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INTRODUCTION
Biological nitrogen fixation, in which microorganisms reduce
dinitrogen ·to ammonia, is a very important process, since it is
the predominant natural source of nitrogen for agricultural crop
production. Due to diazotrophy's agronomic importance, one needs
basic knowledge of it at molecular and genetic level to solve the
present and future problems of nitrogen inputs effectively. One
of the major problems in the manipulation of nitrogen fixing genes
has been the inhibition of this process by oxygen in many of the
diazotrophs. However, Azotobacter vinelandii, a member of Azoto
bacteriaceae family has a unique capacity to fix nitrogen in the
presence of oxygen. A. vinelandii which is widely distributed in
soil, is also known for its multiple copies of genome, 10-40 copies
per cell (Sadoff et ~ 1979). Recently it has also been proposed
that A. vinelandii possesses an "alternative pathway" (Bishop et
~ 1980) for reducing dinitrogen, which operates in the absence
of molybdenpm. Inspite of all these special features, very little
is known about its nif genetics, due to lack of genetic techniques.
However, lot of work has been carried out on the physiology of
nitrogen fixation and the structure and function of its nitrogenase
(Yates, 1974; Eady and Postgate, 1974; Mortenson et al., 1979;
Robson and Postgate, 1980; Eady, 1981). Development of recombinant
DNA techniques in recent years has made it possible to overcome
some of the technical barriers in the analysis of nif genetics in
A. vinelandii, It should now be possible to reveal the molecular
mechanisms involved in aerobic nitrogen fixation and the alternative
pathway.
- 2 -
The organization and expression of nitrogen fixation (nif)
genes has been thoroughly studied in Klebsiella pneumoniae, a
free living, microaerophilic diazotroph, due to its close relationship,
both physiolgically and genetically with Escherichia coli (Dixon,
1984; Cannon et ~ 1985). The cloned nif DNA fragments from
this organism has been used to identify and isolate nif genes from
other diazotrophic bacteria. The knowledge about nif genetics
in K. pneumoniae has been referred extensively in comparative
studies. Hence, some important features of nif genes in K. pneu
moniae will be discussed.
Nif genetics of Klebsiella pneumoniae :
Nitrogenase enzyme complex which catalyzes the reduction
of dinitrogen to ammonia in diazotrphs, is composed of two proteins,
component I and component II. Component I (Mo-Fe protein or
dini trogenase) is a tetramer of two different polypeptides (c(l. p.J
and component II (Fe protein) is dimer of identical subunits. In
_!5_:_ pneumoniae component I has an average molecular weight (M W)
of 220,000 daltons and each subunit oC or p has a MW of 56,000
daltons. Whereas, the component II (Fe-S protein or dinitrogenase
reductase) has a MW of 66,000 and each subunit 34,000 daltons.
(Eady and Smith, 1979). Generally the reduced component II transfers
electrons to component I which is dependent upon MgA TP hydrolysis.
Component I in turn reduces the dinitrogen (Mortenson and Thornley,
I 979).
- 3 -
Initial genetic studies revealed that ni f genes were located
close to histidine (his) operon (Streicher ~ ~ 1971; Dixon
and postgate, 1971) on K. pneumoniae genome and that when
his operon and adjacent DNA were transferred by conjugation
into~ coli, the reciepient gained ability to fix nitrogen (Dixon
and Postgate, 1972). Using classical genetic techniques an
F' WN68) was constructed in~ coli (cannon ~ ~ 1976) carrying
Klebsiella his and ni f genes. Dixon ~ ~ (1976) constructed
a promiscuous his-ni f plasmid pRD1 (formerly called RP41)
derived by recombination between FN68 and the P-type drug
resistance (Kanamycin, tetracycline and carbenicilliit~ plasmid
RP4. Later when molecular cloning techniques were developed
pRD1 was used as source for cloning the K. pneumoniae genes.
