activity. The N-terminus of the purified
protein could also be sequenced.
The lack of complementation by pRU706, which contains just the iolD gene
and the complementation by pRU713, which contains all three genes,
indicates that the mutation in iolD is polar on the downstream genes, iolE and
137
iolB. Therefore, at least two of these three genes probably share the same
promoter and one or both of these genes is essential for myo-inositol
catabolism. This concurs with data obtained from B. subtilis that iolE and iolB
are essential for myo-inositol catabolism, although the specific functions of the
genes are not known (Yoshida et al., 1997). It is not currently possible to
predict their role in myo-inositol utilisation, as other homologues in the
databases have no known function. The requirement for iolE and iolB could
also be tested by point mutation of the individual genes. It is not known
whether orf1 is required for myo-inositol utilisation, although testcode analysis
indicated that this gene is unlikely to be coding again. This could be tested by
mutation of the gene.
The deduced amino acid sequence of the DNA interrupted by Tn5-lacZ in
RU361 had homology with methylmalonate semialdehyde dehydrogenases,
including IolA of B. subtilis. Based on sequence homology, the putative
function of the protein is malonic semialdehyde oxidative decarboxylase, an
enzyme active in the final stages of the myo-inositol breakdown pathway. To
determine whether just iolA would be required to restore the ability to utilise
myo-inositol to RU361, the putative gene could be obtained by PCR
amplification and conjugated into RU361 to see whether it would restore the
ability to utilise myo-inositol. Due to time constraints, this was not attempted.
Further sequencing of the complementing cosmid pRU3111 revealed the
presence of several putative genes. Upstream of iolA was a putative gene
whose deduced amino acid sequence had homology to gluconokinases.
138
However, there was no significant homology with IolC of the B. subtilis iol
operon, which is postulated to be a gluconokinase (Yoshida et al., 1997).
Whether this putative gene is involved in myo-inositol breakdown could be
ascertained by mutation of the gene in 3841 to see if myo-inositol catabolism
is knocked out.
Downstream of the iolA gene was a putative gene whose deduced amino acid
sequence had homology to Aau3 of S. meliloti. This is required for utilisation
of PHB cycle intermediates. PHB is one of the principal carbon storage
compounds in nodules. Other proteins in the databases that have identity
with the putative gene have no known function. It is not thought that this gene
has any role in myo-inositol utilisation due to its lack of homology with known
iol genes, but this could also be investigated by mutation of the gene in 3841.
Sequencing of other subclones of pRU3111 revealed ORFs whose deduced
amino acid sequence had strong homology with components of the DPP
operon. These proteins are not expected to be involved in myo-inositol
utilisation. Hence, they were not investigated further.
A complementing cosmid was not obtained for RU307, so no information was
obtained for genes surrounding the Tn5 insertion. Transduction of RU307
showed that both the myo-inositol and the toxic escape phenotypes were
tightly linked to the transposon insertion. Without further sequence
information and a complementing cosmid, it was not possible to elucidate the
molecular basis of the glutamic acid gamma hydrazide phenotype.
139
It was hypothesised that RU307 might be impaired in the ability to utilise myo-
inositol because the deduced amino acid sequence of the putative gene
upstream of the Tn5 mutation had homology with putative rhizopine and D-
galactose binding proteins. A Pseudomonas species myo-inositol binding
protein also has homology to D-galactose binding proteins (Deshusses and
Belet, 1984) and a Pseudomonas sp. JD34 myo-inositol uptake mutant was
impaired in growth on myo-inositol, but not completely unable to grow. This
evidence strongly suggests that RU307 is mutated in an uptake system for
myo-inositol.
Uptake experiments using radio-labelled myo-inositol showed that 3841 has
an inducible system for the uptake of myo-inositol that the mutants RU360,
RU361 and RU307 do not express. This transport system in R.
leguminosarum bv. viciae is myo-inositol specific, as there was no inhibition in
3841 by an excess of a variety of different carbon compounds, including D-
galactose. The ATP binding component of ABC transport systems are highly
conserved, so it was not unexpected that the transport system was not
actually a D-galactose transporter, despite the high homology. If the transport
system were responsible for transporting glutamic acid gamma hydrazide into
the cell, then an excess of this compound would have been expected to block
transport of myo-inositol in 3841, but it did not. Therefore, the uptake system
mutated in RU307 is not a high affinity glutamic acid gamma hydrazide
transport system. It is not possible from results gained during this project to
explain the ability of RU307 to grow in the presence of glutamic acid gamma
hydrazide.
140
The inducible system for uptake of myo-inositol by 3841 was repressed by the
presence of pyruvate in the growth medium. This suggests that myo-inositol
is not preferentially utilised when other carbon sources are present. However,
this might be specific to pyruvate, as uptake was not tested in the presence of
other sugars. The mutants were all able to transport glucose into the cell at
the same rate as 3841, indicating that there was no deficiency in uptake of
this sugar.
There was a low rate of uptake of myo-inositol into the cell in all the strains
when grown on pyruvate. This suggests the presence of a constitutive system
for myo-inositol transport which may not necessarily be myo-inositol specific.
There are two uptake systems in P. putida, one high affinity system involving
a periplasmic binding protein and a low affinity system not associated with a
binding protein (Reber et al., 1977). The presence of this second system may
explain the slow growth by RU307 on myo-inositol. As the rate of uptake was
so much lower, the growth rate was also greatly reduced. Therefore, it is
likely that RU307 is mutated in a myo-inositol uptake system. If one or more
of the final breakdown products of myo-inositol induce uptake, this would
explain why there was no induction of uptake in RU360 and RU361 as they
were blocked in the breakdown of myo-inositol. To locate the second uptake
system would require a second mutation to be introduced into RU307.
Bahar et al. (1998) reported that 3841 can catabolise rhizopines when a
plasmid containing moc genes is introduced. Although there is a rhizopine
binding gene (mocB) amongst the moc genes, there is no known transport
141
system. Therefore, it was hypothesised that rhizopines entered the cell using
a chromosomally encoded transport system. This could be the system
mutated in RU307. The moc gene plasmid could be introduced into RU307
and the strain tested for the ability to grow on rhizopines as the sole carbon
and nitrogen source. It is postulated that RU307 will grow very slowly, or not
at all.
In order to fully elucidate the myo-inositol catabolic pathway in R.
leguminosarum bv. viciae, several more genes need to be identified. An
attempt was made to complement a S. meliloti myo-inositol dehydrogenase
(idh) mutant using the 3841 cosmid library in the hope of isolating a region of
DNA containing a R. leguminosarum bv. viciae myo-inositol dehydrogenase
gene. However, this was unsuccessful. Another strategy would be PCR
amplification using primers designed from the S. meliloti idh gene. This might
also be possible using primers from the B. subtilis myo-inositol
dehydrogenase gene iolG, as based on results gained in this project, this
gene is likely to have high homology with R. leguminosarum bv. viciae genes.
CHAPTER 4 - NODULATION AND RHIZOSPHERE GROWTH ................ 142
4.1 Introduction .......................................................................................... 143
4.2 Results .................................................................................................. 145
4.2.1. Rate of Nodulation ................................................................................... 145
4.2.1.1 Dry Weight of Vetch Plants ................................................................ 149
4.2.2 Acetylene Reduction Assay on Pea Nodules............................................ 151
142
4.2.3 Plant Dry Weights for Pea......................................................................... 155
4.2.4 Nodule Number and Mass for Pea............................................................ 157
4.2.5 Competition for Nodulation......................................................................... 161
4.2.5.1 Competitive Ability of the Complemented Mutants............................. 170
4.2.6 Measurement of Nodule Co-Residence .................................................... 173
4.2.7 Average Number of Nodules per Vetch Plant ........................................... 176
4.2.9 Rhizosphere Growth Assay................................................................... 186
4.2.10 Analysis of a nodC-phoA Fusion............................................................. 192
4.3 Discussion............................................................................................. 195
142
Chapter 4 - Nodulation and Rhizosphere Growth
143
4.1 Introduction
myo-Inositol is present in soil and in the rhizosphere (Lynch et al., 1958,
McKercher and Anderson, 1968, Sulochana, 1962, Yoshida, 1940, Wood and
Stanway, 2000). It is also found in plant tissues, including legume root
nodules (Kouchi and Yoneyama, 1984, 1986, Skøt and Egsgaard, 1984,
Streeter, 1987; Streeter and Salminen, 1985). There are many micro-
organisms competing in the rhizosphere for resources. Therefore, there may
be a competitive advantage for bacteria that are able to utilise myo-inositol or
derivatives such as rhizopines.
The results of research carried out to date are mixed. Heinrich et al. (1999)
reported that a rhizopine producing strain of S. meliloti occupied more nodules
than a non-producing strain when co-inoculated into the rhizosphere of alfalfa.
The competitive advantage remained over a period of four years. However,
Bosworth et al (1994) and Scupham et al. (1996) found that disrupting a locus
of inositol genes sometimes enhanced competitive ability. Raggio et al.
(1959) found that supplementation of the growth medium of bean with
mesoinositol (myo-inositol) resulted in an increase in the percentage of
nodulated roots and nodules per root of R. leguminosarum bv. phaseoli. The
reasons for these effects are unknown.
In Chapter 3, it was shown that the myo-inositol mutants are able to grow in
vitro on different carbon sources at the same rate as 3841 and that the
presence of myo-inositol in the culture media does not inhibit growth. RU360
144
was previously shown to nodulate vetch and reduce acetylene at the same
rate as 3841 (Poole et al., 1994). It was decided to test whether the myo-
inositol mutants RU360, RU361 and RU307 would be at a competitive
disadvantage when co-inoculated into a plant rhizosphere with 3841 in order
to determine whether the ability to utilise myo-inositol is important for
rhizosphere growth and survival.
Experiments were carried out in the rhizosphere of pea (P. sativum) and vetch
(V. sativa) plants, both of which R. leguminosarum bv. viciae nodulates.
Vetch was used primarily as the plants are smaller and so are easier to
cultivate in large numbers. It would also have been impractical to analyse
every nodule on pea plants. The plants were grown in sterile vermiculite or
water agar with a defined nutrient solution, to ensure that no exogenous
factors could affect results and to ensure standardisation between
experiments.
Several aspects of growth in the rhizosphere were considered for the mutants
and 3841. These were the ability to grow in the rhizosphere, nodulate plants,
nitrogen fixation rates, average plant mass, average number of nodules and
nodule mass per plant and the time taken for nodules to form. The ability of
the mutants to successfully form nodules when co-inoculated into the
rhizosphere with 3841 was also investigated. The ability to respond to plant
signals was measured using the nodC gene fused to the phoA gene.
145
4.2 Results
4.2.1. Rate of Nodulation
It was shown previously that RU360 can nodulate vetch plants (Poole et al.,
1994). This needed to be established for the mutants RU361 and RU307 and
whether they were affected in the rate or number of nodule formation
compared with 3841. Vetch seedlings were grown in boiling tubes containing
0.2% water agar supplemented with nitrogen-free rooting solution. This
enabled nodule formation to be observed without disturbing the plants.
Representative plants and roots inoculated with each strain are shown in
Figure 4.1 and Figure 4.2.
Seven day-old seedlings were inoculated with 103 cfu of RU360, RU361,
RU307 or 3841. Each strain was inoculated onto eight plants. Eight plants
were uninoculated. Nodules were first observed on at least one plant per
replicate four days post-inoculation (Figure 4.3). Nodule numbers increased
rapidly until 11 days post-inoculation after which there was just a slight
increase in nodules of RU361 and 3841 17 days post-inoculation. The
increase in nodule numbers was similar for the mutants and wildtype
throughout the experiment. No nodules were observed on any of the
uninoculated plants. The plants were harvested 21 days post-inoculation.
The data were subjected to Analysis of Variance (Appendix 4.1). There was
no significant difference in the overall average number of nodules per plant
146
(P>0.1). All nodules appeared pink in colour, which signifies the presence of
leghaemoglobin and suggests that the nodules were actively fixing nitrogen.
Figure 4.1 Vetch Plants Grown in Water Agar
A representative vetch plant inoculated with each strain as indicated. The plants
were harvested three weeks post-inoculation.
147
Figure 4.2 Roots of Vetch Plants
RU360
RU361
RU307
3841
Roots of a representative vetch plant inoculated with each strain as
indicated. Nodules are indicated by arrows.
148
Figure 4.3 Average Number of Nodules per Plant Formed on Vetch
Each point represents the mean of eight plants.
149
4.2.1.1 Dry Weight of Vetch Plants
Twenty-one days post-inoculation, the shoots of the plants were
harvested, dried and weighed to see if there were any differences
between plants inoculated with different strains (Figure 4.4). The data
were subjected to Analysis of Variance (Appendix 4.2). The
uninoculated plants weighed significantly less than plants that were
inoculated (P<0.05). There was no significant difference in average dry
weight between plants inoculated with the mutant strains or 3841
(P>0.1).
150
Figure 4.4 Average Plant Dry Weight for Vetch
Each result represents the mean of eight vetch plants with standard deviation.
151
4.2.2 Acetylene Reduction Assay on Pea Nodules
The results presented above indicate that the mutants are able to form pink
nodules on plants. In order to confirm that the nodules were effective, it was
necessary to determine whether nitrogen fixation was taking place. The
acetylene reduction assay was used to indirectly measure nitrogenase activity
of bacteroids in nodules. The nitrogenase enzyme is able to reduce acetylene
to ethylene at a rate that is proportional to the amount of nitrogen that can be
fixed, giving results in moles of acetylene reduced. This is not a direct
measurement and there are many factors which may cause erroneous results
(reviewed in Giller, 1987). However, it is useful for establishing that the
nitrogenase enzyme is active and is easier and cheaper than direct
measurement techniques.
Fifteen pea plants were inoculated with 106 cfu of 3841, RU361 or RU307.
Peas were chosen for this experiment as vetch are so small that precise
measurements per plant would be more difficult to obtain. Representative
plants are shown in Figure 4.5. Three uninoculated plants were randomly
placed amongst the inoculated plants to test that cross-contamination did not
occur. The plants were harvested four weeks post-inoculation. Each plant
had pink nodules on the roots, confirming the ability of the mutants to nodulate
peas. The uninoculated plants had no nodules and were not analysed further.
The acetylene reduction assay was then performed. The plants were assayed
in five jars, each containing three plants. The average amount of acetylene
reduced per plant was then calculated (Figure 4.6).
152
The data were subjected to Analysis of Variance (Appendix 4.3). There was
no significant difference in the rates of acetylene reduction between RU361
and 3841 (P>0.1). Strain RU307 reduced more acetylene than RU361 and
3841 but this was not statistically significant (0.1>P>0.05). It was shown
previously that RU360 reduced acetylene at the same rate as 3841, so this
was not measured again (Poole et al., 1994).
153
Figure 4.5 Pea Plants Used for the Acetylene Reduction Assay
Representative pea plants inoculated with each strain as indicated.
154
Figure 4.6 Acetylene Reduction Rates
Each series represents the average result of 15 pea plants assayed in
duplicate or triplicate with standard deviation.
155
4.2.3 Plant Dry Weights for Pea
Plant dry weights were obtained for the pea plants that were used in the
acetylene reduction assay (Figure 4.7). The data were subjected to Analysis
of Variance (Appendix 4.4). There was no significant difference in plant dry
weight between 3841, RU361 and RU307 (P>0.1).
156
Figure 4.7 Average Pea Dry Weight
Each series represents the average of 15 pea plants with standard deviation.
157
4.2.4 Nodule Number and Mass for Pea
The data for average total nodule number per plant and average total wet
nodule mass per plant were subjected to Analysis of Variance (Appendix 4.5,
Appendix 4.6). The mutants RU361 and RU307 formed significantly more
nodules per plant than 3841 (P<0.05) (Figure 4.8). However, there was no
significant difference in the average total nodule mass per plant between the
mutants and 3841 (P>0.1) (Figure 4.9). These data suggest that nodules
formed by the mutants are smaller and less efficient at nitrogen fixation but
that the plants compensated by permitting more nodules to be formed. This is
in contrast to vetch, where nodule numbers were the same (Figure 4.3).
