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Copyright 0 1984 by the Genetics Society of America
ISOLATION AND GENETIC ANALYSIS O F CAULOBACTER MUTANTS DEFECTIVE
IN CELL SHAPE AND MEMBRANE
LIPID SYNTHESIS
DAVID A. HODGSON,* PENNY SHAWt AND LUCILLE SHAPIRO'
*Department of Molecular Biology, Division of Biological
Sciences, Albert Einstein College of Medicine; *Department of
Biochemistry, Stanford University Medical School, Stanford,
Calfornia 94305;
+Biogen, Geneva, Switzerland
Manuscript received April 2, 1984 Revised copy accepted August
17, 1984
ABSTRACT
In this paper we report the isolation, characterization and
genetic analysis of several C. crescentus mutants altered in
membrane lipid synthesis. One of these, a fatty acid bradytroph,
AE6002, was shown to be due to a mutation in thefatA gene. In
addition to the presence of the fatA506 mutation, this strain was
found to contain two other mutations, one of which caused the
production of a water-soluble brown-orange pigment (pigA) and
another which caused formation of helical cells (hclA). Egpression
of the latter two phenotypes re- quired complex media and both were
repressed by glucose. However, the lesions were mapped to loci that
are separated by a substantial distance. The hclA and the fatA
genes mapped close together, possibly implying that comu- tation
had occurred in AE6002. Data are presented that allow the unambigu-
ous identification of a second Fat gene (fatB) in C. crescentus.
The map position of another mutation in membrane lipid biogenesis,
the glycerol-3-P04 auxo- troph gpsA505, was also determined. During
this study theflaZ gene was fine- mapped and the positions of proC
and $changed from the previously reported location.
AULOBACTER crescentus has proven to be a useful organism to
study uni- C cellular differentiation (SHAPIRO, NISEN and ELY 198
1). During a discrete portion of its cell cycle this Gram-negative
bacterium synthesizes and assembles a flagellum and pili at one
pole of the cell. To study the control processes involved in this
cell surface differentiation, many mutants have been isolated and
characterized. BERT ELY and coworkers have identified greater than
30 linkage groups involved in flagellum synthesis (pa), motility
(mot) and che- motaxis (che) (JOHNSON and ELY 1979; JOHNSON, FERBER
and ELY 1983; SHAW et al. 1983). Studies involving some of these
mutants have demonstrated trans control o f p a and che genes as
part of a regulatory hierarchy (BRYAN et al. 1984; OHTA et al.
1984; R. CHAMPER and L. SHAPIRO, unpublished results). These
mutants permit access to the controls involved in the final
expression of a given differentiation event. Analysis of the
initial control events in the differentiation process requires the
isolation of conditional mutations that block the cell cycle at
either swarmer cell or stalk cell formation and are thus unable
Genetics 108: 809-826 December. 1984.
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810 D. A. HODGSON, P. SHAW AND L. SHAPIRO
to form a colony. This problem has been approached by isolating
mutants in known biosynthetic pathways and then determining their
effect on the expres- sion of the cell cycle. Two types of
conditional mutants that lead to a block in the cell cycle have
been isolated. These are temperature-sensitive cell cycle mutants
which are altered in DNA replication and cell division (OSLEY and
NEWTON 1977) and auxotrophic mutants which are altered in membrane
bio- synthesis (CONTRERAS et al. 1979, 1980; HODGSON et al.
1984a,b; LETTS et al. 1982; MANSOUR, HENRY and SHAPIRO 1980, 1981;
SHAPIRO et al. 1982).
In this paper we report the map positions of several genes that
are involved in lipid metabolism in C. crescentus. The gpsA gene,
required for glycerol-3- phosphate dehydrogenase activity
(CONTRERAS, SHAPIRO and HENRY 1978), was shown to reside near the
novr gene and the fatty acid auxotroph, AE6001 (fatB503), and was
mapped near t h e j a E F gene cluster. We also report the
isolation and characterization of a mutant of C. crescentus,
AE6002, which exhibits a partial dependence on fatty acids for
growth. This mutant had a complex phenotype which included the
secretion of a brown pigment, in ad- dition to the formation of
long helical cells when grown in rich media. The expression of both
pigment formation and helical cell formation was shown to be
repressed by glucose. Genetic dissection of AE6002 revealed the
presence of three independent mutations, all of which were
identified and mapped. The partial fatty acid dependence appears to
be due to an allele of thefatA gene. This gene was previously shown
to be involved in the regulation of fatty acid synthesis and
membrane protein synthesis (HODGSON et al. 198413). We show here
that thefatA alleles and another gene involved in the regulation of
fatty acid synthesis, fatB (HODGSON et al. 1984a), map in different
regions of the chromosome.