A series plasmids carrying overlapping restriction DNA fragments
were constructed (Cannon ~ ~ 1977, 1979; Puhler and Klipp,
1981) which were used in the mapping of ni f and in the study
of various nif gene products. Genetic mapping of K. pneumoniae
ni f DNA so far revealed that atleast 17 genes are present
which are contiguous and clustered in a 23 Kb region close
to the his operon. The 17 ni f genes arranged (Fig.1) in the
order his ••• ni f QBALFMUSUXNEYKDHJ •••• ShiA and are organised
in 7 or 8 operons (Dixon, 1984). Most ·of the nif genes are
transcribed in the direction of his operon except nif F and
nif J which are transcribed in the opposite orientation. Table
lists the molecular weights and functions of various gene
products (Cannon et ~ 1985). ni f H codes for the
component II, ni f D and ni f K code for o( and J3 subunits
respectively of component I. These three genes and another gene
his n 1f
DG Q 8 A L F M V S UXN E y K D H J genetic map \ r I I II I I I , I I I R R R H RX X R R: H R X restriction ~I · pCRAIO
map
I I R R
pGR Ill pMC2 r-t R R
pGRII2 H X
R R pWK25
R R I
pGRII3 II· R R·
pSA30
pCRA37 R R
, pGRII9
R pCMI
R R R
------R R H H
K, obase po1rs
" 5 10 15 20 25 30 35 '-'
Fig. I. Genetic and physical map of K. pneumoniae his nif region
(Ausubel ~ ~. 1982).
Gene
Q
B
A
L
F
M
v s u X
N
E
y
K
D
H
J
TABLE I
nif gene products and their function in K. pneumoniae
(From Cannon et al., 1985)
Mol. wt. of product (xl03 daltons)
Unknown
49
57
45
19
28
42
45
25
18
50
40
24
60
56
35
120
Function
Mo uptake
FeMoCo Synthesis
Transcription activation
Transcription repression
Flavodoxin subunit
Kp2 processing
FeMoCo synthesis
Kpl processing
Kpl processing
Unknown
FeMoCo synthesis
FeMoCo synthesis
Unknown
Kpl ~ - subunit
Kpl ~- subunit
Kp2 subunit
Pyruva te-Fla vodoxin-oxido-reduc-
tase subunit.
- 4 -
nif Y (whose function is not known) are transcribed as a single
operon from a promoter located upstream of nif H. nif M is involved
in the processing of component II and the mutations in nif M produce
inactive component II. The nif B, nif V, nif S, nif U, nif N and
nif E are involved in the synthesis of Fe-Mo cofactor of component
I nif Q may be involved in the Mo uptake. Function of nif X is
not clearly known. nif F and nif J encode components of a specific
electron transport pathway to nitrogenase. The nif F product is
a flavodoxin whereas nif J product derives reducing power from
anaerobic metabolism as a pyruvate: flavodoxin oxidoreductase.
nif A gene product activates the transcription of all other nif
genes and nif L is involved in the repression of nitrogenase synthesis
in the presence of oxygen and ammonia. nif A and nif L are trans-
cribed as a single operon from a promoter located upstream of
nif L.
Unique feature of the nif genes IS the unusual primary struc
ture of their promoters (Ow et al., 1983; Beynon et al., 1983). -- -- -- --
The nif promoters have an atypical consensus sequences 5'
-CTGGCACN; TTGCA-3' between positions -27 and -11 rather
than the characteristic sequence at -35 and -10 found in most
of the bacterial promoters (Rosenberg and Court, 1979; Hawley
and McClure, 1983).
Regulation of nif genes :
Nitrogenase synthesis was known to be repressed by fixed nitrogen
- 5 -
(Tubb and Postgate, 1973) and by the presence of Oxygen (Eady
et al., 1978). However, the exact mechanism of this control was
elucidated only after the development of lac fusion techniques.
where the (d -galactosidase gene (without its promoter) is cloned
downstream of the regulatory elements of the gene of interest
Expression of these regulatory elements is monitored by assaying
p -galactosidase activity. Most of the results discussed below have
been obtained using this powerful technique (Casadaban et ~
1980).
The nif genes are regulated at two levels. The first level
involves a centralized nitrogen regulation system (ntr system)
mediated by the products of ntr A (~F), ntr B ~L) and ntrC
(~G). The second level is specific control of nif operon mediated
by the products of nifL and nifA .