158
Figure 4.8 Total Average Nodule Number per Pea Plant
Each series represents the average of 15 pea plants with standard deviation.
159
Figure 4.9 Total Average Nodule Mass per Pea Plant
Each series represents the average result of 15 pea plants with standard
deviation.
160
161
4.2.5 Competition for Nodulation
The results presented above indicate that the mutants nodulate vetch and pea
plants and fix nitrogen in nodules on pea as effectively as 3841. It was
decided to examine whether the mutants would be just as competent at
initiating nodulation when co-inoculated with 3841 into the rhizosphere of host
plants, or whether 3841 would have a competitive advantage, due to its ability
to utilise myo-inositol.
The mutants RU360 and RU361 were co-inoculated with 3841 into the
rhizosphere of pea in equal numbers. Each strain was also inoculated alone.
The inocula were each applied to four or five plants. An inoculum size of 103
cfu was chosen to enable growth in the rhizosphere prior to nodulation. Four
plants were uninoculated, to test whether cross-contamination occurred.
Twelve nodules were harvested from each plant six weeks post-inoculation
and stabbed onto TY agar containing different combinations of antibiotics, to
distinguish between nodules containing the mutants and 3841 (Figure 4.10).
None of the uninoculated plants had any nodules. Representative colonies
were then streaked onto streaked onto AMA containing 10mM myo-inositol to
confirm the phenotype of each strain.
When inoculated alone, all of the nodules contained the strain inoculated, as
defined by the ability to grow in the presence of the appropriate antibiotics.
These results signify that there was no curing of antibiotic resistance genes
from the strains. When co-inoculated in equal numbers with 3841, RU360
162
occupied 12.5% (6 of 48) of the nodules. At least one nodule containing
RU360 was obtained from each plant harvested. RU361 occupied none of the
nodules when co-inoculated with 3841. These data indicate that 3841 has a
substantial advantage over the mutants in forming nodules when co-
inoculated.
163
Figure 4.10 Pea Nodule Occupancy
1 = uninoculated, 2 = 3841 103 cfu, 3 = RU360 103 cfu, 4 = RU361 103 cfu, 5 = 3841
103 cfu + RU360 103 cfu, 6 = 3841 103 cfu + RU361 103 cfu.
164
The competition experiment was carried out on vetch plants, with equal
numbers of 3841 with RU360, RU361 and RU307 (103 cfu) co-inoculated into
the rhizosphere. Based on the results obtained on peas, it was anticipated
that 3841 would have a competitive advantage. Therefore, plants were also
co-inoculated with 10-fold and 100-fold more of the mutants than 3841 (104
cfu and 105 cfu) to investigate whether the mutants would occupy
proportionately more nodules. Each inoculum was applied to at least six
plants. All the nodules from each plant were harvested five weeks post-
inoculation.
As expected, when inoculated alone, all nodules contained the appropriate
strain (Figure 4.11). When co-inoculated in equal numbers with RU360, 3841
dominated the nodules, occupying 97.6% (82 of 84) of nodules. The nodules
containing RU360 were obtained from two plants. Strain 3841 maintained the
dominance when higher numbers of RU360 were inoculated as it occupied
99.4% (175 of 176) of nodules when RU360 was in 10-fold excess. When
RU360 was in 100-fold excess, 95.9% (186 of 194) of nodules were occupied
by 3841. Nodules containing RU360 were obtained from three plants.
The same trend was observed with RU361 co-inoculated with 3841 (Figure
4.12). No nodules contained RU361 when co-inoculated in equal numbers (0
of 116). The dominance was maintained by 3841 when higher numbers of
RU361 were inoculated. Strain 3841 occupied 89.5% (171 of 191) of nodules
when RU361 was in 10-fold excess. At least one nodule containing RU361
was obtained from each plant harvested. When RU361 was in 100-fold
165
excess, 98.1% (156 of 159) of nodules were occupied by 3841. Nodules
containing RU361 were obtained from two plants.
166
Figure 4.11 Nodule Occupancy of Vetch by RU360 and 3841
1 = uninoculated, 2 = 3841 103 cfu, 3 = RU360 103 cfu, 4 = 3841 103 cfu + RU360 103
cfu, 5 = 3841 103 cfu + RU360 104 cfu, 6 = 3841 103 cfu + RU360 105 cfu.
167
Figure 4.12 Nodule Occupancy of Vetch by RU361 and 3841
1 = uninoculated, 2 = 3841 103 cfu, 3 = RU361 103 cfu, 4 = 3841 103 cfu + RU361 103
cfu, 5 = 3841 103 cfu + RU361 104 cfu, 6 = 3841 103 cfu + RU361 105 cfu.
168
The dominance exhibited by 3841 when co-inoculated with RU360 and
RU361 was not maintained when co-inoculated with RU307 (Figure 4.13). At
least nine plants were treated with each inoculum. When co-inoculated in
equal numbers, RU307 occupied 47.1% (81 of 172) of the nodules. When
inoculated at 10-fold and 100-fold higher than 3841, RU307 occupied 69.3%
(131 of 189) and 80.9% (148 of 183) of the nodules respectively. At least one
nodule containing RU307 was obtained from each plant harvested.
The data presented above indicate that the myo-inositol catabolic mutants
RU360 and RU361 suffer a severe disadvantage in competition for nodulation
when co-inoculated with 3841. However, RU307, which is not a complete
myo-inositol mutant, is competitive when co-inoculated with 3841. This
suggests that there is a requirement for the ability to utilise myo-inositol in
order to be competitive for nodulation.
169
Figure 4.13 Nodule Occupancy of Vetch by RU307 and 3841
1 = uninoculated, 2 = 3841 103 cfu, 3 = RU307 103 cfu, 4 = 3841 103 cfu + RU37 103
cfu, 5 = 3841 103 cfu + RU307 104 cfu, 6 = 3841 103 cfu + RU307 105 cfu.
170
4.2.5.1 Competitive Ability of the Complemented Mutants
It was shown in Chapter 3 that the mutants RU360 and RU361 are
complemented by cosmids containing the region of DNA interrupted by Tn5-
lacZ, so that the ability to grow on myo-inositol is restored. The
complemented mutants grew at similar rates to 3841 on glucose and myo-
inositol in vitro, indicating that the presence of a cosmid did not have any
adverse effects on growth. Therefore, it was postulated that the
complemented mutants would not be at a competitive disadvantage when co-
inoculated with 3841.
The complemented mutants RU360/pRU3078, RU360/pRU3079 and
RU361/pRU3111 were co-inoculated in equal numbers with 3841 (103 cfu)
onto five or six vetch plants. All nodules were harvested from the plants five
weeks post-inoculation. After stabbing of the nodules onto TY agar plus
appropriate antibiotics, it became apparent that the cosmids were not always
retained by the mutants (Table 4.1). When inoculated alone, 24.5% of
nodules contained RU360/pRU3078, 74.2% contained RU360/pRU3079 and
60% contained RU361/pRU3111. The remainder of the nodules contained
the appropriate mutant without a cosmid. Plasmids based on pLAFR1 are
known to be unstable in the absence of selection pressure (Long et al., 1982).
When the nodules were harvested from plants that had been co-inoculated
with a complemented mutant and 3841, none of the nodules contained
RU360/pRU3078 or RU360/pRU3079 and 6.6% contained RU361/pRU3111.
171
However, 20.8% of nodules from plants inoculated with RU360/pRU3078 and
3841 contained RU360, 31.6% of nodules from plants inoculated with
RU360/pRU3079 and 3841 contained RU360 and 8.9% of nodules from
plants inoculated with RU361/pRU3111 and 3841 contained RU361 (Table
4.1).
The above numbers are considerably higher than those obtained for the
mutants without a complementing cosmid when co-inoculated with 3841.
Presumably, the complemented mutants were able to compete with 3841
more successfully and were able to form some nodules before the cosmid
was lost. However, there was variability in the results. Strain RU360 was
considerably more successful at occupying nodules when complemented than
RU361. Although not conclusive, these data further suggest that the ability to
utilise myo-inositol is important for the ability to compete successfully for
nodulation.
172
Table 4.1 Cosmid Stability in the Rhizosphere
inoculum no. nodules
containing mutant (%)
no. nodules containing
mutant + cosmid (%)
RU360/pRU3078 53 (100) 13 of 53 (24.5)
RU360/pRU3078 + 3841 10 of 48 (20.8) 0
RU360/pRU3079 62 (100) 46 of 62 (74.2)
RU360/pRU3079 + 3841 18 of 57 (31.6) 0
RU361/pRU3111 70 (100) 42 of 70 (60)
RU361/pRU3111 + 3841 4 of 45 (8.9) 3 of 45 (6.7)
Figures represent nodules harvested from five or six vetch plants.
173
4.2.6 Measurement of Nodule Co-Residence
To determine the phenotype of bacteria in nodules from plants that were
inoculated with mixtures of 3841 and mutants, whole nodules were stabbed
successively onto TY agar, TY agar plus streptomycin, TY agar plus
streptomycin and kanamycin and (if appropriate) TY agar plus streptomcyin,
kanamycin and tetracycline. Nodules may theoretically contain a mixture of
strains. Nodules whose contents grew on streptomycin but not on kanamcyin
or tetracycline clearly only contained 3841. However nodules whose contents
grew on kanamycin could also contain 3841 in addition to a mutant strain.
Therefore, the dominance of 3841 may actually be higher than the results of
the above experiments have already shown.
To test for co-residence of different strains, vetch nodule squashes that grew
on kanamycin were streaked from the original master plate without antibiotics
to determine the phenotype of individual colonies. Six individual colonies
were then sub-streaked onto plates containing appropriate combinations of
antibiotics. The results from experiments with different RU360 and RU361
inoculum sizes were combined, due to the limited number of nodules obtained
that contained kanamycin resistant bacteria. From vetch inoculated with
RU307, nodules were only tested from the plants that had received equal
inocula of RU307 and 3841.
The same procedure was also applied to two nodules from plants inoculated
with each strain alone. The results show that when the mutant was inoculated
174
alone, all colonies were kanamycin resistance and that when 3841 was
inoculated alone, it did not acquire kanamycin resistance. Therefore, curing of
the kanamycin gene was not responsible for colonies being obtained from
mixed nodule squashes that were not kanamycin resistant. A representative
number of colonies were also streaked onto myo-inositol, to confirm the
phenotype.
The results indicate that 3841 was found in most of the nodules that contained
the mutants, although the mutants were predominant (Table 4.2). Therefore,
the advantage that 3841 may be even greater than suggested by data
presented earlier. In addition, RU307 does not appear to be equally as
competitive as 3841. However, only twelve nodules containing RU307, which
might not have been a large enough sample to give a true representation of
nodule occupancy.
175
Table 4.2 Individual Nodule Occupancy
inoculum details strain no. colonies % of colonies frequency of
nodules
occupied
3841 3841 12 100 2/2
RU360 RU360 12 100 2/2
RU361 RU361 12 100 2/2
RU307 RU307 12 100 2/2
RU360 +3841 3841
RU360
16
56
22
78
7/12
12/12
RU361 +3841 3841
RU361
7
65
9
91
7/12
12/12
RU307 + 3841 3841
RU307
14
58
19
81
9/12
12
The results represent six colonies streaked from each nodule.
Only nodules that contained kanamycin resistant bacteria were investigated. It
should be noted that such nodules were rare, due to the dominance of 3841.
176
4.2.7 Average Number of Nodules per Vetch Plant
It was shown in Section 4.2.1 that vetch inoculated with the mutants alone
formed the same number of nodules as plants inoculated with 3841. The
average number of nodules per plant when mixed inocula were applied was
also noted to see if the higher overall inoculum resulted in more nodules being
formed (Figure 4.14, Figure 4.15, Figure 4.16). The plants were harvested
five weeks post-inoculation.
The data were subjected to Analysis of Variance (Appendix 4.7). There was
no significant difference in nodule numbers per plant between RU360,
RU360/pRU3078, RU360/pRU3079 and 3841 inoculated alone and co-
inoculated in equal numbers (P>0.1). There were however, significantly more
nodules when RU360 was co-inoculated in 10-fold and 100-fold excess
(P<0.05), although there was no significant difference in nodule numbers
between the 10-fold and 100-fold experiments (Figure 4.14).
There was no significant difference (P>0.1) in the number of nodules when
RU361, RU361/pRU3111 and 3841 were inoculated singly or co-inoculated in
equal numbers (Appendix 4.8). When RU361 was co-inoculated in 10-fold
excess, there were significantly more nodules formed than when RU361,
RU361/pRU3111 and 3841 were inoculated alone and when RU361/pRU3111
was co-inoculated with 3841 (P<0.05). There was no significant difference
between RU361 co-inoculated in equal numbers, 10-fold or 100-fold excess
with 3841 (Figure 4.15).
177
For plants co-inoculated with RU307 in equal numbers, 10-fold and 100-fold
excess with 3841, there were significantly more nodules formed than when
either strain was inoculated alone (P<0.05) (Appendix 4.9). There was also a
significant increase in nodule numbers between RU307 co-inoculated in equal
numbers and 10-fold excess (P<0.05) but there was no significant difference
between RU307 co-inoculated in equal numbers and 100-fold excess (P>0.1).
The results indicate that the plants were not disadvantaged in forming nodules
when inoculated with the mutants. More nodules were formed when all the
mutants were co-inoculated with 3841 in 10-fold and 100-fold excess. This
was probably due to the higher inoculum size and indicates that the mutants
are able to elicit nodule formation when co-inoculated with 3841. With
RU307, this led to a higher proportion of nodules containing the mutant.
However, with RU360 and RU361, the additional nodules formed did not
contain the mutants but 3841. This suggests that even though the myo-
inositol catabolic mutants can elicit nodule formation, they either do not gain
access to the nodules, or competition from 3841 causes them be lost from the
nodule.
178
Figure 4.14 Average Number of Nodules per Vetch Plant Inoculated with
RU360 and 3841
Each result represents the average of at least six plants with standard deviation.
10x and 100x signify that RU360 was inoculated in 10-fold and 100-fold excess
respectively.
179
Figure 4.15 Average Number of Nodules per Vetch Plant Inoculated with
RU361 and 3841
Each result represents the average of at least six plants with standard deviation.
10x and 100x signify that RU361 was inoculated in 10-fold and 100-fold excess
respectively.
180
Figure 4.16 Average Number of Nodules per Vetch Plant Inoculated with
RU307 and 3841.
Each result represents the average of at least six plants with standard deviation.
10x and 100x signify that RU307 was inoculated in 10-fold and 100-fold excess
respectively.
181
4.2.8 Average Plant Dry Weights for Vetch
The average dry weights were also obtained for vetch plants from each
inoculum five weeks post-inoculation. The results of plants inoculated with
RU360, RU360/pRU3078, RU360/pRU3079 and 3841 are shown in Figure
4.17. The data were subjected to Analysis of Variance (Appendix 4.10).
There was a significant difference in the weight of the uninoculated plants and
the inoculated plants (P<0.05). There was no significant difference in the
average dry weight of plants inoculated with RU360, RU360/pRU3078,
RU360/pRU3079 and 3841 either alone or when co-inoculated (P>0.1).
The average dry weight of plants inoculated with RU361, RU361/pRU3111
and 3841 are shown in Figure 4.18. The data were subjected to Analysis of
Variance. There was a significant difference in the weight of the uninoculated
plants and the inoculated plants (P<0.05). There was no significant difference
in the average dry weight of plants inoculated with RU361, RU361/pRU3111,
RU361 and 3841 co-inoculated in equal numbers or with RU361 in 100-fold
excess (P>0.1). However, the average dry weight of plants inoculated with
3841 and RU361 in 10-fold excess were significantly higher than the other
plants (P<0.05).