MATERIALS AND METHODS
Bacterial strains and growth conditions: C. crescentus CBI 5
(AE5000) and derivative strains (Table 1) were grown at 30" in
peptone yeast extract (PYE) broth (POINDEXTER 1964) or in BMG
buffered minimal medium with 0.5% glucose (Contreras, SHAPIRO and
HENRY 1978). Unsupple- mented media contained 1 % Tergitol NP-40.
Supplemented media contained either a mixture of 0.5% Tween 4 0 +
0.5% Tween 80 (TW) or 1 % Tergitol NP-40 plus 1 mM oleic acid (TO)
or other fatty acids, as indicated. PYE or BMG agar plates were
supplemented with either 0.4 mg/ml of Brij 58 (B) or with 0.4 mg/ml
of Brij 58 + 0.1 mg/ml of oleic acid (BO) or with Tween.
Cultures were shifted from one medium to another by collecting
cells in a Falcon sterile dis- posable filtration unit (0.45 pm)
and washing them with twice the volume plus Tergitol to remove
residual fatty acid. The filter was then transferred to a tube
containing a small volume of medium and gently vortexed, and the
washed cells were inoculated into the appropriate medium.
Isolation of mutants: AE6002 (pzgA50I hclA50l fatA506) was
obtained by N-methyl-"-nitro- nitrosoguanidine (NTG) mutagenesis of
AE5000. A midlog phase culture of AE5000 in PYE broth was added to
an equal volume of NTG in PYE (400 pg/ml) and shaken at 30" for 7 0
min. The cells were then collected by centrifugation and washed
with PYE and grown to saturation in PYE at 30" (CONTRERAS et al .
1979). The survivors were plated on BMG Tween plates, incubated at
30" and replica plated on BMG plates with and without Tween. A
colony that grew on BMG plus Tween, but not on BMG, was purified
and chosen for further study (AE6002). Another fatty acid-
requiring strain, AE6001 (fatB503), was obtained in a similar
manner, except that the mutagenesis and subsequent growth were
carried out in BMG plus Tergitol and oleic acid instead of PYE and
the mutagenesis was for 30 min instead of 70 min. AE6001, unlike
AE6000 (HODGSON et al .
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MEMBRANE MUTANTS IN CAULOBACTER 81 1
1984b) and AE6002, did not grow on PYE plates without oleic
acid. When plated for single colonies on PYE, AE6001 (fatB503)
formed microcolonies. Revertants of AE6001 occurred at a frequency
of and were clearly distinguishable as normal-sized colonies. The
isolation of AE5168 (gpsA505) has already been described (CONTRERAS
et al. 1979).
Materials: Oleic acid (95% pure) was obtained from Fisher; Brij
58 was obtained from Pierce; Tween 40, Tween 80, Tergitol NP-40 and
oleic acid (99% pure) were purchased from Sigma Chemical Company.
All other fatty acids (99% or greater purity) were obtained from
Supelco, Inc., Bellefonte, Pennsylvania.
Construction of donor strains by conjugation: Double conjugal
donor strains were needed to po- sition genes by three-factor
crosses. Some donor strains were constructed by bCr30-mediated
general transduction (ELY and JOHNSON 1977). However, in some cases
the genes of interest, pigA and pheA, gpsA and leuA, or gpsA and
metF were not contransducible. Therefore, RP4-mediated conjugation
(BARRETT et al. 1982a,b) had to be used (HOWSON et al. 1984b).
First the Tn5- marked gene (pheA, leuA or metF) was transduced into
the cell containing the marker of interest (pigA or gpsA). The
plasmid pVSl (a kanamycin-sensitive derivative of RP4) was then
conjugated from E. coli NC9412 into the double mutants. Two
counterselectable markers were needed to use these strains as
donors. Therefore, they were mated with a strain containing a drug
resistance marker (streptomycin for ts104 or rifampicin for ts140)
and a ts marker that was distal to the Tn5-marked gene (ts104 in
the case of pheA, and ts140 for leuA and me@). The drug resistance
and ts markers were chosen so that when the resultant donor strains
were selected against in further crosses there was no interference
in the recombination frequencies. The matings were carried out at
room temperature so as not to select against the
temperature-sensitive mutation. The recombinants were selected on
PYE plates with kanamycin plus streptomycin (PigA) or rif- ampicin
(me@ or leuA). The new donors were also tested for possession of
the Tn5-marked gene, pVS1, and pigA or gpsA. These strains were
then used as normal donors with the ts markers and either the
rifampicin sensitivity (PigA) or streptomycin sensitivity ( gpsA)
as the counterselective markers.