The nitrogen assimilation is controlled by ntr system in all
the enteric bacteria (Magasanik, 1982). The ntr genes of K. pneu-
moniae have also been cloned and found to have organization similar
to that of ..£:.._ coli (Espin et ~ 1982; de Bruiin and Ausubel, 1983;
Merrick and Stwart, 1985). The ntr B and ntrC genes are linked
to R!..!:!A (structural gene for glutamine synthetase) and form a
single operon, which is transcribed from ntr B to ntrC either from
their own promoter or by readthrough transcription from the stronger
~A promoter. ntr B o.na ntr C gene products of K. pneumoniae
have a MW of 31,000 daltons and 54,000 daltons respectively (Espin
- 6 -
et al., 1982). ntr A gene is not linked to ~A ntr BC and its gene
product has a MW of 75,000 daltons (de Brujijn and Ausubel, 1983).
The expression of ntr A is independent of nitrogen status of the
cell (Merrick and Stewart, 1985).
Fig. 2 shows the current model of how ntr genes control
the transcription of nif genes (Cannon ~ al., 1985). At low levels
(4- mM) expression of ~A/ntr BC is greatly enhanced
from the ntr promoter preceeding ~A, due to autogenous activation
by ntrC gene product in concert with ntr A. ntrC protein activates
the transcription from nifA promoter and other operons like hut
(histidine utilization), ~(proline utilization) aut (arginine utilization).
The nifA gene product in turn activates the transcription from
all other ni f operons and again ntr A is necessary for this activation,
while nifL product is maintained in an inactive form (Buchanan
Wollaston et ~ 1981). In the presence of high levels of NH4(>
20 mM), ntrC in conjunction with ntrB product represse s the trans
cription of nifLA promoter (Drummond et al, 1983). This regulation
is not affected by oxygen.
The second level of regulation, which occurs at intermediate
levels of NHl) 4- mM) and at low levels of dissolved oxygen(>o.l
uM) involves nifL product, where repressor form of nifL is predo
minant and most likely inactivates the nifA protein.
The role of nifA was clarified by constructing plasmids which
Act1ve---~
1nact1ve
nlfl ndA c:=J
t nl r C c===J
t Inactive
ntrB ginA r==J
N
_. ----- P3-----P2-PI
!\ Low N
All other nd operons
nfr and nJf promoters
t j Mod 1 11 e d R N A-p-o -1 y-fTl-e -r o-s-e~j
t S1gmo subunll
t n IrA
Fig. 2 Regulation of nif genes (Cannon ~ ~. 198 5).
- 7 -
express this gene constitutively (Buchanan ~ ~ 1981 ), when
these plasm ids were present, ni f expression was not subjected
to repression by NH4 or 02
and it was also independent of
mutations in ntr genes. It was also found that less amount
of nifA was enough for the activation. Thus showing, ntr genes
activated ni f expression which is inhibited in repressing condi
tions (+NH4
or o2
) bythe interaction of nifA with nifL. These
experiments also demonstrated that lack of nitrogenase activity
in vivo at 37°C was due to the inactivation of nifA.
The nifLA operon is also subjected to autoregulation
by ni fA gene product. In concert with ntrA gene product,ni fA
protein can substitute for the ntrC gene product and activate
the operons of other nitrogen metabolism genes, .9!!:!_A, hut,
~ and cut (Ow and Ausubel, 1983; Drummond ~ ~ 1983).
However, ntrC, ntrA cannot activate nifH promoterofKlebsiella.
This led to the proposal that ntrA could act as an alternate
sigma factor for RNA polymerase (Beynon ~ ~ 1983; deBruijn
and Ausbel, 1983). Since the nif promoters have an atypical
structure, modification of RNA polymerase might be necessary
to allow transcription at these promoters. Merrick and Gibbins
(1985) sequenced the ntrA gene and identified some sequences
which are similar to those found in known site specific DNA
binding domains present in five sigma factors from I:_ coli
and B. subtils and may be involved in the recognition of -35
and -10 promoter sequences (-27 and -11 incaseofnifpromoters).