The average plant dry weight of plants inoculated with 3841 and RU307 are
shown in Figure 4.19. The data were subjected to Analysis of Variance.
There was a significant difference in the weight of the uninoculated plants and
the inoculated plants (P<0.05). There was no significant difference in the
182
average dry weight of plants inoculated with RU307 and 3841 either alone or
when co-inoculated in equal numbers or with RU307 in 10-fold excess
(P>0.1). However, the average dry weight of plants inoculated with 3841 co-
inoculated with RU307 in 100-fold excess was significantly higher than plants
inoculated with either RU361 or 3841 alone (P<0.05).
These results show that even though more nodules were formed when higher
mutant numbers were applied to plants, there was no obvious correlation with
plant dry weight. All plants treated with all inocula weighed the same for
RU360, but plants treated with a 10-fold excess of RU361 weighed more,
although plants inoculated with 100-fold excess did not. With RU307, the
plants inoculated with 100-fold excess weighed more, but plants inoculated
with 10-fold excess did not. Therefore, the data are inconclusive as to
whether more nodules result in greater plant dry weight. However, it can be
concluded that plants inoculated with the mutants are not disadvantaged for
growth compared to plants inoculated with 3841.
183
Figure 4.17 Average Plant Dry Weights for Vetch inoculated with RU360
and 3841.
Each series represents the average of at least six plants with standard deviation.
10x and 100x signify that RU360 was inoculated in 10-fold and 100-fold excess.
184
Figure 4.18 Average Plant Dry Weights for Vetch Inoculated with RU361
and 3841
Each series represents the mean of at least six plants, with standard deviation.
10x and 100x signify that RU361 was inoculated in 10-fold and 100-fold excess.
185
Figure 4.19 Average Plant Dry Weights for Vetch Inoculated with RU307
and 3841
Each series represents the mean of at least six plants, with standard deviation.
10x and 100x signify that RU307 was inoculated in 10-fold and 100-fold excess.
186
4.2.9 Rhizosphere Growth Assay
It was shown in Section 4.2.1 that nodules are formed on plants at the same
time, whether inoculated with the mutants or 3841. In Section 3.2.1 it was
shown that the mutants and 3841 have the same growth rates in vitro.
However, conditions in the rhizosphere may be completely different to those in
vitro and if 3841 has a growth advantage at the early stages of rhizosphere
colonisation, this might be responsible for domination of the nodules.
This hypothesis was tested by measuring the growth of RU360, RU361,
RU307 and 3841 in the rhizosphere of vetch in the eight days immediately
following inoculation (Figure 4.20, Figure 4.21, Figure 4.22). This assay gives
an indication of colony numbers for the entire plant system, so bacteria
associated with plant roots as well as those in the rhizosphere were
harvested. The data were subjected to Analysis of Variance (Appendix 4.13).
When inoculated singly, all strains grew over eight days. Strain RU360
attained the highest colony numbers and RU307 the lowest, but the
differences were not significant (0.1>P>0.05).
The results for RU360 and 3841 are shown in Figure 4.20. Numbers of both
strains increased steadily over six days. The data after eight days were
subjected to Analysis of Variance (Appendix 4.14). The numbers of both
RU360 and 3841 when co-inoculated were not significantly different, but
RU360 numbers were significantly lower when co-inoculated than when
inoculated alone (P<0.05).
187
The results for RU361 and 3841 are shown in Figure 4.21. Both strains grew
well over six days. The data after eight days were subjected to Analysis of
Variance (Appendix 4.15). There was no significant difference in numbers of
RU361 and 3841 inoculated alone, or for 3841 when co-inoculated, but
numbers of RU361 when co-inoculated were significantly lower (P<0.05).
Numbers of RU361 were consistently lower throughout the study. As RU361
grew well over the time period, there may have been an inoculation error.
The results for RU307 and 3841 are shown in Figure 4.22. Both strains grew
over six days when inoculated alone and when co-inoculated. The data after
eight days were subjected to Analysis of Variance (Appendix 4.16). When co-
inoculated, numbers of 3841 were significantly higher than RU307 inoculated
alone or co-inoculated (P<0.05). There was no significant difference between
3841 inoculated alone or co-inoculated (P>0.1), or for RU307 inoculated alone
or co-inoculated (P>0.1).
The data indicate that the mutants were all able to grow in the rhizosphere in
the eight days following inoculation. When inoculated into the rhizosphere
alone, numbers of RU360 were highest, but this was not significant
(P>0.05<0.1). However, when co-inoculated, numbers of 3841 were
significantly higher (P>0.05) than the mutants. Overall, all strains grew well in
the rhizosphere and based on these observations, if 3841 has a competitive
advantage purely due to a higher growth rate, then RU307 would also be
expected to be at a competitive disadvantage for nodulation. In addition, if
188
successful nodule occupancy were purely due a growth advantage, when the
mutants were co-inoculated in 10- and 100-fold excess, they would have been
expected to dominate the nodules.
189
Figure 4.20 Growth of RU360 and 3841 Recovered from the Rhizosphere
Each point represents the mean of at least four replicates.
190
Figure 4.21 Growth of RU361 and 3841 Recovered from the Rhizosphere
Each point represents the mean of at least four replicates.
191
Figure 4.22 Growth of RU307 and 3841 Recovered from the Rhizosphere
Each point represents the mean of at least four replicates.
192
4.2.10 Analysis of a nodC-phoA Fusion
Strains 3841 has a clear competitive advantage for nodulation over RU360
and RU361. The ability to utilise myo-inositol as a carbon source does not
appear to be responsible for the competitive advantage, as the mutants were
all able to grow well in the rhizosphere in the absence of exogenous carbon.
However, RU360 and RU361 may not able to respond to signals given out by
plants as effectively as 3841 because a breakdown product of myo-inositol is
required, so that nodules are formed slightly more slowly than by 3841.
Nodulation (nod) genes encode proteins that are required for the formation of
nodules on legumes. Their expression is induced by components of plant cell
exudates. The nodC gene encodes an N-acetyl-glucosaminyl transferase
involved in the formation of lipo-chito-oligosaccharide Nod factors that initiate
nodule morphogenesis in legumes (Geremia et al., 1994, Spaink et al., 1994,
see Section 1.2.1). If the mutants are unable to respond as effectively to plant
signals as 3841, this may be reflected by reduced expression of nodC.
Therefore, the ability of 3841 and the mutants to express nodC in response to
a plant product was assessed in vitro.
A nodC-phoA fusion (pIJ1687) was conjugated into the mutants and 3841.
Expression of NodC was assessed by measuring the activity of a promoter-
less alkaline phosphatase (PhoA), encoded by the phoA gene fused to the
nodC gene. Hesperetin was the plant product used to induce expression of
nodC. The mutants and 3841 were assayed for alkaline phosphatase activity
193
in the presence and absence of hesperetin when grown on pyruvate and
pyruvate plus myo-inositol. Strain 3841 was also tested when grown on myo-
inositol only (Table 4.3).
Without induction by hesperetin, the activity of alkaline phosphatase was very
low by all the strains tested, on all carbon sources. In the presence of
hesperetin, activity of alkaline phosphatase was greatly increased for all
strains on all carbon sources. The data were subjected to Analysis of
Variance (Appendix 4.17). Highest activity was for RU360 grown on pyruvate
plus myo-inositol and 3841 grown on myo-inositol. The activity of RU361
grown on pyruvate plus myo-inositol was lower than that of the other strains,
but this was not significant (0.01>P>0.05). These data indicate that the
mutants are able to express nodC in response to plant products in vitro as
effectively as 3841.
194
Table 4.3 Induction of the nodC Gene
activity of alkaline phosphatase in nmol mg-1 min-1
strain pyruvate pyruvate + myo-
inositol
myo-inositol
RU360 uninduced 258.84 ± 98.5 112.16 ± 8.16 N/A
induced 4911.58 ± 245.37 5531.9 ± 291.46 N/A
RU361 uninduced 100.37 ± 7.08 152.37 ± 8.52 N/A
induced 3560.47 ± 154.26 3142.91 ± 53.41 N/A
RU307 uninduced 122.79 ± 10.43 157.66 ± 3.59 N/A
induced 4510.98 ± 63.04 4591.21 ± 602.29 N/A
3841 uninduced 59.48 ± 6.35 151.18 ± 39.14 227.24 ± 16.28
induced 4138.48 ± 507.28 4220.79 ± 370.75 5370.43 ± 1707.11
Each result represents the mean of at least two cultures assayed in triplicate.
N/A = not assayed.
195
4.3 Discussion
The mutants RU360, RU361 and RU307 were all able to form nodules on pea
and vetch plants. There was no significant difference between the total
numbers of nodules formed on vetch plants inoculated with the mutants and
3841 alone, or the time taken for them to form. There was also no difference
in plant dry weight between the mutants and 3841, indicating that the mutants
provided as much fixed nitrogen to the host plants as 3841. It is possible that
3841 formed nodule initials more quickly than the mutants. These are not
visible to the naked eye and would have required the use of staining
techniques. Due to time constraints, this was not attempted.
All the nodules formed on plants inoculated with the mutants and 3841
appeared pink. This indicates the presence of leghaemoglobin and suggests
that the nodules were fixing nitrogen. The acetylene reduction assay
confirmed this for pea nodules containing RU361, RU307 and 3841 and
showed that nitrogenase activity was similar for all three strains. This was
previously shown to be the case for RU360 (Poole et al., 1994).
Higher numbers of nodules were formed on peas inoculated with RU361 and
RU307 than 3841, although total nodule mass and plant dry weights were the
same. These data suggest that peas inoculated with the mutants formed
nodules that were not quite as effective at nitrogen fixation as those
inoculated with 3841. Presumably, the plants compensated for this by
directing the formation of additional nodules. Vetch are considerably smaller
196
than peas so they require less fixed nitrogen and hence fewer nodules.
Therefore, a small reduction in effectiveness per nodule may not affect the
plant as much, so that there would be no noticeable difference in nodule
number per plant. However, it is not possible to conclude from this study that
the mutants are less effective at nodulating vetch than 3841 when inoculated
alone.
As vetch inoculated with the mutants and 3841 formed nodules at the same
rate, bacteroids from pea nodules fixed nitrogen at the same rate and plant
dry weights were the same for both vetch and pea, it was expected that the
mutants would be equally as competitive as 3841 for nodule formation when
co-inoculated with 3841. However, co-inoculation of peas and vetch with
RU360 or RU361 and 3841 resulted in massive domination of nodules by
3841, even when the mutants were inoculated in 10-fold and 100-fold excess.
These data indicate a role for myo-inositol in nodulation competitiveness,
which has not been shown before.
Bosworth et al. (1994) and Scupham et al. (1996) showed that interruption of
the inositol locus of S. meliloti was sometimes beneficial to host plants.
However, the effects of disruption of the myo-inositol locus were not
consistent, suggesting that there is a role for myo-inositol in S. meliloti. There
also appears to be a role for rhizopines, as Gordon et al. (1996) showed that
the rhizopine-producing strain S. meliloti L5-30 had a competitive advantage
for nodulation of lucerne (Medicago sativa) in soil when co-inoculated with a
mutant strain, even though when inoculated alone, the mutant had a similar
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rate of growth and nodulation to the wild type. The mutant occupied less than
30% of nodules. This competitive advantage remained four years after
inoculation, even though there had been turnover of nodules in that time
(Heinrich et al., 1999).
The data from the competition experiments using RU360/pRU3078,
RU360/pRU3079 and RU361/pRU3111 indicate that had the cosmids been
stable in the environment, 3841 might not have had a competitive advantage.
The transposon inserted into two completely different loci in RU360 and
RU361, which suggests that it was the restoration of the ability to utilise myo-
inositol, rather than the presence of other beneficial regions on the cosmid
that was responsible for restoring the competitive advantage. However, to
eliminate the possibility that independent factors on the cosmids were
responsible, a more stable vector should be used. In Section 5.2.7, the pOT1
plasmid was shown to be stable in pea root nodules containing 3841, so this
vector should be suitable. In Section 3.2.5.1.1, the cloning of iol genes from
pRU3078 into pOT1 was described. The region of DNA required for
complementation of RU361 could also be cloned into pOT1 and the
competition experiment repeated. It is postulated that the complemented
mutants would be as competitive as 3841.
Strain RU307 was able to compete successfully for nodulation when co-
inoculated onto vetch with 3841. The nodule occupancy tests imply that 3841
may be slightly more competitive as the majority of nodules from the mixed
inocula experiments contained 3841. However, this experiment needs to be
198
repeated with a much larger sample size, to confirm the results. Strain RU307
differs from RU360 and RU361 in that it is not a complete myo-inositol mutant,
but is mutated in an uptake system for myo-inositol (c.f. Section 3.2.5). There
is no apparent deficiency in the myo-inositol catabolic pathway as the first two
enzymes in the catabolic pathway were induced when RU307 was grown on
myo-inositol as the sole carbon source (c.f. Section 4 or 5). The requirement
for myo-inositol was probably not as a carbon source because RU307 was
competitive, despite the fact that its growth rate on myo-inositol is only a
quarter of that of 3841 in vitro.
It is clear that there were other carbon sources present in the system, since
RU360, RU361, RU307 and 3841 all grew in the rhizosphere. Substrate
exhaustion was also unlikely to be a factor, as the strains all grew well over
eight days, by which time nodules had begun to form. myo-Inositol did not
have an inhibitory effect on the mutants in vitro. However, there was
catabolite repression by other carbon compounds of some myo-inositol
catabolic enzymes and the myo-inositol uptake system in vitro (c.f. Section 3
and Section 5). This suggests that even if myo-inositol were present in the
rhizosphere, catabolite repression might occur, so other sugars would be used
preferentially and 3841 would not gain any competitive advantage by being
able to utilise myo-inositol.
In addition, the mutants and 3841 all grew well in the rhizosphere over eight
days, although 3841 grew slightly better when co-inoculated. If the
dominance of 3841 is purely because of a growth advantage in the
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rhizosphere, then RU307 should also have been at a competitive
disadvantage. In addition, the competitive disadvantage should have been
overcome when RU360 and RU361 were co-inoculated in 10- and 100-fold
excess as the mutants growth should swamp that of 3841.
These results are in contrast to those of Wood and Stanway (2000), who
showed that in soil solution, levels of myo-inositol decreased concomitantly
with the increase of four different strains of rhizobia, which suggests that myo-
inositol was used preferentially. However, the soils that were used contained
myo-inositol as the dominant sugar. This is not the case in other soils that
have been studied (c.f. Section 1.3.3.2) and so myo-inositol might only be
used preferentially when it is present in such a large quantity that catabolite
repression by other sugars is not significant. However, all experiments were
carried out in sterile vermiculite in this project, which may give very different
results to experiments carried out in soil.
Higher numbers of nodules per vetch plant were observed when RU360 and
RU361 were co-inoculated in 10-fold and 100-fold excess with 3841.
However, the majority of bacteria recovered from these nodules were still
3841 and there was no increase in plant dry weight, indicating that there was
not more nitrogen fixation occurring. These data indicate that the plants were
able to sense the additional rhizobia and produce signals that the rhiobia
could respond to. However, the mutants were still not able to get into the
nodule. This confirms that the dominance of the wild type is not because it
grows faster in the rhizosphere. Therefore, 3841 must be gaining an
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advantage over the mutants, either at the commencement of infection or
during early infection events.
Many more infection threads are formed than go onto make nodules (Bauer,
1981) and the mechanisms through which the plant controls which infection
threads become nodules have not yet been identified. Oresnik et al. (1998)
studied a rhamnose catabolic mutant. The authors postulated that as plant
cell walls are continuous with infection threads, rhamnogalacturonan subunits
or degradation products may be present in infection threads. Depending on
when the mechanism that results in infection threads being aborted occurs,
proportionately more infection threads formed by the mutant may be aborted if
the mutant strain grew more slowly because of its inability to utilise rhamnose,
resulting in a majority of nodules containing the wild type.