Scoring of mutant alleles: Possession of gpsA506, fatA506 or
fatB503 was scored as the inability of replicated patches of cells
to grow on BMG plates lacking 1 mM glycerol plus 1 mM glycerol
phosphate, 0.5% Tween 40 plus 0.5% Tween 80 or 0.4% Brij 58 plus
0.1% Fisher oleic acid, respectively. Possession of pigA501 was
scored by picking cells from a master plate onto PYE plates (20
clones/plate). After 2-3 days at 30" the production of pigment
could be tentatively scored. These plates were then replicated onto
additional PYE plates and pigment production by the small patch of
cells was again scored after 2 days. Any colonies that gave
conflicting results were repicked and replicated until scoring was
unambiguous. If colonies were too close together the diffusible
pigment was taken up by Pig- cells and false positives were
obtained. Helical cell formation (hclA501) was scored by direct
microscopic examination of cells picked from the master plate into
a drop of water on a microscope slide. Possession of hexB was
scored as the inability to utilize glucose. Instead, xylose (0.3%)
was used as a carbon source (B. ELY, personal communication). Other
mutant phenotypes were scored as detailed elsewhere (BARRET-T et
al. 1982b; CONTRERAS et al. 1980; HODCSON et al. 1984b).
Electron microscopy: Samples were prepared for electron
microscopy by placing a drop of cell suspension onto a
carbon-coated parlodion grid for 120 sec. The cells were stained
with 1% (w/ v) phosphotungstic acid for 1 sec and then visualized
in a JOEL JEMIOOS electron microscope at 80 kV.
RESULTS
Growth requirements of AE6002 and the derivative strain AE5568:
The mutant strain AE6002, isolated from cultures of AE5000 treated
with NTG, was iden- tified as a leaky fatty acid auxotroph, and
upon restreaking on PYE plates supplemented with Tween it was found
to excrete copious amounts of a brown- ish pink soluble pigment.
Pigment formation was observed only in rich PYE
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812 D. A. HODGSON, P. SHAW AND L. SHAPIRO
TABLE 1
List of strains
Strain Genotype
C. crescentus CB 15 AE5000 AE5 168 AE5438 AE5445 AE5448 AE5449
AE5458
AE5463
AE5467 AE5470 AE547 1
AE5479 AE5480 AE5487 AE5489
AE5492 AE5498 AE55 12 AE55 13 AE55 14 AE55 17 AE5522 AE5527
AE5531 AE5533 AE5536 AE5537 AE5541 AE5542 AE5552 AE5555
AE5556
AE5561 AE5563 AE5567 AE5568 AE5577 AE5660 AE5661 AE6000 AE600
1
Wild type gpsA505 (RP4 tet[Am]) gpsA505 r i f503 (RP4 t e t
[AmJ) gpsA505 $503 ' pheA108::Tn5 pig.4501 hclA5OI fatA506
serA104jlaZ102 nov-105 rij-506 pheAlO8 :Tn5 pigAS01 hclA501
fatA506
cysD137:Tn5 hclA501 pigAS01 fatA506 riy502
metB123:Tn5 gpsA505 ny503 ts104 (pVS1) cysEI40::TnS pigA501
hclA5OI fatA506
79-502 jZaE178::Tn5 ts-104 (pVS1) hexBII6 rijf-508 thrAlO
rij-509 pheAlO8::Tn5 pigA501 ts-104 str-153
j laEl78::Tn5 hexB116 rif508 cysD137::Tn5 hexBII6 rif508 gpsA505
cysD137::Tn5 cysD137::TnZ hclA5OI gpsA505 str-507 ts-140 rij-510
cysD137::Tn5 thrA 101 hclA5OI cysE14O::TnS hclA5OI ts-104 (pVS1)
cysD137::Tn5flaF132 ts-104 (pVS1) metF127::Tn5 gpsA505 leuA I3 I :
:Tn5 gpsA 505 metF127::Tn5 gpsA505 (pVS 1) leuA131::Tn5 gPsA505