- 8 -
It has also been shown that nucleotides -72 to -184 in K. pneu
moniae ni fH promoter is necessary for the activation by ni fA
(Buck et ~ 1985). Deletion of these sequences relieves the
inhibitory effect on nif gene expression by multiple copies
of nifH, nifU and nifB promoters and it can also be activated
weakly by ntrC (Buck ~ ~ 1986). This upstream sequence
seems to be a specialisation among certain ni f promoters,
because no such effect was found in case of ni fl. promoter,
thus permitting the efficient activation byregulatory proteins
functionally homologous to ni fA.
Azotobacter vinelandii
Nitrogenase complex of A. vinelandii is also composed
of two proteins, component I and component II (Bulen and
Leconte, 1966). Component has an average MW of 245,000
daltons, containing 2 atoms of Mo per molecule. The two sub
units and have an average MW of 61 ,ODD daltons each (Swisher
~ ~ 1977; Shah and Brill, 1977). Molybednum was found asso
ciated with component I also as a co factor, FeMoCo (Nagatani
et ~ 1974) which contained Fe, S and Mo in the ratio 8:
6: 1 (Shah and Brill, 1977). Component, II (MW 60,000) is a
dimer formed of two identical subunits of 31,200 daltons each
containing 289 aminoacids (Hausinger and Howard, 1980).
Nitrogenase complex of A. vinelandii J..!:!. vivo or J..!:!. vitro crude
extracts is more resistant to oxygen (Bullen ~ ~ 1964) than
that of other diazotrophs. Different mechanisms have been
- 9 -
proposed to explain the oxygen tolerance of A. vinelandii
nitrogenase. The more widely accepted one has been respiratory
protection supported by conformational protection (Postgate
et §.1 1981 ). During aerobic growth, nitrogenase is protected
by high rate of respiration, which scavenges the excess oxygen.
When 0 2 concentration is strong enough to overcome the rate
of respiration, nitrogenase forms a complex with a protective
protein and in this conformation nitrogenase is inactive but
remains undamaged. When the oxygen concentration is lowered,
the complex dissociates the nitrogenase becomes active.
Shethana ~ ~ (1966) isolated an FeS protein also called 'Shethna
protein' (Fe-S proteinii) of MW 24,000 daltons, which in its
oxidized form binds to nitrogenase and might act as the protec
tive agent during the conformational protection (Scherings,
1977).
Another important physiological factor which i fluences
the nitrogen fixation is fixed ammonia. In A. vinelandii ammonia
or any easily assimilated nitrogen source represses the synthesis
of nitrogenase enzyme (Shah et §.1 1972). However, the extent
of inhibition of nitrogenase activity was found to be variable,
anywhere between 30-100% inhibition, depending on growth
conditions (Eady, 1981 ). Klugkist and Haaker (1984) explained
these variable results. Inhibition of nitrogenase activity by
NH4
CI is dependent upon : [a] concentration of dissolved
02
in the medium: under extreme concentrations of oxygen
(very low or very high) nitrogenase activity is low and inhibition
by NH4
CJ is very strong. Whereas, at optimal concentrations
- 10 -
of oxygen, nitrogenase activity was maximum and inhibition
by NH4
CJ is small. [b] pH of the medium: inhibition by NH4
C I
was very strong at low pH values. [c] stage of growth: at
the end of growth, nitrogenase activity is more strongly inhibited
than at an early phase of growth.
Genome Organization :
Genetic studies of A. vinelandii was hampered due to
lack of auxotrophic mutants. Though nif mutants could be
isolated at high frequency, it was difficult to isolate auxotrophic
mutants. Very few auxotrophic mutants were isolated,adenine
(Mishra and Wyss, 1969), purine and pyrimidine auxotrophs(Page
and Sadoff, 1976) and mutants lacing sulfate reductase. The
difficulty in the isolation of mutants was attributed to genetic
redundancy in the form of an unusually high chromosome copy
number, 10-40 copies per cell (Sadoff, 1979).