The myo-inositol oxidation pathway is the major pathway providing sugar
residues for cell wall synthesis during germination of bean (Sasaki and
Nagahashi, 1990). It is also present in other complex plant lipids and in plant
cell membranes as phosphoinositides (Loewus, 1990). In addition, the
presence of myo-inositol in nodules, including those of pea, has been widely
reported (Kouchi and Yoneyama, 1984, 1986, Skøt and Egsgaard, 1984,
Streeter, 1987; Streeter and Salminen, 1985).
Therefore, the theory postulated for rhamnose utilisation might also be
applicable to myo-inositol. Although there mutants were able to grow well in
the rhizosphere, the composition of available nutrients in the infection thread
201
might be very different to the rhizosphere. Strain RU307 would be expected
to be disadvantaged because of its poor growth rate on myo-inositol, but if
other nutrients are limited, it might be able to grow well enough to ensure its
infection thread are not disproportionately aborted.
Another possibility is that the presence of myo-inositol or one of its derivatives
acts as a signal to Rhizobium, encouraging infection thread development or
the formation of nodules. myo-Inositol is likely to be present in areas that
nodules are developing because of its role in cell wall synthesis. myo-Inositol
might also be present near developing nodules as it used to transport auxins,
which are also found at the sight of developing nodules. Perhaps myo-inositol
acts as a signal, highlighting the areas where nodules are developing. If 3841
can respond to plant signals and produce infection threads more quickly than
RU360 or RU361, then the host plant may favour the formation of full nodules
containing 3841 when there is a mixed inoculum.
It has been shown that when two sides of a root are spatially separated and
inoculated at different time intervals, nodulation is prevented on the side
inoculated 24 hours later (Kosslak et al., 1984, Sargent et al., 1987). The
suppression of nodulation increases as the time interval between the two
inoculated sides increases (Kosslak et al., 1983), indicating that if two strains
were co-inoculated and one could initiate nodulation more quickly, then that
strain would have a clear competitive advantage.
202
When inoculated alone, there was no competition from other strains, so
nodules always contained the mutants. However, it is possible that the
nodules were actually formed more slowly than on plants inoculated with
3841. If this difference were measured in hours then this would not have
been observed in the experiments conducted in this project as the plants were
assessed once a day. In a future experiment, 3841 could be inoculated into
the rhizosphere of plants at different time intervals after the mutants to see if
this would allow the mutants to overcome the disadvantage.
It was shown that when bean roots were inoculated with R. phaseoli and
cultured in a growth medium supplemented with myo-inositol, the percentage
of roots nodulated and the number of nodules per root increased (Raggio et
al., 1959). Another future experiment could investigate whether this occurs
with vetch or peas and if there are any differences between plants inoculated
with the mutants and 3841.
To determine the longevity of the mutants in the nodules, nodules could be
harvested at different times post-inoculation. The nodules studied in the
competition experiments were all harvested approximately five weeks post-
inoculation. The few nodules that contained RU360 or RU361 from plants co-
inoculated with two strains, usually also contained small numbers of 3841.
This was not due to curing of the kanamycin resistance gene, but may have
been due to intra-nodule competition between the mutants and 3841.
203
If nodules were harvested at different intervals after formation, there might be
a difference in nodule composition and the numbers occupied by each strain
might be more even. This would determine whether the mutants initiated
formation of nodules as successfully as 3841 but were then displaced by
3841, or whether 3841 has an advantage at the earliest stages of nodule
formation. Harvesting the nodules earlier may also result in less instability of
the complementing cosmids. This experiment would complement the pre-
inoculation experiment suggested above.
The response of nodC of the mutants and 3841 to hesperetin was measured
in order to test whether the ability to catabolise myo-inositol is important for
signalling between plants and Rhizobium. The responses of the different
strains were similar, which suggests that the mutants were not inhibited in
their response to plant signals, as did the increased nodule numbers when the
mutants were inoculated in excess. However, there are several Nod factors
and as it was not practical to study them all, it is possible that other Nod
factors are not as effective in the mutants.
It is not known whether the bacteroids were also a mix of two strains, as the
composition of undifferentiated bacteria obtained from nodules might not truly
reflect the genotype of the bacteroids. A more effective way to study nodule
composition would be to use a reporter gene that can be studied in planta,
such as green fluorescent protein (GFP). Several types of GFP are available
that give different colour responses under UV light. The genes encoding the
different types of GFP could be linked to a gene that is constitutively
204
expressed, either by integration into the chromosome, or on a stable plasmid.
The GFP fusions could be introduced into the mutants and 3841, then the
nodule contents could be examined under UV light to identify the phenotype
of bacteroids and free-living bacteria in the infection threads.
To further investigate the role of myo-inositol in competition for nodulation,
mutants in different parts of the degradation pathway need to be isolated and
tested. This is crucial in order to determine whether different components of
the pathway are required. To determine whether myo-inositol is important for
competition in all legume symbioses it would be necessary to see if the effects
were in the same in different Rhizobium species and their corresponding host
plants. It would also be useful to obtain mutants in pathways that lead to myo-
inositol derivatives, such as inositol phosphates or rhizopines to try to
determine the specific role of myo-inositol in competition for nodulation.
CHAPTER 5 - REGULATION OF MYO-INOSITOL CATABOLISM ........... 204
5.1 Introduction .......................................................................................... 205
5.2 Results .................................................................................................. 208
5.2.1 Isolation of a myo-inositol Specific Promoter ............................................ 208
5.2.2 Sequence Analysis of the myo-Inositol Inducible Promoter ...................... 209
5.2.3 Induction of the myo-Inositol Inducible Promoter ...................................... 218
5.2.4 Catabolite Repression............................................................................... 223
5.2.5 Relationship to myo-Inositol Mutants ........................................................ 228
5.2.6 Expression in the Rhizosphere and Nodules ............................................ 230
5.2.7 Stability of pRU601 in the Rhizosphere .................................................... 231
205
5.2.8 Construction of a Specific myo-Inositol Inducible GFP-UV Promoter Probe
............................................................................................................................ 232
5.2.9 RU360 β-Galactosidase Assay ................................................................. 236
5.2.10 myo-Inositol Catabolic Enzyme Assays .................................................. 238
5.3 Discussion............................................................................................. 241
204
Chapter 5 - Regulation of myo-inositol catabolism
205
5.1 Introduction
In order to understand the regulation of the pathway of myo-inositol
catabolism, the myo-inositol mutants and 3841 were tested for their
ability to utilise the first two enzymes in the proposed myo-inositol
catabolic pathway. It had been shown previously that RU360 was not
able to induce these enzymes, but that 3841 could (Poole et al., 1994).
To identify additional genes involved in myo-inositol utilisation to those
described in Chapter 3, attempts were made to isolate myo-inositol
inducible promoters, using Green Fluorescent Protein (GFP) as an
indicator. GFP is a small protein that fluoresces when excited by UV or
short-wave blue light. It was originally isolated from the bioluminescent
jellyfish Aequorea victoria. As no exogenous cofactors are required for
fluorescence, the gene for GFP can be placed under the control of a
heterologous promoter and expression studied with no apparent
interference with cell growth and function (Chalfie et al., 1994). There
is limited autofluorescence at the same wavelengths by biological
material, enabling study of GFP in the presence of plants or micro-
organisms.
A R. leguminosarum bv. viciae DNA promoter-probe library consisting
of random fragments fused to a promoter-less gfpuv gene in the
plasmid pOT1, was previously constructed in this laboratory (Schofield,
1999). Plasmid pOT1 is a 5278bp, broad host range plasmid that
206
contains a gene encoding gentamycin resistance. It is based on the
plasmid pBBR1-MCS5 and contains the promoterless gfpuv gene
between two unique transcriptional termination sites, to prevent
expression of GFP-UV by promoters on the plasmid. The gfpuv gene
was used because it is 18 times more fluorescent than the wildtype gfp
gene but retains the same excitation and emission maxima (Crameri et
al., 1996).
Approximately 15,000 promoter fragments were generated in pOT1,
with an average size of 1.56kb. These fragments were estimated to
represent approximately 40% of all Rhizobium promoters. The
fragments were cloned into the SalI site of pOT1, which was destroyed
in the process. In order to select for promoters that were not induced in
standard laboratory conditions, the library was screened on AMA and
AMS containing glucose and ammonia as the sole carbon and nitrogen
sources respectively. Approximately 71% of the colonies did not
express GFP-UV in these conditions and contained a Rhizobium
chromosomal DNA fragment. These colonies were selected and
stocked as a library. This library has previously yielded defined
promoter fragments, identified by their ability to induce expression of
GFP-UV (Schofield, 1999).
The plasmid pOT1 containing the promoter-less gfpuv gene was also
used to make defined myo-inositol inducible promoter-probe vectors,
using the region interrupted in RU360. By fusing the promoter region of
207
the iolD gene to a promoter-less gfpuv gene it was hoped that induction
of myo-inositol catabolic genes in the rhizosphere and nodules could be
studied.
208
5.2 Results
5.2.1 Isolation of a myo-inositol Specific Promoter
Approximately 10,000 colonies of the promoter-probe library in 3841
were plated onto AMA containing 10mM myo-inositol as the sole
carbon source. Seventeen colonies expressing GFP-UV were
identified on a transilluminator at a wavelength of 420nm. These
colonies were streaked onto AMA containing other carbon sources
(glucose, pyruvate, mannitol, sorbitol, fructose, glycerol), to determine
whether the promoter was specific to myo-inositol. Four colonies were
isolated that only expressed GFP-UV in the presence of myo-inositol
and therefore contained a myo-inositol specific promoter. Sequencing
showed that all four colonies contained the same 2048bp fragment in
pOT1. The plasmid containing this fragment was named pRU601 and
the promoter contained in the 2048bp fragment was designated iolXp.
209
5.2.2 Sequence Analysis of the myo-Inositol Inducible Promoter
The iolXp fragment was sequenced in the 5’ and 3’ direction (Figure
5.1, Figure 5.3) in order to try to identify the promoter responsible for
expression of the gfpuv gene. BlastX analysis of the deduced amino
acid sequence revealed a putative ORF of 660bp, which showed
homology with the transcriptional regulatory protein of several two-
component sensor-regulator systems. The ORF was designated orf1
(Figure 5.1, Figure 5.3). The closest match was 45% identity (p-value
of 1 x 10-36) with a putative transcriptional regulator from Bordetella
pertussis. There was also 39% identity (p-value of 3 x 10-31) with the
FeuP protein of R. leguminosarum bv. viciae. FeuP is involved in the
regulation of iron uptake (Yeoman et al., 1997). The orf1 gene
encodes a predicted protein of 220 amino acids, with a molecular
weight of 23988 and a P.I of 5.4.
Regulatory proteins such as FeuP are known to feature an acid pocket
(Parkinson and Kofoid, 1992). The residues in FeuP that correspond to
glutamic acid (amino acid 7), aspartic acid (amino acids 8, 9, 51) are
thought to form part of the acid pocket into which the side chain of
lysine (amino acid 101) protrudes (Yeoman et al., 1997). These
residues are conserved among similar regulatory proteins (Parkinson
and Kofoid, 1992), including in the putative Orf1 protein (Figure 5.3).
The orf1 gene is transcribed divergently to the promoter-less gfpuv
210
gene. Therefore, it is not possible that the promoter for orf1 is
responsible for expression of the gfpuv gene.
The remainder of the iolXp fragment did not have homology with any
known sequences in the Genbank or EMBL databases. Therefore,
testcode analysis was performed. This indicated that apart from a
small region of approximately 100bp, the sequence is likely to be
coding (Figure 5.2). Three further putative ORFs were subsequently
identified.
An ORF of 636bp was designated orf2 and is transcribed in the same
direction as the promoter-less gfpuv gene (Figure 5.1, Figure 5.3). The
orf2 gene encodes a predicted protein of 212 amino acids, with a
molecular weight of 22986.2 and a P.I of 5.6. An ORF of 729bp was
designated orf3 and overlaps orf2 in a different frame, but is also
transcibed in the same direction as the promoter-less gfpuv gene
(Figure 5.1, Figure 5.3). The orf3 gene encodes a predicted protein of
243 amino acids, with a molecular weight of 25965.2 and a P.I of 10.3.
The fourth putative ORF is 88bp and was designated orf4. It is at the
end of the 2048bp insert and is not complete. It is immediately
upstream of the promoter-less gfpuv gene and is transcribed in the
same direction (Figure 5.1, Figure 5.3).
211
Figure 5.1 pRU601 Insert
pRU6017326 bp
GFP-UV
replication
m obilis ationgentam ycin res is tance
orf2
orf1
orf3orf4
rbs-gfp
termination (omega)
termination (pharmacia)
Not I (320)
Sst I (3260)
Eco R I (3061)
Eco R I (3240)Pac I (4012)
Pac I (6090)
PstI (3999)
PstI (4737)
212
Figure 5.2 Coding Regions of pRU601
Testcode analysis of iolXp. Sequence that is in the top portion of the graph is likely
to be coding. Sequence in the middle portion is ambiguous and sequence in the
bottom portion is unlikely to be coding.
T = Testcode.