(pVS1) cysD137::TnZ pigA501 fatA506 metFI27::Tn5 gpsA505 ts-140
riff-510
leuA131::TnS gpsA505 ts-140 rf-510
cysDZ37::Tn5 thrA101 ts-104 (pVS1) cysD137::Tn5 hclA5Ol ts-104
(pVS1) lacAIOl::Tn5 ts-104 (pVS1) cysD137::Tn5 fatA506 purAl01
str-509 aroBlO2 str-605 nov-501 metB123::Tn5 gpsA505 rij-503 ( p V
S I ) fatAS01 fatB503
(PVS 1 )
(PVS 1 )
(PVS 1 )
(PVS 1 )
Derivative or source (donor X recipient)
CONTRERAS et al. (1 979) AE5 168 spontaneous AE5438 spontaneous
SC1300 X AE6002 T " SC849 spontaneous NC9412 X AE5448 Cb
HODCSON et al. (1 984b)
SC1287 X AE5445 HODGSON et al. (1 984b) SC 149 1 X AE6008 C
HODGSON et al. (1 984b) SC 144 spontaneous SC 126 spontaneous
AE5458 X SC913 C
AE5679 X AE5680 C SC 1382 X AE5480 C AE5 168 spontaneous AE5463
X AE5000 T AE5463 X AE5000 T AE5512 spontaneous SC1609 spontaneous
AE5463 X SC126 T AE5471 X AE5470 T HODCSON et al. (1 98413) SC1321
X AE5512 T SC1490 X AE5512 T NC9412 X AE5536 C NC9412 X AE5537 C
AE5533 X AE6008 C AE5541 X AE5522 C
AE5542 X AE5522 C
AE5527 X AE5670 T HODGSON et al. (1 984b) HODGSON et al. (1
984b) AE5552 X AE5000 T SCI 19 spontaneous SC1756 spontaneous
NC9412 X AE5467 C HODGSON et al. (1 984b) AE5000 NTG mutagene-
sis
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MEMBRANE MUTANTS IN CAULOBACTER 813
TABLE 1 -Continued
Strain
AE6002
AE6005 AE6006 AE6007 AE6008 SC119 SC126 SC144 SC827 SC849 SC913
SCI 140 SC1287 SC 1288 SC 1293 SC 1294 SC 1300 SC1321 SC1382 s c 1
4 1 7 SC1424 SC 1490 SC1491 SC 1609 SC 1708 SC1756
~ ~ ~ ~~~
Derivative or source Genotype (donor X recipient)
pigA501 hclA5Ol fatA506
fatB503 str-501 fatB503 ry-501 pigAS01 hclA5Ol fatA506 str-502
pigA501 hclA5Ol fatA506 $502 purA 101 thrA 10 1 hexBll6 proClO6
str-141 serA 104 flaZ102 nov-105 ts-104 str-153 lacA 101 ::Tn5
str-152 metB123::Tn5 ts-104 str-153 (pVSI) hisB137:Tn5 ts-104
str-153 (pVSI) trpK107:Tnfi ts-104 str-153 (pVS1) cysB108::Tn5
ts-104 str-153 (pVSI) pheAlOB::Tn5 ts-104 str-153 (pVS1)
metF127::Tn5 ts-140 (pVS1) cysD137::Tn5 ts-104 str-153 (pVS1)
serAll3::TnZ ts-104 (pVS1) hisD1?6::Tn5 ts-104 (pVS3) leuA131 ::Tn5
ts-140 (pVS 1) cysEl4O::Tn5 ts-140 str-153 (pVS1) ts-140 cysD
137::Tn5flaF132 ry- 181 aroBlO2 str-605
AE5000 NTG mutagene-
AE6001 spontaneous AE6001 spontaneous AE6002 spontaneous AE6002
spontaneous BARRETT et al. (1 982b) JOHNSON and ELY (1 977) JOHNSON
and ELY (1977) BARRETT et al. (1982b) BARRETT et al. (1982b)
BARRETT et al. (198213) BARRETT et al. (1982b) BARRETT et al.
(1982b) BARRETT et al. (1982b) BARRETT et al. (1 982b) BARRETT et
al. (1 982b) BARRETT et al. (1982b) BARRETT et al. (1 982b) BARRETT
et al. (198213) BARRETT et al. (1982b) BARRETT et al. (1982b)
BARRETT et al. (1982b) BARRETT et al. (1982b) BARRETT et al.
(1982b) BARRETT et al. (1982b) BARRETT et al. (1982b)
sis
E. coli NC94 12 hsdR hsdM supE44 thr- leuB6 lacy BARRETT et al.
(1982b)
tonA2l thi-1 (pVS1)
a T = transduction. See MATERIALS AND METHODS. * C =
conjugation.
medium and not in minimal medium. The growth requirements of
AE6002 were similar to a fatty acid auxotroph, AE6000 (futASOl),
previously described (HODGSON et al. 1984b; LETTS et al. 1982).