Sadoff et al
A. vine Iandi i ce lis
(1971) found that exponentially growing
14 contain as much as 15x1 0 g DNA per
cell which is 40 times more than present in an~ coli cell,where-
asin cyst form, the amount of DNA was 10 times more than
that of an ~ coli cell. Studies with purified chromosomal DNA
(Sadoff ~ ~ 1979) showed that average G+C content was
65%. The denaturation kinetics follow second order kinetics in the
first 60 min. Cot 1/2value is similar to that of E.coli DNA. Even
the average sedimentation coefficient (1600 svedberg units) was
similar to that of E.coli chromosome(1300 svedberg units). These
data suggested that A. vinelandii genome might exist in 40 copies per
cell in mid-exponential phase where as it excists in 10 copies per
- 11 -
cell in cyst form (Sadoff et ~ 1979). Later Terzaghi (1980) found
that the rate of survival of UV irradiated A. vinelandii cells was
similar to that of ..£:._ coli cells, suggesting that all the copies may
not be biologically functional.
Genetics of nif in Azotobacter vinelandii
The niC mutants isolated in earlier stages of work (Wyss
and Wyss, 1950; Green et ~ 1953) could not be characterized
due to Jack of purified nitrogenase proteins and efficient transfor
mation system. Later several stable nif mutants were obtained
after mutagenesis with nitrosoguanidine and enrichment with peni
cillin (Shah et ~ 1973). These mutants have been well characterized
by titrating the extracts of niC strains with purified components
of nitrogenase, determining the amount of antigenic cross reactive
material for each component in the extract, locating the nitrogenase
components on polyacrylamide gels by an iron staining (Brill et
~ 1974) tecpnique and by electron paramagnetic resonance spectros
copy of whole cells, which indicates the presence or absence of
a signal (g = 3.65) unique to component I. Table 2 shows the charac
teristics of each mutant strain (Shah et ~ 1973; Brill et ~ 1974).
Mutants UW 1 and UW2 do not synthesize component I and component
II, therefore, mutation may be regulatory in nature. Mutants UW3
and UWlO make inactive component I, whereas in strains UW6
and UW38 component I was not detected. UW91 makes inactive
component II. In UW45 component I is inactive but it can be activa
ted by the addition of FeMoCo to the crude extracts (Nagatani
Strain
Wild type
UWl
UW2
UW3
UW6
UWlO
UW38
UW45
UW91
TABLE 2
nif- mutants of A. vinelandii (Bishop et ~ 1977)
Activity
I+II+
I II
I II
I II
(II+
(II+
(II++
I+II+
1+1(
CRM
I+II+
(II-,
I II
I+I(
(II+
I+II+
(II+
I+II+
I+II+
Derepreseed activity
+
- 12 -
et ~ 1974) Later it was found that UW38, which lacks component
I activity makes more than 5-8 fold component II than the wild
type (Shah ~ ~ 1974). nif+ revertants of this strain synthesized
equivalent amounts of both the components, suggesting that the
releaxed control of component II and the nif- phenotype were caused
by a single mutation. Using strain UW2 some mutants derepressed
for NH 4 were obtained, which make 1 o5 fold more nitrogenase
in the presence of NH4 than the wild type (Gordon and Brill, 1972).
Using a transformation procedure described by Page and
Sadoff (1976), some of the nif mutations were mapped (Bishp and
Brill, 1977; Bishop et ~ 1977) and figure 3 shows the genetic
map obtained. Mutations nif-1 and nif-2 are tightly linked and
may be located in the same gene. Mutation nif-45 was closer to
nif-1 and nif-2 than to any other mutations. Mutations nif-6 and
nif-38 appeared to be tightly linked to each other but they can
be separated by recombination. !\.4wte.tigr:~s .!:!.!!_ 91 ~ .!:!.!!_ l 0 ~
~ lecaliz.eEI +H ~ ~ rec9FR9ir:~e.tier:~. Mutations nif-91 and nif-10
may be localized in the same region. From these results they con
cluded that nif genes do not fall into one cluster as in Klebsiella
pneumoniae and may be spread over a large region of A. vinelandii
genome. It may be pointed out that crude chromosomal DNA was
used for transformation in these experiments and the linkage observ
ed between two mutations could as well be due to the large size
of the transforming DNA.
Phenotype 1-11---;----+----------~+---~----~--+--
Mutation nif-1 nif-45 nif-6 nif-10 nd-91 nif-3 n if -2 nif-38
Fig. 3. Genetic map of niC mutants.