orf1 orf2 orf3 orf4
0 500 1000 1500 2000
bp
14
12
10
8
6
4
T
213
Figure 5.3 DNA Sequence of pRU601 Insert
1 TCCTCATGGC CGTCCCAGAC GATAGCCGAG CCCGCGCTCG GTCTCGATCA AGGAGTACCG GCAGGGTCTG CTATCGGCTC GGGCGCGAGC CAGAGCTAGT -2 P R G L R Y G L G R E T E I 51 CATGAGTACC GAGCTTCTTG CGCAACCGGC TCACATGGAC CTCGATGGCG GTACTCATGG CTCGAAGAAC GCGTTGGCCG AGTGTACCTG GAGCTACCGC -2 H T G L K K R L R S V H V E I A 101 TTGCTGTCGA TTTCCCTATC GAAGGAATAG AGCCGCTCTT CGAGCTGCGC AACGACAGCT AAAGGGATAG CTTCCTTATC TCGGCGAGAA GCTCGACGCG -2 N S D I E R D F S Y L R E E L Q A 151 CTTCGAAAGA AGCTGGCCGG GACGTTGCAG GAAGGCCTCA AACAGGACCC GAAGCTTTCT TCGACCGGCC CTGCAACGTC CTTCCGGAGT TTGTCCTGGG -2 K S L L Q G P R Q L F A E F L V 201 ATTCCCGCGC CGTTAGAACA ATGGCCTTGC CATTCAGAGT GATGCTTCTG TAAGGGCGCG GCAATCTTGT TACCGGAACG GTAAGTCTCA CTACGAAGAC -2 E R A T L V I A K G N L T I S R 251 GCGGCGAGAT CGATGGCGAG CGGGCCAAGC GTGATGTTGG GATTGGGATT CGCCGCTCTA GCTACCGCTC GCCCGGTTCG CACTACAACC CTAACCCTAA -2 A A L D I A L P G L T I N P N P N 301 GCCGGCATAA CGGCGCGCCA CCGACCCGAT CCGCGCCGAA AGCTCCGCAA CGGCCGTATT GCCGCGCGGT GGCTGGGCTA GGCGCGGCTT TCGAGGCGTT -2 G A Y R R A V S G I R A S L E A 351 GATCGAAGGG CTTCACCATG TAATCGTCAG CGCCGGCATT CAATCCGGCA CTAGCTTCCC GAAGTGGTAC ATTAGCAGTC GCGGCCGTAA GTTAGGCCGT -2 D F P K V M Y D D A G A N L G A * 401 ATACGGTCAG TGACCTGGTC GAGCGCTGTC AGGATCATGA CCGGCGTCAC TATGCCAGTC ACTGGACCAG CTCGCGACAG TCCTAGTACT GGCCGCAGTG -2 I R D T V Q D L A T L I M V P T V 451 GTCGCCGCGC CCACGGAACC CCTTCAGAAA GGGAATGCCA AGCCCATCGG CAGCGGCGCG GGTGCGTTGG GGAAGTCTTT CCCTTACGGT TCGGGTAGCC -2 D G R G R L G K L F P I G L G D P 501 GCAGCATCAG ATCGAGCAGC ACCAGATCAT AAGCAGCCGC AACGATTGCA CGTCGTAGTC TAGCTCGTCG TGGTCTAGTA TTCGTCGGCG TTGCTAACGT -2 L M L D L L V L D Y A A A V I A * 551 TCGCCCGCTT GGTCGAGACG GTTCACCCAA TCGACGGAAT GGCCGTCGGC AGCGGGCGAA CCAGCTCTGC CAAGTGGGTT AGCTGCCTTA CCGGCAGCCG -2 D G A Q D L R N V W D V S H G D A
214
601 TGCGATCTGG TCGCGAATGG CCGCGCCCAG CGCCGTATCG TCCTCGATTA ACGCTAGACC AGCGCTTACC GGCGCGGGTC GCGGCATAGC AGGAGCTAAT -2 A I Q D R I A A G L A T D D E I L * * * 651 GTAGAACCCG CATCGCGTCC CTCCCTCCCT CTCTCTCTTT CGTCTCCTAG CATCTTGGGC GTAGCGCAGG GAGGGAGGGA GAGAGAGAAA GCAGAGGATC -2 L V R M orf1 701 TCCACCTGCC AACTTACGGG AAGCTGAAGC AATGTCGCGC GGTTTGTCAG AGGTGGACGG TTGAATGCCC TTCGACTTCG TTACAGCGCG CCAAACAGTC 751 GCGCCGGTCA GCTTGGAACG CAATTGATCA GACCGAAATG AGAGGAAACA CGCGGCCAGT CGAACCTTGC GTTAACTAGT CTGGCTTTAC
TCTCCTTTGT
orf2 +3 M K T G T L P I L A L A V M A 801 AGAATGAAAA CCGGCACCTT GCCCATCTTG GCGCTCGCCG TCATGGCCGT TCTTACTTTT GGCCGTGGAA CGGGTAGAAC CGCGAGCGGC AGTACCGGCA +3 T S A Q A R D D C D V P I S N W 851 GACATCCGCT CAGGCAAGAG ACGACTGCGA CGTTCCGATC AGCAATTGGA CTGTAGGCGA GTCCGTTCTC TGCTGACGCT GCAAGGCTAG TCGTTAACCT +3 K T H A A V R A M A G Q R G W T L 901 AGACGCATGC GGCGGTTCGC GCCATGGCCG GGCAACGAGG TTGGACGCTG TCTGCGTACG CCGCCAAGCG CGGTACCGGC CCGTTGCTCC AACCTGCGAC +3 K R I K I D D G C Y E L Q G T D 951 AAGCGCATCA AGATCGATGA CGGTTGTTAC GAACTCCAGG GCACCGACAA TTCGCGTAGT TCTAGCTACT GCCAACAATG CTTGAGGTCC CGTGGCTGTT +3 D G R R F E A K I D P V T L E V 1001 GGACGGCCGT CGCTTCGAAG CGAAAATCGA TCCGGTGACG CTTGAAGTCA CCTGCCGGCA GCGAAGCTTC GCTTTTAGCT AGGCCACTGC GAACTTCAGT +3 I Q L D E R P E D R R A P P H K G 1051 TCCAACTCGA TGAACGCCCC GAAGATCGCC GAGCCCCCCC CCACAAAGGT AGGTTGAGCT ACTTGCGGGG CTTCTAGCGG CTCGGGGGGG GGTGTTTCCA +3 S Q L R S L M T Y R K F L L A A 1101 AGCCAACTGA GGTCACTTAT GACGTACCGC AAATTTCTTC TGGCCGCTTG TCGGTTGACT CCAGTGAATA CTGCATGGCG TTTAAAGAAG ACCGGCGAAC +3 A G I G F L A P L G S L A M P F 1151 CGCCGGCATC GGCTTTCTCG CACCCCTCGG CAGTCTGGCC ATGCCGTTCC GCGGCCGTAG CCGAAAGAGC GTGGGGAGCC GTCAGACCGG TACGGCAAGG orf3 +3 P A P E S A A A G A V P A G Y Q E +1 V P P P A P Y Q P A I K
215
1201 CCGCACCGGA GAGTGCCGCC GCCGGCGCCG TACCAGCCGG CTATCAAGAG GGCGTGGCCT CTCACGGCGG CGGCCGCGGC ATGGTCGGCC GATAGTTCTC +3 S E D D N R K E E H R L R H D D +1 A R T I I A R R S I A S V T T T 1251 AGCGAGGACG ATAATCGCAA GGAGGAGCAT CGCCTCCGTC ACGACGACGG TCGCTCCTGC TATTAGCGTT CCTCCTCGTA GCGGAGGCAG TGCTGCTGCC +3 R Y A A D D C E G E D D D A C S +1 A V T P P M T A K A R M T M P A V 1301 CCGTTACGCC GCCGATGACT GCGAAGGCGA GGATGACGAT GCCTGCAGTC GGCAATGCGG CGGCTACTGA CGCTTCCGCT CCTACTGCTA CGGACGTCAG +3 R G G G G Q Q Q N A T P P S N G L +1 E A A A G S N K T P P R P A T A 1351 GAGGCGGCGG CGGGCAGCAA CAAAACGCCA CCCCGCCCAG CAACGGCCTT CTCCGCCGCC GCCCGTCGTT GTTTTGCGGT GGGGCGGGTC GTTGCCGGAA +3 F T P G S R P R V Q T N +1 S R L V P G R G S R R T E P S S 1401 TTCACGCCTG GTTCCAGGCC GCGGGTCCAG ACGAACTGAA CCGTCAAGTC AAGTGCGGAC CAAGGTCCGG CGCCCAGGTC TGCTTGACTT GGCAGTTCAG +1 Q R R I S M R M K S L I A M L A V 1451 AAAGGAGAAT ATCGATGAGA ATGAAATCCC TTATCGCCAT GCTTGCAGTC TTTCCTCTTA TAGCTACTCT TACTTTAGGG AATAGCGGTA CGAACGTCAG +1 T T A L T V P G L A M A R E V T 1501 ACCACGGCCC TTACCGTTCC CGGCCTTGCC ATGGCGCGCG AAGTGACCTT TGGTGCCGGG AATGGCAAGG GCCGGAACGG TACCGCGCGC TTCACTGGAA +1 T T N M R N Y G G D G A Y L A Y 1551 CACCACCAAC ATGCGCAACT ACGGCGGGGA TGGTGCCTAC CTTGCCTATT GTGGTGGTTG TACGCGTTGA TGCCGCCCCT ACCACGGATG GAACGGATAA
+1 Y V T D A Q G K Y V G S L W M A G 1601 ACGTCACCGA TGCGCAGGGC AAATATGTCG GCAGCCTTTG GATGGCGGGC TGCAGTGGCT ACGCGTCCCG TTTATACAGC CGTCGGAAAC CTACCGCCCG +1 G K T R Y Y E H L T G W Y R A T 1651 GGCAAGACCA GGTATTACGA GCATCTCACT GGCTGGTACC GCGCCACCGG CCGTTCTGGT CCATAATGCT CGTAGAGTGA CCGACCATGG CGCGGTGGCC +1 G N A A E I N G I T G A S V G A 1701 TGGCAATGCC GCCGAGATCA ACGGCATCAC GGGTGCCAGC GTTGGTGCCG ACCGTTACGG CGGCTCTAGT TGCCGTAGTG CCCACGGTCG CAACCACGGC
+1 G R S L K V T V D L A D T L F D A 1751 GCCGTTCACT CAAGGTCACG GTCGATCTTG CCGATACGCT GTTCGATGCC CGGCAAGTGA GTTCCAGTGC CAGCTAGAAC GGCTATGCGA CAAGCTACGG +1 G Y Q L H I D S A V E D M R D S 1801 GGCTATCAGC TTCACATCGA CTCCGCCGTG GAAGACATGC GCGACAGCCC CCGATAGTCG AAGTGTAGCT GAGGCGGCAC CTTCTGTACG CGCTGTCGGG
216
217
+1 N E I V V P L T S T G S G Q K L 1851 AAACGAGATC GTGGTGCCGC TGACATCGAC CGGCTCGGGG CAGAAGTTGA TTTGCTCTAG CACCACGGCG ACTGTAGCTG GCCGAGCCCC GTCTTCAACT +1 K G T R Y I A A F S Y A R 1901 AGGGCACGCG CTACATCGCC GCCTTCAGCT ATGCGCGTTG AGCGGGAAGG TCCCGTGCGC GATGTAGCGG CGGAAGTCGA TACGCGCAAC TCGCCCTTCC orf4 +1 M T R S L H R W F G L I G 1951 ACGCTCGCCA TGACGCGCTC GCTTCACCGC TGGTTTGGGC TAATCGGCTC TGCGAGCGGT ACTGCGCGAG CGAAGTGGCG ACCAAACCCG ATTAGCCGAG +1 V L L S V V A L S G A A L S I 2001 GGTGCTTCTC AGCGTCGTCG CGCTGAGCGG AGCCGCACTG TCGATCGA CCACGAAGAG TCGCAGCAGC GCGACTCGCC TCGGCGTGAC AGCTAGCT
Letters in blue or red indicate deduced amino acids. For meaning of
abbreviations, see Appendix 3.1. Start and stop codons for each ORF
are bold and underlined. Putative SD sequences are bold. The
conserved residues of the deduced amino acid sequence of orf1 that
correspond to glutamic acid (amino acid 7), aspartic acid (amino acids
8, 9, 51) and lysine (amino acid 101) are indicated by asterisks.
218
5.2.3 Induction of the myo-Inositol Inducible Promoter
The iolXp fragment was isolated because it was responsible for myo-
inositol dependent induction of expression of GFP-UV. In order to
determine whether this induction is linked to myo-inositol catabolism,
expression was measured after the plasmid was conjugated into the
mutants RU360/pRU601, RU361/pRU601 and RU307/pRU601. These
strains, along with 3841/pRU601, were tested for GFP-UV expression
after overnight growth in the presence of different carbon sources
(Figure 5.4, Figure 5.5).
GFP-UV expression was calculated in relation to the growth of the
cultures, giving specific fluorescence (V). V was calculated using the
equation A-X/B-Y where A is the fluorescence of the sample, X is
fluorescence of the blank well, B is the OD630nm of the sample and Y is
the OD630nm of the blank well. For all strains tested, GFP-UV
expression was barely detectable when grown on pyruvate or pyruvate
plus myo-inositol, glucose, mannitol, sorbitol and fructose (Figure 5.4,
5.5, 5.6). Induction of expression of GFP-UV only occurred in 10mM
myo-inositol for 3841/pRU601 (Figure 5.4) and RU307/pRU601
(Figure5.6). The expression by RU307/pRU601 was only a quarter of
the expression of 3841/pRU601 (specific fluorescence of 11314 and
447735 respectively) (Figure 5.6).
219
These results indicate that there is a clear need for a functional myo-
inositol catabolic pathway in order to induce expression of GFP-UV and
that the promoter is specifically induced by myo-inositol.
220
Figure 5.4 Expression of GFP-UV by 3841/pRU601
P = 20mM pyruvate, PI = 20mM pyruvate + 10mM myo-inositol, I = 10mM myo-
inositol, G = 10mM glucose, M = 10mM mannitol, S = 10mM sorbitol, F = 10mM
fructose.
Each value represents the mean of three cultures, with standard deviation.
221
Figure 5.5 Expression of GFP-UV by RU360/pRU601 and RU361/pRU601
P = 20mM pyruvate, PI = 20mM pyruvate + 10mM myo-inositol, I = 10mM myo-
inositol, G = 10mM glucose, M = 10mM mannitol, S = 10mM sorbitol, F = 10mM
fructose. Each value represents the mean of three cultures, with standard deviation.
RU360 = dark blue, RU361 = light blue.
222
Figure 5.6 Expression of GFP-UV by RU307/pRU601
P = 20mM pyruvate, PI = 20mM pyruvate + 10mM myo-inositol, I = 10mM myo-
inositol, G = 10mM glucose, M = 10mM mannitol, S = 10mM sorbitol, F = 10mM
fructose.
Each value represents the mean of three cultures, with standard deviation.
223
5.2.4 Catabolite Repression
Induction of GFP-UV expression by 10mM myo-inositol was repressed
in 3841/pRU601 by the addition of 20mM pyruvate (Figure 5.4).
Therefore, the effect of combinations of myo-inositol and other carbon
sources (pyruvate, malate, succinate, glucose) on induction of GFP-UV
expression was tested. (Figure 5.7). The results indicate that catabolite
repression occurs. The extent of repression was dependent on the
concentration of the additional substrate. Addition of each of the four
carbon sources repressed induction of GFP-UV expression at a
concentration of 1mM, but the inhibition was considerably less than at
10mM.
224
Figure 5.7 Catabolite Repression of myo-Inositol Inducible GFP-UV
Expression in 3841/pRU601
I = 10mM myo-inositol, P = pyruvate, S = succinate, M = malate, G = glucose.
Each value represents the mean of three cultures, with standard deviation.
225
The kinetics of induction of GFP-UV expression after exposure to myo-
inositol were measured in 3841/pRU601. Cells grown overnight in
glucose were resuspended in different carbon sources and
fluorescence was measured every 10 minutes for 830 minutes (Figure
5.8). Induction of expression of GFP-UV was considered to occur
when the specific fluorescence was in excess of 1500V, as this is
higher than expression in the absence of myo-inositol (Figure 5.4).
Induction of expression of GFP-UV began at 150 minutes for pRU601
in 3841 in the presence of 10mM myo-inositol and 1mM myo-inositol.
When there was a combination of 10mM myo-inositol with 1mM of a
different carbon source, induction of expression of GFP-UV was
severely delayed. Expression of GFP-UV began after 200 minutes in
10mM myo-inositol plus 1mM pyruvate, 330 minutes in 10mM myo-
inositol plus 1mM malate, 360 minutes 10mM myo-inositol plus 1mM
succinate and 380 minutes in 10mM myo-inositol plus 1mM glucose.
With all carbon sources, the level of expression increased over the time
period measured.
There was no further increase in expression in 1mM myo-inositol after
830 minutes, due to the culture no longer growing, presumably
because of substrate exhaustion. There was much higher overall
expression in 10mM myo-inositol alone than when in combination with
other carbon sources, although at the end of the time period, the
amount of expression in the presence of 1mM pyruvate was almost as
226
much as in 1mM myo-inositol alone. This was probably due to
exhaustion of pyruvate in the growth medium, which is a three-carbon
compound.
There was no appreciable induction of expression of GFP-UV over the
time measured when 3841/pRU601 was grown in 10mM pyruvate,
glucose, succinate and malate as the sole carbon source. There was
also no appreciable expression when there was a combination of
10mM amounts of the above carbon sources and 10mM myo-inositol.
For clarity on the graph, only the data for 10mM pyruvate is shown
(Figure 5.8).
227
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
0 200 400 600 800time (mins)
spec
ific
fluor
esce
nce
(V)
1mM I 10mM I 1mM P + 10mM I1mM G + 10mM I 1mM S + 10mM I 1mM M + 10mM I10mM P
Figure 5.8 GFP-UV Expression Over Time by 3841/pRU601
I = myo-inositol, P = pyruvate, G = glucose, S = succinate, M = malate.
Each set of data represents the mean of three cultures of 3841/pRU601, with
standard deviation.
228
5.2.5 Relationship to myo-Inositol Mutants
The iolXp fragment was extracted from the pOT1 vector as a 2078bp
PacI fragment with flanking pOT1 DNA. This fragment was then used
as a probe for Southern hybridisation studies with the complementing
cosmids pRU3078, pRU3079 and pRU3111 digested with three
different restriction enzymes, EcoRI, PstI and SalI. Plasmid pRU601
was digested with PacI, giving a 2078bp fragment and a 5246bp
fragment, containing the pOT1 vector (Figure 5.9).
The probe bound to both pRU601 fragments that had been PacI
digested. The binding of the probe to the larger fragment may have
been due to partial digestion by PacI of pRU601. Incomplete digestion
with PacI has been observed previously in the laboratory (P.S. Poole,
pers. comm.).
There was no hybridisation of the fragment to the cosmids. These data
indicate that iolXp is a distinct locus to the myo-inositol utilisation loci
identified in this project. This was not a surprise, as the genes mutated
in RU360 and RU361 are distinct loci and there are clearly other genes
involved in myo-inositol utilisation in Rhizobium that have yet to be
identified.
229
Figure 5.10 Southern Hybridisation of pRU601
0.8% agarose gel showing DNA bands stained with ethidium bromide
visualised on a UV transilluminator.
Southern blot with of above agarose gel.
1 = 1kb ladder, 2 = pRU3078 EcoRI, 3 = pRU3079 EcoRI, 4 = pRU3111 EcoRI, 5 =
pRU3078 PstI, 6 = pRU3079 PstI, 7 = pRU3111 PstI, 8 = pRU3078 SalI, 9 =
pRU3079 SalI, 10 = pRU3111 SalI, 11 = pRU601 PacI.
←5.246kb
←2.078kb
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
←5.246kb
←2.078kb
5kb→
2kb→ 3kb→
230
5.2.6 Expression in the Rhizosphere and Nodules
There is a clear pattern of induction of the myo-inositol inducible
promoter in vitro. To investigate the importance of myo-inositol in the
rhizosphere, expression of GFP-UV by the inoXp fragment was studied
in the rhizosphere and nodules. P. sativum and V. sativa plants were
inoculated with pRU601/3841 at a density of 106 cfu per plant. Three
individual plants were harvested 1, 2, 3 and 4 days post-inoculation.
The rhizosphere bacteria were isolated and examined under a
microscope for expression of GFP-UV. Less than 1% of bacteria
observed in the rhizosphere expressed GFP-UV, indicating that the
promoter was not induced by conditions in the rhizosphere. The
bacteria that were thought to fluoresce might have been contaminating
plant particles that autofluoresced and were mistaken for GFP-UV-
expressing rhizobia.
Four weeks post-inoculation, five or six nodules were harvested from
three P. sativum plants, surface sterilised and crushed under a
microscope to examine expression of GFP-UV in bacteroids. In
contrast to the results gained from free-living bacteria in the
rhizosphere, virtually all bacteroids observed expressed GFP-UV,
indicating widespread induction of the promoter in nodules. However, it
was not possible to quantify the amount of GPF-UV expression, so it is
difficult to conclude that massive induction of expression occurred.
231
5.2.7 Stability of pRU601 in the Rhizosphere
It was not practical to apply gentamycin to the rhizosphere to guarantee the
retention of pOT1 plasmid by 3841. It was therefore necessary to assess the
stability of the plasmid in the absence of selective pressure. The 16 nodules
harvested from three P. sativum plants in Section 5.2.6 were streaked onto TY
agar, with and without gentamycin. Bacteria from 13 of the 16 nodules were
gentamycin resistant (81%). Six individual colonies were streaked from each
of the TY agar plates without gentamycin, onto AMA containing 10mM
glucose or 10mM myo-inositol, plus gentamycin to select for pOT1. Of the
resulting 78 colonies, 37 (47.5%) retained gentamycin resistance and
expressed GFP-UV when grown on myo-inositol but not when grown on
glucose (Table 5.1).
Table 5.1 Stability of pRU601 in the Rhizosphere
plant nodule number of gentamycin resistant colonies (of six)
A 1 3 2 1 3 2 4 2 5 1 6 0 B 1 5 2 6 3 1 4 5 5 6 C 1 1 2 0 3 3
232
4 1 5 0
5.2.8 Construction of a Specific myo-Inositol Inducible GFP-UV
Promoter Probe
The gene interrupted in RU360 was selected as a candidate for a defined
promoter probe, in order to study the induction of myo-inositol catabolic genes
in the rhizosphere and nodules. The promoter probe was made by PCR
amplification of the complementing cosmid pRU3078 (Figure 5.10) and cloned
into pOT1 containing the promoter-less gfpuv gene. This vector was chosen
because GFP-UV expression had been successfully studied previously in vitro
and in vivo.
Primers p213 and p214 were designed to amplify a 743bp fragment
containing the beginning of iolD along with 350bp upstream of the putative
start codon of iolD. It was hoped that this would contain the promoter region
for the gene. The restriction enzyme sites PstI and SpeI were included in the
primers p213 and p214 respectively, to enable the fragment to be cloned into
pOT1 immediately upstream of and in the same direction as the gfpuv gene.
The resulting plasmid was named pRU703. Plasmids pRU706 and pRU713
were also cloned in the same direction as the gfpuv gene, so that they too
could be used for promoter probe studies (c.f. Section 3.2.5.1.1). Sequencing
confirmed the correct orientation of the PCR fragments in pRU703, pRU706
and pRU713.
233
Plasmids pRU703, pRU706 and pRU713 were conjugated into RU360,
RU361, RU307 and 3841 to examine induction of the gfpuv gene. None of
the promoter probe plasmids expressed GFP-UV in the presence of myo-
inositol or pyruvate (Figure 5.11). There is clearly a functional promoter
present in the probes, as pRU713 complemented RU360, restoring the ability
to grow on myo-inositol. Therefore, there may have been a problem with the
cloning strategy. Co-workers in the laboratory have also found that the gfpuv
gene is not expressed in plasmids constructed by cloning into the SpeI site of
the pOT1 plasmid (P.S.Poole, pers. comm.). This site is immediately adjacent
to the ribosomal binding site of the gfpuv gene so perhaps ribosome binding is
disrupted when DNA is cloned into this site in the plasmid.
Figure 5.10 Promoter-Probe Vectors of iolD
iolD iolE iolB orf1
PstI (1938)
EcoR I (2115)
EcoR I (3013)SalI (754)SalI (5448)SstI (955)
SstI (4072)
1kb
pRU703
pRU706
pRU713
234
The 5.606kb region sequenced from pRU3078 with arrows detailing the
sequence encompassed by the subclones.
235
Figure 5.11 Expression of GFP-UV in Promoter Probe Vectors
Each series represents the mean of three replicates. Plasmids in 3841 and RU307
were grown on 10mM myo-inositol, plasmids in RU360 and RU361 on 10mM myo-
inositol plus 20mM pyruvate.
236
5.2.9 RU360 β-Galactosidase Assay
The lack of induction of GFP-UV in the promoter-probes pRU703, pRU706
and pRU713 suggests either that the genes interrupted in RU360 are not
inducible by myo-inositol or that there was a problem with the cloning strategy.
It was possible to test whether the genes are myo-inositol inducible because
Tn5-lacZ in RU360 contains the promoter-less reporter gene lacZ, which
codes for β-galactosidase and it is transcribed in the same direction as the
interrupted genes.
It was postulated that the promoter for iolD would induce expression of the
lacZ gene in the presence of myo-inositol. However, when β-galactosidase
activity was measured in RU360, the fusion was not active (Poole et al.,
1994). It was decided to repeat this assay and also to test whether the fusion
was active in RU360 when complemented by pRU3078 and pRU3079 (Table
5.2). Strain RU361 was not tested, as Tn5-lacZ is transcribed in the opposite
direction to the interrupted gene.
The data show that β-galactosidase activity was very low in RU360 grown on
20mM pyruvate. The presence of 10mM myo-inositol in the growth medium
did not cause induction of expression of lacZ. When complemented with the
cosmids pRU3078 and pRU3079, there were 2-fold and 5-fold increases
respectively in the activity of lacZ when myo-inositol was present in addition to
pyruvate. When RU360/pRU3078 and RU360/pRU3079 were grown on myo-
237
inositol as the sole carbon source, there was an increase in expression of lacZ
of 12-fold and 20-fold respectively. These data indicate that the genes
interrupted by Tn5-lacZ in RU360 are myo-inositol inducible, but that a
functional catabolic pathway is required for induction and pyruvate represses
induction.
Table 5.2 β-Galactosidase Activity of RU360
activity in the presence of different carbon sources nmol/mg/min
strain pyruvate pyruvate + myo-inositol
myo-inositol
RU360 160.3 ± 15.5 132.2 ± 4.1 N/A RU360/pRU3078 125.7445466 264.8042469 1453.597377 RU360/pRU3079 90.0 ± 4.3 459.4 ± 35.3 1871.41653 ±
315.9069 Each value represents the mean of three cultures with standard deviation.
238
5.2.10 myo-Inositol Catabolic Enzyme Assays
It has been shown previously that the myo-inositol catabolic enzymes, myo-
inositol dehydrogenase and 2-keto-myo-inositol dehydratase are not induced
in free-living RU360 or bacteroids of 3841 (Poole et al., 1994). To determine
whether the enzymes are inducible in free-living RU361 and RU307, this
experiment was carried out with these two mutants grown overnight in 20mM
pyruvate or 20mM pyruvate plus 10mM myo-inositol. RU360 and 3841 were
tested as controls (Table 5.3).
Bacteroids of 3841 were also tested. Poole et al. (1994) isolated bacteroids
under air by differential centrifugation. However, the nitrogenase enzyme is
inactivated in the presence of oxygen and it is possible that the bacteroids
were damaged by the isolation procedure. Therefore, bacteroids were used
that had been isolated anaerobically by a co-worker in the laboratory.
The data indicate that the first two enzymes in the myo-inositol breakdown
pathway were not induced in 3841 or the mutants when grown on pyruvate as
the sole carbon source. There was an 18-fold increase in myo-inositol
dehydrogenase activity and a 50-fold increase in 2-keto-myo-inositol
dehydratase activity by 3841 when grown on myo-inositol. There was some
catabolite repression of the dehydratase, as when grown on pyruvate plus
myo-inositol, activity was only 45% of the activity when grown on myo-inositol
alone (Table 5.3).
239
There was a 7-fold increase in activity of both enzymes in RU307 when grown
on myo-inositol as the sole carbon source. There was a reduction when
RU307 was grown on pyruvate plus myo-inositol as the activities were only
51% and 52% of the activities when grown on myo-inositol alone. Overall, the
activities in RU307 were 43% and 14% of the activities in 3841.
There was no induction of myo-inositol dehydrogenase by RU360 and RU361
of the dehydrogenase when grown on pyruvate plus myo-inositol. There was
some induction of 2-keto-myo-inositol dehydratase by RU360 and RU361
when grown on pyruvate plus myo-inositol, but the activities were only 16%
and 15% respectively of the amount of activity of 3841 grown on pyruvate plus
myo-inositol. There was no induction in the bacteroids. The bacteroids were
positive for alanine dehydrogenase activity, which indicated that the
bacteroids were not damaged by the isolation process.
240
Table 5.3 Activity of myo-Inositol Catabolic Enzymes
µmol min-1 mg-1 protein
Strain carbon source myo-inositol
dehydrogenase
2-keto-myo-inositol
dehydratase
RU360 pyruvate 0.04 ± 0.001 0.014 ± 0
pyruvate + myo-
inositol 0.01 ± .002 0.094 ± 0.005
RU361 pyruvate 0.04 ± 0.006 0.038 ± 0.011
pyruvate + myo-
inositol 0.05 ± 0.017 0.085 ± 0.003
RU307 pyruvate 0.025 ± 0.001 0.025 ± 0.01
pyruvate + myo-
inositol 0.09 ± 0.018 0.096 ± 0.004
myo-inositol 0.175 ± 0.01 0.184 ± 0.01
3841 pyruvate 0.023 ± .005 0.026 ± 0.02
pyruvate + myo-
inositol 0.404 ± 0.101 0.579 ± 0.104
myo-inositol 0.409 ± 0.11 1.295 ± 0.446
3841
bacteroids
N/A 0.003 ± 0.001 0.013 ± 0.005
Mean of at least two cultures assayed in triplicate, with standard deviation.
241
5.3 Discussion
The iolXp fragment contains a promoter that is specifically induced by
myo-inositol. It is not possible that the promoter for the regulatory gene
orf1 induces expression of the GFP-UV gene, as it is transcribed
divergently. Therefore, a promoter from one of the other three ORFs
must be responsible for the myo-inositol dependent induction of GFP-
UV. In order to test this hypothesis promoter probes could be made.
Nested deletion could be used to identify the smallest region necessary
for induction of GFP-UV expression.
The ORFs could be tested to determine whether they encode genes
involved in myo-inositol utilisation by transposon mutagenesis of each
individual ORF. It is not expected that the ORFs encode genes
involved in myo-inositol utilisation, because there was no homology
with known myo-inositol utilisation genes, although orf4 is too small a
region to be certain, but they could encode regulators, the structure of
which are not known. If orf4 were shown to be responsible for the myo-
inositol dependent expression of GFP-UV, then iolXp could be used as
a probe to obtain a larger clone. Inverse PCR could be used to identify
the entire gene.
Based on sequence homology of the deduced protein, orf1 is thought to
encode the response regulator component of a two-component
regulatory system. Such systems allow bacteria to make adaptive
242
responses to changes in their environment. The most homologous
protein was FeuP of R. leguminosarum bv. viciae, which is involved in
the regulation of iron uptake (Yeoman et al., 1997). The deduced
product of orf1 contained conserved residues that are characteristic of
similar regulatory proteins, although there was only 39% identity to
FeuP. Therefore, orf1 is not likely to encode a protein that is
functionally equivalent to FeuP. To deduce the function of orf1, a
transposon mutant could be made so that the phenotype could be
determined. It is postulated that downstream of orf1 will be a gene
encoding the sensor regulator component. This could be ascertained
by constructing a primer to read further downstream in the
chromosomal DNA. To test whether this regulatory system affects
myo-inositol metabolism, a mutant would be required.
When conjugated into the myo-inositol mutants, the promoter present in
iolXp was not induced in any of the strains grown on pyruvate or
pyruvate plus myo-inositol. There was induction by RU307/pRU601
grown on myo-inositol although expression was only a quarter that of
3841/pRU601. The promoter is probably not induced by myo-inositol
alone, as some expression would also be expected in all the strains
grown on pyruvate plus myo-inositol, including RU360/pRU601 and
RU361/pRU601 because they are able to transport some myo-inositol
into the cell.
243
It can be hypothesised that the promoter responds to a breakdown
product of myo-inositol. The growth rate of RU307 on myo-inositol is
approximately a quarter the rate of 3841, due to reduced uptake of
myo-inositol by RU307 into the cell. Strain 3841 has an inducible
system for myo-inositol uptake (c.f. Section 3.2.5). If induced by a
breakdown product of myo-inositol, this would explain why the induction
of expression of GFP-UV was lower in RU307 than 3841. To ascertain
the compound that induces iolXp, the induction of expression of GFP-
UV by 3841/pRU601 could be measured following addition of
intermediates in the myo-inositol breakdown pathway.
The myo-inositol dependent induction of expression of GFP-UV was
repressed by other carbon compounds. This suggests that other
carbon sources are utilised preferentially to myo-inositol and that
catabolite repression occurs. When measured over time, the level of
GFP-UV expression increased, which may reflect the catabolite
repressors being removed from the growth media and utilisation of
myo-inositol beginning.
The iolXp promoter could be a powerful tool in the identification of
genes involved in myo-inositol utilisation. If iolXp were conjugated into
a 3841 transposon library and the resulting conjugants spread onto
AMA containing 10mM myo-inositol as the sole carbon source, it would
be expected that most colonies would glow. Any that did not would be
likely to be mutated in genes involved in induction of the promoter.
244
This simple screening method could also be used to identify mutants
that have escaped catabolic control. If the colonies were plated onto
AMA containing myo-inositol plus other carbon sources such as
glucose and pyruvate, then the colonies would not be expected to glow,
due to catabolite repression. Any colonies that glow would have
escaped this and therefore, may be mutated in genes encoding global
regulators of myo-inositol catabolism.