- 13 -
However, in the past few years some progress has been made
in understanding the organization and expression of A. vinelandii
nif genes. This was largely due to the availability of a good trans-
formation system in A. vinelandii and also the use of molecular
cloning methods.
Transformation system on plates of BNF agar, described
by Page and Sadoff (1976) was modified to allow competence indue-
tion in liquid medium, where competence was induced by growing
A. vinelandii in iron limited BNF medium (Page and vonTigerstrom,
1978). The cells wee readily transformed by crude lysate or purified
DNA (Page and vonTigerstrom, 1979) achieving transformation
-3 -2 frequencies of 10 to 10 . Competence development was found
optimal at pH 7.2 to 7.4- and required restricted aeration of the
culture. It was also found that nitrogen fixing cell were less compe-
tent than those grown with fixed nitrogen because of the increased
demands for reducing power by respiration and nitrogenase (Page,
1982). This transformation procedure was further refined so that
plasmid DNA can be used resulting in transformation at a frequency
4- -2 of 3 x 10- to 5 x 10 depending on the plasm ids used (Glick
et ~ 1985).
Most of the information available to date on A. vinelandii
was obtained using the cloned K. pneumoniae nif DNA fragments.
Initially, Ruvkun and Ausubel (1980) showed that nif structural
genes (nifHDK) of K. pneumoniae hybridized to chromosomal DNA
- 14 -
of many nitrogen fixing bacteria, including A. vine1andii. K. pneu
moniae nifHDK fragment hybridized to 5 fragments of 12.5, 8.1,
3.5, 2.2 and 0.8 Kb in size, with EcoRI digested A. vinelandii (strain
UW 1 0) chromosomal DNA. This indicated that nif genes in A.
vinelandii might be reiterated or they are dispersed on the chromo
some. However, Krol et al. (1982) analyzed nif transcrips by hybridi
zing RNA blots of A. vinelandii with K. pneumoniae nifHDK probe
and demonstrated that nif structural genes in A. vinelandii are
transcribed as a single operon, suggesting that at1east nif structural
genes are clustered. Medhora et al (1983) reported the construction
of a genomic library of A. vinelandii in a cosmid, pHC79, from
which four recombinant cosmids, pMP1, pMP2, pMP3 and pMP4
having sequence homology with K. pneumoniae nifHDK DNA were
isolated. When Bglll digests of these cosmids were probed with
Klebsiella nifHDK fragment, a 22.5 Kb fragment in pMP 1, a 4.3
Kb fragment in pMP2 and pMP3 and a 6 Kb fragment in pMP4
showed hybridization. It was also argued that nif sequence in pMP 1
may exist on a different Bglll restriction fragment of A. vinelandii
chromosome as compared with nif sequences that have been cloned
in pMP2 or pMP3. Therefore, this organism contains more than
one copy of nif sequences.
When pMP1 ~nd pMP2 were used for the marker rescue test
by transformation of nif- mutant UW6 ((II+), pMP2 transformed
UW6 to ampr, nif+ phenotype. Whereas, pMP1 failed to transform
it to nif+ phenotype, though ampr transformants were obtained
- 15 -
(Medhora, 1984 ). Bishop et al. (1985) reported the construction
of a gene library of A. vinelandii in the EcoRI site of pBR325
and isolation of recombinant plasmids pLBl and pLB3, which hybri
dize with nifHDK probe of K. pneumoniae. pLBl and pLB3 contained
2.6 and 1.4 Kb EcoRI fragments respectively. Marker rescue tests
by genetic transformation indicated that pLB1 contained wild type
allele for nif-6 and nif-38 mutations carried by nif strains UW6
and UW38 and pLB3 contained wild type allele for nif-1 0 (UW 10
strain) mutation, indicating that these fragments contained DNA
sequences for atleast part of the nifK (2.6 Kb fragment) and nifD
(1.4 Kb fragment) genes. The authors have also demonstrated that
these two fragments are contiguous on A. vinelandii genome.