The presence of myo-inositol in root exudates and soil has been
reported (Lynch et al., 1958, McKercher and Anderson, 1968,
Sulochana, 1962, Yoshida, 1940, Wood and Stanway, 2000).
However, there was no induction of GFP-UV expression in the
rhizosphere of P. sativum or V. sativa plants, despite evidence that
pRU601 was retained by 3841 in 81% of nodules and so was probably
stable in the rhizosphere. There are obviously other carbon sources
released into the rhizosphere by P. sativum and V. sativa, as the myo-
inositol mutants RU360, RU361 and RU307 were able to grow as well
as 3841 in the rhizosphere of V. sativa plants (c.f. Section 4).
Therefore, there may be catabolite repression of the myo-inositol
inducible promoter occurring in the rhizosphere. It is also possible that
myo-inositol was not released into the rhizosphere in significant
amounts during this experiment. However, the rhizosphere was not
analysed for carbon compounds released.
245
In contrast to the lack of expression in the rhizosphere, there was
widespread expression of GFP-UV by bacteroids in pea nodules. The
presence of myo-inositol, derivatives and other cyclitols in nodules on
several legumes, including P. sativum has been widely documented
(Davis and Nordin 1983, Kouchi and Yoneyama, 1986, Kouchi and
Yoneyama 1984, Skøt and Egsgaard, 1984, Streeter, 1987; Streeter
and Saminen, 1986). Carbon dioxide labelling studies, oxygen
consumption and enzyme activity assays have shown that myo-inositol
and other cyclitols are probably not metabolised in nodules (Davis and
Nordin 1983, Kouchi and Yoneyama, 1986, Streeter and Salminen,
1986, Poole et al., 1994). However, the first two enzymes in the myo-
inositol catabolic pathway were not expressed in pea nodules
containing 3841 (c.f. Section 5.2.10, Poole et al., 1994). Therefore,
myo-inositol catabolism appears to be switched off in the nodule. This
might be due to repression of catabolic enzymes by other carbon
sources, particularly dicarboxylates, which are the preferred carbon
source by bacteroids in nodules.
The lack of induction of the first two enzymes in the myo-inositol catabolic
pathway was confirmed for free-living RU360 and RU361. Sequencing of the
mutated region in the mutants did not reveal any putative genes with
homology to those encoding the first two enzymes in the myo-inositol
catabolic pathway. Therefore, there could not have been a polar effect of the
transposon insertion on these genes. This indicates that there is a global
system of regulation of myo-inositol catabolic genes, where the final
246
breakdown products of the pathway induce expression of the first enzymes.
Strain RU307 did not induce the first two enzymes in the pathway when grown
on pyruvate and myo-inositol, but when grown on myo-inositol alone, the
enzymes were induced at similar rates to that of 3841, indicating that there
was no deficiency in the myo-inositol breakdown pathway.
There are clearly two different systems of regulation that occur in free-living
rhizobia and in bacteroids. There may be a difference in the regulation of
iolXp in bacteroids as the iolXp promoter is expressed in nodules, even though
the catabolic enzymes are not. The product that induces expression of iolXp
might be produced via an alternative pathway that does not utilise the first two
enzymes in the catabolic pathway. Or, there might be another compound
present in the nodule that has not been tested that can also cause expression
of the promoter, such as a derivative of myo-inositol. However, an alternative
explanation relates to the sensitivity of the microscope used to study GFP-UV
expression. It was noted that although RU360/pRU601 does not appear to
express GFP-UV in solid or laboratory media, when cells that had been left to
grow for a week were examined under a microscope, some GPF-UV
expression was observed, although nowhere near as much as 3841/pRU601
(P.S. Poole, pers.comm.). The level of GFP-UV expression observed by
bacteroids in nodules was not quantified, therefore, it is not possible to
conclude that widespread expression was occurring. Therefore, it may have
just been background expression and nothing specific after all.
247
One situation where there is a difference in gene regulation between
free-living rhizobia and bacteroids concerns rhizopines. The expression
of rhizopine synthesis (mos) genes in S. meliloti is controlled by the
symbiotic nitrogen-fixation regulatory system NifA/NtrA (Murphy et al.,
1988). In contrast, free-living rhizobia express rhizopine catabolism
(moc) genes, which are not expressed by bacteroids. Rhizopines can
be utilised by some rhizobia as the sole carbon and nitrogen source,
which may give them a competitive advantage over non-utilising
strains. Alternatively, they might be involved in signalling between
bacteroids and free-living rhizobia. Strain 3841 is not a rhizopine
producing strain, so this process could not be responsible for induction
of iolXp, but there might be other myo-inositol derivatives that have not
yet been identified that may cause expression of GFP-UV in nodules.
A future experiment could examine whether there is induction of
expression of GFP-UV in bacteroids of the myo-inositol catabolic
mutants. If RU360/pRU601, RU361/pRU601 or RU307/pRU601
express GFP-UV in bacteroids, this would indicate that the regulation of
iolXp is different in nodules to free-living rhizobia in the rhizosphere and
that either myo-inositol is being utilised by a different pathway, or other
compounds induce the promoter. However, as less myo-inositol is
transported into the mutants, it might be difficult to view GFP-UV
expression in vivo.
248
The complementation of the RU360 mutation by pRU713 (c.f. Section
3.2.5.1.1) indicates that this fragment contains a functional promoter. The
failure of the promoter probe vectors to induce expression of GFP-UV was
unexpected. Sequencing of the plasmids indicated that there was no
apparent deletion of part of the promoter-less GFP-UV gene although the
insertion site of the PCR fragments in the SpeI site of the pOT1 plasmid was
very close to the start of the gfpuv gene. The presence of a terminator at the
end of pRU713 might explain the lack of GFP-UV expression for this
fragment, but not why there was no expression in pRU703 and pRU706. The
β-galactosidase assay, measuring activity of the lacZ gene of Tn5-lacZ in
RU360 indicated that the interrupted gene was induced by myo-inositol, but
only when the catabolic pathway was complete. Therefore, induction of
expression of 3841/pRU703 and 3841/pRU706 would have been expected.
The most likely explanation is that there was a problem with the cloning
strategy, as co-workers in the laboratory have also found that the gfpuv gene
is not expressed in plasmids constructed by cloning into the SpeI site of the
pOT1 plasmid (P.S.Poole, pers. comm.). This site is immediately adjacent to
the ribosomal binding site of the gfpuv gene so perhaps ribosome binding is
disrupted when DNA is cloned into this site in the plasmid. To overcome this,
the fragments would need to be cloned into a different restriction enzyme site
further upstream of the ribosomal binding site, such as the SalI site. This is
known to produce active fusions as this was the site used for construction of
the Rhizobium library that was used in this project.
249
The lacZ activity of RU360 when complemented by the cosmids pRU3078 and
pRU3079 demonstrates that myo-inositol, or one of its breakdown products,
induces the gene interrupted by Tn5-lacZ. Uptake assays demonstrated that
myo-inositol is taken into the cells by RU360 but at a low rate. There was no
induction in RU360 grown on pyruvate plus myo-inositol, yet there was a small
amount of induction when complemented by the cosmids pRU3078 and
pRU3079. The lower activity by RU360/pRU3078 and RU360/pRU3079 when
grown in the presence of pyruvate was probably due to catabolite repression.
Therefore, once again, a breakdown product of myo-inositol is thought to
induce the promoter.
250
CHAPTER 6 - FINAL DISCUSSION ................................................................. 249
6.1 Conclusions .............................................................................................. 250
6.2 Future Work............................................................................................... 256
BIBLIOGRAPHY ............................................................................................... 260
249
Chapter 6 - Final Discussion
250
6.1 Conclusions
myo-Inositol is ubiquitous throughout nature and is an essential component of
many compounds. Several environmental micro-organisms have been
identified that can utilise myo-inositol as the sole carbon source, including B.
subtilis, K. aerogenes, Pseudomonas species, S. meliloti and R.
leguminosarum. It is likely that there is a specific myo-inositol catabolic
pathway that is conserved between these species.
During this project, three R. leguminosarum bv. viciae myo-inositol mutants
were characterised. Strains RU360 and RU361 were mutated in putative
genes encoding enzymes in the catabolic pathway. Strain RU360 was
mutated in acetolactate synthase (iolD) and RU361 in malonic semialdehyde
oxidative decarboxylase (iolA). Strain RU307 was mutated in a transport
system for myo-inositol (intA). It is clear that the regulation of myo-inositol
catabolism is under control of a global regulatory system, as the catabolic
genes are not arranged in one single operon, yet mutation of the final genes
in the pathway prevented induction of the first enzymes and the transport
system (Int). Based on the probable function of iolD, additional steps were
added to the proposed catabolic pathway. The work in this thesis shows that
myo-inositol catabolism is not complete until acetolactate or a further product
has been formed.
It is not possible to assign roles for iolE or iolB, the two putative genes
downstream of iolD. Complementation of RU360 required iolDEB, showing
251
that one or both of the iolEB genes are required for catabolism. IolE and IolB
of B. subtilis are both required for myo-inositol catabolism, but their functions
are unknown.
The high homology of the genes identified in this project with B. subtilis myo-
inositol catabolic genes indicates that the genes originated from the same
common ancestor. However, the genes are not arranged in similar patterns,
as the B. subtilis genes are arranged in one operon, whereas the R.
leguminosarum bv. viciae genes are located at more than three loci. The
three loci identified are not likely to be widely separated, since cosmids
containing approximately 30kb of DNA were not able to cross-complement. It
is not currently known whether all the genes of R. leguminosarum bv. viciae
are chromosomal as not all of the genes have been identified. There has only
been limited research into myo-inositol utilisation in rhizobia, but the myo-
inositol dehydrogenase gene (idh) in S. meliloti is known to be isolated on the
chromosome. In contrast, in R. trifolii, some, if not all, of the genes are
plasmid-encoded, as are the rhizopine catabolic genes, which are located on
the Sym plasmid in R. leguminosarum bv. viciae and S. meliloti.
The reasons for the separation of the genes are not known. Perhaps the
genes were originally arranged in an operon in a common ancestor, but were
in a promiscuous region, which led to their separation. Alternatively, they may
have all originally been on a plasmid and gradually became integrated into the
chromosome. It is also possible that the genes were separate and came
together in B. subtilis. The genes are likely to be subject to different types and
252
levels of regulation and may even function in more than pathway, which would
also explain their separation. It is not possible to speculate on the reasons for
the spatial separation of the genes, based on the current evidence, but if the
genes are identified in more organisms it might become possible to determine
their evolutionary path.
There are at least two transport systems for myo-inositol in R. leguminosarum
bv. viciae, one of which is inducible and highly specific for myo-inositol uptake.
The presence of a specific pathway suggests that myo-inositol is an important
compound. This system might also be used to transport rhizopines, because
of the high homology of intB with mocB and the fact that only a binding
protein, but not a complete transport system has been identified amongst the
known moc genes. However, uptake was repressed by growth in the
presence of pyruvate, indicating that myo-inositol is not preferentially utilised
as a carbon source by Rhizobium. myo-Inositol has many functions in cells
and so it would be expected that a high steady-state intracellular
concentration would be required to induce the catabolic pathway. Otherwise,
myo-inositol might be used as a carbon source when it is actually required for
biosynthesis of other compounds.
Based on the growth rates in vitro and in vivo, the transposon insertions in
RU360, RU361 and RU307 did not impair growth relative to the wild type. All
three mutants were also able to nodulate plants and fix nitrogen at rates
similar to that of the wild type and the dry weights of the plants were all
similar. However, when co-inoculated with the wild type, RU360 and RU361
253
were at a massive competitive disadvantage for nodulation. However, RU307
was very competitive with the wild type. The disadvantage was thought to be
unlikely to be due to the ability to utilise myo-inositol as a carbon source,
because RU307 grows much more slowly than the wild type in vitro.
However, in the infection threads, it is not known what other factors might be
limiting that enable RU307 to compete successfully with the wild type. In the
rhizosphere, the ability to utilise myo-inositol offered no advantage to 3841
when co-inoculated with the mutants and other carbon compounds may
repress the myo-inositol catabolic enzymes, as occurred in the in vitro studies.
It is possible that in situ, when there are many different types of organisms
competing for resources, the ability to utilise myo-inositol or derivatives such
as rhizopines will be important, as was suggested by the work of Heinrich et
al. (1999). However, the strain used in these experiments is not a rhizopine
utiliser.
Clearly, the ability to respond to myo-inositol is crucial to nodule
competitiveness. It is postulated that the ability to respond to the presence of
myo-inositol might be part of a signalling mechanism between the host plant
and rhizobia as there are likely to be large quantities of myo-inositol and
derivatives in areas of developing nodules. myo-Inositol and derivatives have
been consistently reported in nodules. Their role is thus far unknown, but the
evidence reported in this work and elsewhere indicates that are probably not
utilised as a carbon source. One possibility is that these compounds are
involved in regulating osmotic potential, which is crucial for bacteroids in
nodules.
254
myo-Inositol or derivatives induce the first two enzymes in the catabolic
pathway, as well as iolD and iolXp. The first two enzymes in the catabolic
pathway are not induced in the myo-inositol catabolic mutants RU360 and
RU361, indicating that one or more of the final products induce the first
enzymes, in a feedback mechanism. The expression of Tn5-lacZ also
required a complete pathway and was repressed by pyruvate, indicating that
iolD might be regulated differently to the first two enzymes in the pathway.
Expression of iolXp was also repressed in the catabolic mutants and by the
presence of different carbon compounds in 3841, again indicating that it is a
breakdown product rather than pure myo-inositol that induces these
promoters.
In nodules, bacteroids of 3841/pRU601 were seen to be expressing GFP-UV,
indicating induction of iolXp. The first two enzymes in the catabolic pathway
were not induced in bacteroids. This suggests that there a change in
regulation of myo-inositol genes in bacteroids compared with in free-living
bacteria. However, these results must be treated with caution, as the
microscope is extremely sensitive and so there may not have been true
induction. There are likely to be many other carbon compounds present in the
nodule, including C4-dicarboxylic acids, which are the major energy source of
bacteroids. In free-living bacteria, malate and succinate inhibited myo-inositol
dependent induction of iolXp, but if GFP-UV expression is being induced in the
nodule, the lack of repression in the nodule could be due to a high flux and
low concentration of C4-dicarboxylic acids, which are used as an energy
255
source. There might also be a change in regulation of the genes during
symbiosis that results in myo-inositol being channelled into different
biosynthetic pathways. This could be tested by mutation of key nitrogen
regulatory genes such as nifA and then observing whether that has an effect
on expression of GFP-UV.
To conclude, during this project, it was shown that the ability to utilise myo-
inositol is crucial for nodulation competition in R. leguminosarum bv. viciae.
The specific role of myo-inositol remains unknown. Given the plethora of
roles that myo-inositol plays in cell function in all organisms, it would seem
unlikely that myo-inositol is only important as a carbon source in Rhizobium.
Possible explanations are that it is utilised as a carbon source in infection
threads, that it is involved in signalling between the host plant and bacterium
and that derivatives are involved in regulating osmotic potential in the nodule.
256
6.2 Future Work
There are several experiments that have been identified in each chapter that
are important to further ascertain the role of myo-inositol in competition for
nodulation. However, due to time constraints within this project, the
experiments were not carried out. The experiments are summarised below.
It is crucial to identify all the genes involved in myo-inositol utilisation in order
to elucidate the full catabolic pathway. Although the inducible expression of
the first two enzymes in the pathway is suggestive of rhizobia sharing the
same pathway as B. subtilis, it is not unequivocal proof that the entire pathway
is the same. The mechanism of regulation also needs to be discovered. It is
not currently known whether the genes studied in this project are induced by
myo-inositol or by derivatives. The uptake system was inhibited by other
sugars, as was the iolXp promoter and Tn5-lacZ in RU360. To determine
whether all the genes are chromosomal, 3841 could be cured of plasmids and
then tested to see if it retains the ability to utilise myo-inositol. Another way to
identify genes would be to make PCR probes from the B. subtilis iol genes
and from the S. meliloti idh gene.