Recently, Brigle ~ ~ (1985) reported the isolation of 6
Kb Smal fragment, which carries nifHDK genes. This fragment
was sequenced and the sequence of nif H, D and K genes was
compared with that of other nitrogen fixing bacteria. Sequence
analysis revealed that, the region preceeding nifH contains sequences
of striking homology with the K. pneumoniae nif H promoter (Beynon
et ~ 1983). Since this homology was contained within the consensus
nif promoter region, it might contain nifH promoter of~ vinelandii.
nifH and nifD are separated by 129 bp and 101 bp separate nifD
and nifK. There was no consensus nif promoter sequence in these
intercistronic regions indicating that all the three genes are trans
cribed from the nifH promoter. nifH and nifD sequences end with
tandem stop condons, which along with secondary structures found
- 16 -
in intercistronic region might play regulatory role in differential
expression of the individual gene products.
The search for the nif regulatory genes in A. vinelandii
was carried out by inducing various cloned regulatory elements
of nif genes of K. pneumoniae which were fused to ~·-galactosidase
gene.
When nifA gene of Klebsiella cloned into broad host range
plasmid pKT230, was introduced into A. vinelandii nif mutant
UW 1 ((I(), it could activate the nif genes of A. vinelandii, indicating
the presence of an activator gene, analogous to K. pneumoniae
nifA (Kennedy and Robson, 1983). Drummond and Kennedy (1985)
introduced plasmids carrying nif genes of Klebsiella fused to lacZ
gene into A. vinelandii strains UW (wild type), UW 1 and UW2 (both
(II-). Results of this experiment showed that ntrC and nifL genes
of Klebsie:la had no effect on nif expression in A. vinelandii. Whereas
nifA carried on a low copy number plasmid corrected these mu
tations, Klebsiella nifL-lacZ and nifF-lacZ fusion:; expressed very
strongly and were not repressed by ammonium. However, nifH
lacZ fusion failed to express even in the wild type strain (UW)
under any conditions, except when Klebsiella nifA was also present,
that too weakly. Based on these findings, it was suggested that
nif regulatory mechanism in Azotobacter includes an ntrC like
activator, but the nif specific regulation is less like that of ..!5.:_
pneumoniae. The differences were attributed to the different
- 17 -
physiological conditions under which they fix nitrogen which may
impose certain constraints on the regulatory systems involved.
Alternative pathway for nitrogen fixation :
Almost all the nitrogen fixing bacteria require Mo for the
nitrogenase activity, which was rationalized by the isolation of
an essential FeMoCo (Shah and Brill, 1977). This protein, is proposed
to contain at least part of the active site of component I and
may be involved in N2 binding (Hawkes et al., 1984). Molybdenum
is also involved m the regulation of nitrogenase synthesis (Eady
et ~ 1982). However, several evidences show that in A. vinelandii
there may be another enzyme complex which reduces nitrogen
in the absence of molybdenum. Initial evidence came when some
Nif+ pseudorevertants of nif- strains (UW6, UW 1 0) of A. vinelandii,
which could fix nitrogen in the presence of 1 mM tungsten (W) wu·c. isol<a.~ (Bishop et ¥1 1980). Characterization of these pseudorevertants
revealed that two new ammonia repressible proteins were synthesized
and the conventional nitrogenase proteins were not made. When
wild type A. vinelandii of nif parental strain of above mentioned
pseudorevertants were grown in broth with no added Mo, they
also showed the presence of the two new proteins (Bishop et ~
1982). Based on these evidences, Bishop et al (1980, 1982) proposed
that there is an "alternative pathway" and it is Mo independent.
Another group isolated a resistant mutant (WD2) of wild type A.
vinelandii (UW), which can grow in Burk's nitrogen free medium
- 18 -
containing 10 mM W, whereas, N2 independent growth of wild
type is inhibited by 0.01 mM W (Riddle et ~ 1982). WD2 strain
exhibited 22% of whole cell acetylene reduction activity of the
wild type in broth containing Mo and had lowered affinity for
acetylene. Two dimensional gel electrophoresis and ESR analysis
of cell extracts showed that instead of native nitrogenase proteins,
some new ammonia repressible proteins were formed. Premakumar
et ~ (1984) provided further evidence by demonstrating the presence
of an alternative reductase (component II) in the extracts of nif
mutants U W 1, UW3 and UW9 grown under Mo deficient conditions.
They also showed that "alternative reductase" is repressed by Mo
and W, whereas Vanadium (V) stimulated the synthesis.
These studies, coupled with the hybridisation of multiple
fragments with K. pneumoniae nifHDK fragment (Ruvkun and
Ausubel, 1980; Medhora et ~ , 1983) lend support to the existence
of an "alternative pathway" for nitrogen fixation in A. vinelandii.
Transposon Tn5 mutagenesis :
Transposons, the mobile genetic elements, have been very
useful in the mapping of genes in bacteria. Among them, transposon
Tn5 has been used extensively for mutagenesis. Tn5 is 5700 bp
in size, with two inverted repeats of 1535 bp. The central 2700
bp region confers Kanamycin or neomycin and streptomycin resis
tance (Fig.4). The inverted repeats contain genes necessary for
Tn 5
r r r Km I Nm Sm )
IS50 L IS50 R
I I )I I
I I 1 X p H B p Sm PS X Bm B H p X
II) 0 II) II) 00 <0 M"~t <0 0 0 0 II) 0 00 00 Ol .... N .... 11)00 N ...... <0 00 Ol Ol 'It <0 .... II) ...... II) <0<0 00 0 N II) 0 N .... .... .... N NN N M 'It 'It II) II)
Fig. 4. Restriction map of Tn5.
Bg- Bglll; H-Hindlll; Bm- BamHI; R- EcoRI; P- Pstl;
Sm- Smal.
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transposition (Berg and Berg, 1983). Since the transposon Tn5 does
not have any insertional specificity and the resultant mutants
are very stable and exert strong polarity effects on the downstream
genes in a polycistronic operon, it was the most preferred transposon
(Lupski and deBruijon, 1983).
In general two types of techniques have been used to create
Tn5 mutagenesis :
1. Generalized mutagenesis, where genomic DNA is mutagenized
randomly by introducing a suicide plasmid or phage carrying Tn5.
Selection for Kanamycin or neomycin results in the detection of
transposition of the Tn5 from the suicide vector into the gencrre
of the host and concamitant loss of the vector. In.£:.. coli, defective
)\-phage ( )\ 467) was used as vector. Among suicide plasmids
pSUP2021, which contains ColEI replicon, mob region from RP4
plasmid and tn5 (Simon et ~ 1983) was very useful for mutagenizing
non-enteric bacteria, where colEI replicon was not stable.
2. Site directed mutagenesis : Tn5 was used initially to cons
truct a physical map of the K. pneumoniae nif cluster (Riedel
et ~ 1979). Later Ruvkum and Ausubel (1981) developed a method
for site directed mutagenesis of Rhizobium genome, which is also
called marker exchange. In this technique cloned DNA fragment
in a plasmid was mutagenized with Tn5 and the mutated plasmid
was introduced into the wild type bacterium. Another plasmid
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which was incompatible with this plasmid was introduced and selec
tion was made for the incoming plasmid and Tn5. This resulted
in the transfer of Tn5 insertion from the plasmid to genome by
homologous recombination and simultaneous loss of the vector.
Position of Tn5 on chromosome is confirmed by hybridizing blots
of restriction enzyme digested genomic DNA with 32P-Iabelled
ins~. Using this technique, they mapped the symbiotic genes of
Rhizobium melioloti (Ruvkun et ~ 1982). Later, this method of
site directed mutagenesis was used extensively to map the nif
genes in many nitrogen fixing bacteria.
Aim of the present work is to subclone the DNA fragments
of cosmids isolated from A. vinelandii gene library and have se
quences homologous to K. pneumoniae nifHDKY DNA (Medhora
et ~ 1983) in mobilizable broad host range plasmids (Ditta et
~ 1980, 1985), to identify the nif genes present on these fragements
by complementation of various nif mutants of A. vinelandii and
to map the nif genes using transposon Tn5 mutagenesis (Ditta,
1985). The aim is also to find whether nif genes in A. vinelandii
are present in multiple copies, if so, whether they are functional.
These studies might provide some insight into the organization
and expression of nif genes in A. vinelandii.