It is also important to determine the exact regions required to restore the
ability to utilise myo-inositol to the mutants and to eliminate the possibility of
polar effects on downstream genes caused by transposon insertion. This is
particularly crucial for RU307, as the area around the insertion has not been
fully sequenced, due to the lack of a complementing cosmid.
257
It would also be useful to confirm the specificity of the myo-inositol
uptake system, using myo-inositol derivatives, such as rhizopines,
cyclitols and plant myo-inositol compounds. The whole region around
the uptake system also needs to be elucidated, by complementation of
RU307, or by constructing primers to extend the sequence further using
chromosomal DNA. This should also help to determine the nature of
the glutamic acid gamma hydrazide phenotype. By identifying the
cause of the toxic escape phenotype, it should be possible to eliminate
it as the cause of the competitiveness of RU307.
It is also crucial to discover the function of the gene(s) that the iolXp
promoter regulates or is a part of. If the myo-inositol dependent
expression of GFP-UV is regulated differently in free-living rhizobia and
bacteroids, this may indicate a novel role for the gene(s) regulated by
this promoter. Determining which genes are regulated could lead to
further understanding of the role of myo-inositol in nodules. This could
be achieved by making mutants and screening for loss of the ability to
induce expression of iolXp when grown on myo-inositol, or by screening
for switching on of iolXp when grown on other carbon sources.
To ascertain whether 3841 has an advantage in nodulation because it
induces nodulation more quickly, analysis could be carried out in
planta, using staining techniques to study nodule initiation. Timed
inoculation experiments could also be carried out. The nodule
258
occupancy studies should also be repeated, using larger sample sizes,
to see if RU307 was at some disadvantage. By making complementing
subclones of the cosmids that are stable in the rhizosphere, it should
be possible to test more accurately whether restoration of the ability to
utilise myo-inositol restored competitive ability to the mutants. This
would also eliminate the possibility that independent regions of the
cosmids compensated for the lack of the ability to utilise myo-inositol.
Strain RU307 also needs to be tested to see if it is able to utilise
rhizopines as the sole carbon source when the moc genes are
introduced into this strain. It is postulated that growth will be very slow,
if at all, because in RU307, the second, low affinity system may not
necessarily transport rhizopines as well as myo-inositol.
By studying the earlier processes of infection of plants by rhizobia, it
should be possible to track the progress of the mutants and 3841,
possibly by using GFP-UV. The aim would be to identify the exact
stage at which 3841 gains a competitive advantage over the myo-
inositol mutants. It is also necessary to further check the
competitiveness of RU307, to determine whether it is equally
competitive as 3841, as that would be an indicator that it is not the
ability to utilise myo-inositol as a carbon source.
259
Ultimately, the work carried out on R. leguminosarum bv. viciae could be
repeated in other species of Rhizobium, in order to determine whether myo-
inositol is important in all rhizobia.
260
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APPENDIX
313
Appendix 3.1 The Genetic Code
codon amino acid letter ttt, ttc phenylalanine F tta, ttg, ctt, ctc, cta, ctg leucine L tct, tcc, tca, tcg, agt, cgc serine S tat, tac tyrosine Y taa, tag, tga TERMINATION N/A tgt, tgc cysteine C cct, ccc, cca, ccg proline P tgg tryptophan W cat, cac histidine H caa, cag glutamine Q cgt, cgc, cga, cgg, aga, agg arginine R att, atc, ata isoleucine I atg methionine M act, acc, aca, acg threonine T aat, aac asparagine N aaa, aag lysine K gtt, gtc, gta, gtg valine V gct, gcc, gca, gcg alanine A gat, gac aspartic acid D gaa, gag glutamic acid E ggt, ggc, gga, ggg glycine G Start codons are highlighted in bold.
314
Appendix 3.2 Analysis of variance table for myo-inositol uptake by 3841,
RU360, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 4687.295 9 520.811 212.41 <0.001
Within Groups 46.586 19 2.452
Total 4733.881 28
L.S.D. = 2.676
Appendix 3.3 Analysis of variance table for glucose uptake by 3841,
RU360, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 517.84 9 57.54 1.78 0.161
Within Groups 453.06 14 32.36
Total 970.90 23
L.S.D. = 9.96
315
Appendix 3.4 Analysis of variance table for myo-inositol uptake by 3841
in the presence of excess sugar
Source of
Variation
SS df MS VR P value
Between Groups 3969.23 7 567.03 13.32 <0.001
Within Groups 680.87 16 42.55
Total 4650.1 23
L.S.D. = 11.29
Appendix 3.5 Analysis of variance table for glucose uptake by 3841 in
the presence of excess sugar
Source of
Variation
SS df MS VR P value
Between Groups 879.57 7 125.65 5.56 0.002
Within Groups 361.31 16 22.58
Total 1240.88 23
L.S.D. = 8.225
316
Appendix 4.1 Analysis of variance table for nodule number on V. sativa
plants inoculated with 3841, RU360, RU361 and RU307.
Source of
Variation
SS df MS VR P value
Between Groups 2.94 3 0.98 0.08 0.972
Within Groups 341 27 12.63
Total 343.94 30
L.S.D = 3.646
Appendix 4.2 Analysis of variance table for dry weight of V. sativa
plants inoculated with 3841, RU360, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 0.0083 4 0.0021 7.39 <0.001
Within Groups 0.0089 32 0.0003
Total 0.0172 36
L.S.D. = 0.0184
317
Appendix 4.3 Analysis of variance table for Acetylene Reduction Assay
of P. sativum plants inoculated with 3841, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 0.191E-05 2 0.956E-06 2.63 0.088
Within Groups 0.116E-04 32 0.363E-06
Total 0.135E-04 34
L.S.D. = 0.00055
Appendix 4.4 Analysis of variance table for dry weight of P. sativum
plants inoculated with 3841, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 0.06109 2 0.03054 1.27 0.316
Within Groups 0.28841 12 0.02403
Total 0.34950 14
L.S.D. = 0.2136
318
Appendix 4.5 Analysis of variance table for nodule number on P.
sativum plants inoculated with 3841, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 2784.6 2 1392.3 12.48 0.001
Within Groups 1339.2 12 111.6
Total 4123.8 14
L.S.D. = 14.56
Appendix 4.6 Analysis of variance table for nodule mass of P. sativum
plants inoculated with 3841, RU361 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 0.000085 2 0.000042 0.07 0.933
Within Groups 0.007246 12 0.000604
Total 0.007333 14
L.S.D. = 0.03387
319
Appendix 4.7 Analysis of variance table for nodule numbers of V. sativa
when inoculated with 3841, RU360, RU360/pRU3078 and RU360/pRU3079
Source of
Variation
SS df MS VR P value
Between Groups
Within Groups
Total
L.S.D. =
Appendix 4.8 Analysis of variance table for nodule numbers of V. sativa
when inoculated with 3841, RU361, RU361/pRU3111
Source of
Variation
SS df MS VR P value
Between Groups 588.75 6 98.13 4.04 0.002
Within Groups 1215.56 50 24.31
Total 1808.32 56
L.S.D. = 5.114
320
Appendix 4.9 Analysis of variance table for nodule numbers of V. sativa
when inoculated with 3841 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 1373.09 4 343.27 11.69 <0.001
Within Groups 1585.48 54 29.36
Total 2958.58 58
L.S.D. = 3.435
Appendix 4.10 Analysis of variance table for dry weight of V. sativa
plants when inoculated with 3841, RU360, RU360/pRU3078 and
RU360/pRU3079
Source of
Variation
SS df MS VR P value
Between Groups 0.033277 9 0.003698 11.13 <0.001
Within Groups 0.016272 49 0.000332
Total 0.049551 58
L.S.D. = 0.02218
321
Appendix 4.11 Analysis of variance table for dry weight of V. sativa
plants when inoculated with 3841, RU361, RU361/pRU3111
Source of
Variation
SS df MS VR P value
Between Groups 0.031418 7 0.004488 19.36 <0.001
Within Groups 0.009039 39 0.000231
Total 0.040457 46
L.S.D. = 0.01865
Appendix 4.12 Analysis of variance table for dry weight of V. sativa
plants when inoculated with 3841 and RU307
Source of
Variation
SS df MS VR P value
Between Groups 0.050328 5 0.010066 17.96 <0.001
Within Groups 0.021863 39 0.000561
Total 0.072192 44
L.S.D. = 0.01321
322
Appendix 4.13 Analysis of variance table for colony numbers of 3841,
RU360, RU361 and RU307 recovered from the rhizosphere
Source of
Variation
SS df MS VR P value
Between Groups 0.8493 3 0.2831 2.81 0.075
Within Groups 1.5105 15 0.1007
Total 2.2943 18
L.S.D. = 0.4278
Appendix 4.14 Analysis of variance table for colony numbers of RU360
and 3841 recovered from the rhizosphere when co-inoculated.
Source of
Variation
SS df MS VR P value
Between Groups 2.4151 3 .805 3.59 .041
Within Groups 3.1418 14 .2244
Total 5.3866 17
L.S.D. = 0.682
323
Appendix 4.15 Analysis of variance table for colony numbers of RU361
and 3841 recovered from the rhizosphere when co-inoculated.
Source of
Variation
SS df MS VR P value
Between Groups 4.95223 3 1.65074 21.42 <0.001
Within Groups 1.23285 16 .07705
Total 6.18508 19
L.S.D. = 0.3722
Appendix 4.16 Analysis of variance table for colony numbers of RU307
and 3841 recovered from the rhizosphere when co-inoculated.
Source of
Variation
SS df MS VR P value
Between Groups 2.04601 3 0.682 9.99 <0.001
Within Groups 1.09195 16 0.06825
Total 3.13796 19
L.S.D. 0.3503
324
Appendix 4.17 Analysis of variance table for alkaline phosphatase
assay of RU360, RU361, RU307 and 3841 containing a nodC-phoA
fusion.
Source of
Variation
SS df MS VR P value
Between Groups 1111087 8 1388859 4.05 0.018
Within Groups 3767624 11 342511
Total 1487849 19
L.S.D. = 1175.9
249
Chapter 3 graphs added by PSP as an appendix
250
0
5
10
15
20
25
30
35
40
3841 P 3841 PI 3841 I 360 P 360 PI 361 P 361 PI 307 P 307 PI 307 I
strain
upta
ke o
f glu
cose
nm
ol/m
g/m
in
251
0
10
20
30
40
50
60
3841 P 3841 PI 3841 I 360 P 360 PI 361 P 361 PI 307 P 307 PI 307 I
strain
upta
ke o
f myo
-inos
itol n
mol
/mg/
min
252
0
10
20
30
40
50
60
myo-inositol mannitol sorbitol glucose fructose pyruvate glutamicacid
gamma-hydrazide
galactose
carbon compound
upta
ke o
f myo
-inos
itol n
mol
/mg/
min
253
0
5
10
15
20
25
30
35
myo-inositol mannitol sorbitol glucose fructose pyruvate glutamicacid gammahydrazide
galactose
carbon compound
upta
ke o
f glu
cose
nm
ol/m
g/m
in
249
0
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
day
aver
age
num
ber o
f nod
ules
RU360 RU361 RU307 3841
250
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
uninoculated 3841 RU360 RU361 RU307strain
aver
age
wei
ght p
er p
lant
(g)
251
252
0
0.5
1
1.5
2
2.5
3
3.5
4
3841 RU361 RU307
strain
mic
ro m
oles
eth
ylen
e h-1
per
pla
nt
253
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
3841 RU361 RU307
strain
aver
age
plan
t wei
ght (
g)
254
0
10
20
30
40
50
60
70
80
90
100
3841 RU361 RU307
strain
aver
age
tota
l nod
ule
num
ber p
er p
lant
255
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
3841 RU361 RU307
strain
aver
age
tota
l nod
ule
mas
s pe
r pla
nt
256
0
20
40
60
80
100
1 2 3 4 5 6inoculum
% n
odul
es o
ccup
ied
3841 RU360 RU361
257
0
20
40
60
80
100
1 2 3 4 5 6
inoculum
% n
odul
es o
ccup
ied
3841 RU360
258
0
20
40
60
80
100
1 2 3 4 5 6
inoculum
% n
odul
es o
ccup
ied
3841 RU361
259
0
20
40
60
80
100
1 2 3 4 5 6
inoculum
% n
odul
es o
ccup
ied
3841 RU307
260
0
5
10
15
20
25
30
3841
RU360
RU360/p
RU3078
RU360/p
RU3079
3841
+ RU36
0
3841
+ RU36
0/pRU30
78
3841
+ RU36
0/pRU30
7938
41 +
RU360 1
0x38
41 +
RU360 1
00x
inoculum
aver
age
no. n
odul
es p
er p
lant
261
0
5
10
15
20
25
3841
RU361
RU361/p
RU3111
3841
+ RU36
1
3841
+ RU36
1/pRU31
11
3841
+ RU36
1 10x
3841
+ RU36
1 100
x
inoculum
aver
age
num
ber o
f nod
ules
per
pla
nt
262
0
5
10
15
20
25
30
35
3841
RU307
3841
+ RU30
7
3841
+ RU30
7 10x
3841
+ RU30
7 100
x
inoculum
aver
age
num
ber o
f nod
ules
per
pla
nt
263
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
unino
culat
ed
3841
RU360
RU360/p
RU3078
RU360/p
RU3079
3841
+ RU36
0
3841
+ RU36
0/pRU30
78
3841
+ RU36
0/pRU30
7938
41 +
RU360 1
0x38
41 +
RU360 1
00x
inoculum
aver
age
plan
t dry
wei
ght (
g)
264
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
unino
culat
ed
3841
RU361
RU361/p
RU3111
3841
+ RU36
1
3841
+ RU36
1/pRU31
1138
41 +
RU361 1
0x38
41 +
RU361 1
00x
inoculum
aver
age
plan
t dry
wei
ght (
g)
265
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
unino
culat
ed
3841
RU307
3841
+ RU30
738
41 +
RU307 1
0x38
41 +
RU307 1
00x
inoculum
aver
age
plan
t dry
wei
ght (
g)
266
Graphs chapter 4 added by PSP as an appendix
267
0
1
2
3
4
5
6
7
0 2 4 6 8
day of harvest
log1
0 cf
u pe
r rhi
zosp
here
RU361 3841 RU361 of RU361-3841 3841 of RU361-3841
268
0
1
2
3
4
5
6
7
0 2 4 6 8
day of harvest
log1
0 cf
u pe
r rhi
zosp
here
RU307 3841 RU307 of RU307-3841 3841 of RU307-3841
269
Graphs Chapter 5 added by PSP as an appendix
270
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
P PI I G M S F
carbon source
spec
ific
fluor
esce
nce
(V)
271
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
P PI G M S F P PI G M S F
RU360 carbon source RU361
spec
ific
fluor
esce
nce
(V)
272
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
P PI I G M S F
carbon source
spec
ific
fluor
esce
nce
(V)
273
0
5000
10000
15000
20000
25000
30000
I 20mMP + I
10mMP + I
5mM P+ I
1mM P+ I
10mMS + I
1mM S+ I
10mMM + I
1mM M+ I
10mMG + I
1mM G+ I
carbon source
spec
ific
fluor
esce
nce
(V)
274
0
5000
10000
15000
20000
25000
30000
3841
/pRU60
138
41/pR
U703
3841
/pRU70
738
41pR
U713
RU360/p
RU703
RU360/p
RU707
RU360/p
RU713
RU361/p
RU703
RU361/p
RU707
RU361/p
RU713
RU307/p
RU703
RU307/p
RU713
RU307/p
RU713
strain
spec
ific
fluor
esce
nce
(V)
275
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