Both AE6002 and AE6000 grew well on PYE without additional fatty
acids and growth on minimal medium occurred in the presence of
either fatty acids or biotin. The partial dependence of AE6002 on
exogenous fatty acid was exhibited by slow growth on minimal medium
in the absence of supplement. AE6002 grew with a doubling time of
2.5 hr in the presence and 4 hr in the absence of oleic acid. This
compares with the doubling time of 3 hr, independent of the
presence of oleic acid, of the parental strain AE5000.
A derivative of AE6002 was constructed (AE5568) which separated
the Fat- phenotype from the pigment and helical cell phenotype.
Various compounds were tested as supplements of AE5568 growing on
BMG solid medium (Table 2). The growth requirements of AE5568 were
compared to those exhibited
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814 D. A. HODGSON, P. SHAW AND L. SHAPIRO
TABLE 2
Supplementation ofAE6001 (fatB503), AE6000 (fatA50 1) and AE5568
(fat-506) ~ ~~
AE6001" AE6000 AE5568b Additions (fafB503) (fafA501)
(faf-506)
- - - None - N T
NT + +
Glycerol (1 mM) - Acetate (0.4%) - - Biotin (2 ng/ml) -
Unsaturated fatty acids'
Linolenic acid (1 8:3 A', A'*, AI5) + + NT - - Linoleic acid (1
8:2, A', A'')
Vaccinic acid (1 8: 1, A") + + + Oleic acid (1 8: 1, A') + + +
Palmitoleic acid (1 6: 1, A') + + +
+
Elaidic acid (1 8: 1, A') + + NT Saturated fatty acidsd
Stearic acid (1 8:O) - NT NT Palmitic acid (1 6:O) - NT N T
Myristic acid (14:O) - NT NT
Scored as to the ability of cells to grow in liquid BMGTO, then
washed with BMGT to grow (+) or not (-) in liquid BMGT + supplement
or, in the case of saturated fatty acids, lipid PYE + Tergitol (1
%).
Scored as the ability of cells to grow more rigorously (+) on
solid BMGB plates plus supplement after replication from patches
grown on a PYE plate. (-) indicates lack of stimulation.
All fatty acids were added at 1 mM. Saturated fatty acids would
not dissolve in BMGT but would in PYE + Tergitol
(1 %), hence AE6000 and AE5568 could not be tested, N T = not
tested.
by two other fatty acid auxotrophs, AE6000 ( fa tA) and AE6001 (
fa tB) (HODG- SON et al . 1984a,b). The growth requirements of
AE5568 were the same as for AE6000 ( fa tA) . All but one of the
unsaturated fatty acids (linoleic acid) could stimulate growth.
Furthermore, biotin, a vitamin previously shown to
supplementfatA5OI-containing strains (HODGSON et a l . 1984b), was
an effective supplement for AE5568. AE6001 ( fa tB) differed from
both AE6000 (&A) and AE5568 in that biotin had no stimulatory
effect.
Pigment and helical cell formation: Examination of AE6002 in the
light mi- croscope showed that it formed helical cells in complex
media (PYE) but not minimal media (BMG). Cell morphology was then
analyzed by electron mi- croscopy, and it was found that cells
grown in PYE formed helices which were a dozen or more cell lengths
long (Figure 1). Even single- and double-length cells showed a
distinct helical twist (Figure la). The mutant cells all appeared
to be thinner than wild-type cells grown in PYE. When AE6002 was
grown in BMG (Figure Ib) they were indistinguishable from wild-type
cells grown in minimal medium. A similar media dependence was noted
concerning the pro- duction of a water-soluble, rapidly diffusable
brownish orange pigment. The pigment was produced in copious
amounts on PYE but not at all on BMG. Because minimal media used
glucose as the sole carbon source, the possibility arises that both
of these phenomena are glucose repressible.
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MEMBRANE MUTANTS IN CAULORACTER 815
n
FIGURE 1.-Electron micrographs of C. cresccntw AE6002 grown in
(a) or (c) rich PYE media, (b) minimal BMG media and (d) PYE media
plus 0.5% glucose. Bar = 5 pm. X 3600.
T o test this possibility AE6002 cells were grown in PYE in the
presence and absence of glucose (Figure I C and d, respectively).
Helical cell formation was repressed in the presence of glucose.
Other potential carbon sources were tested for their ability to
repress helical cell formation and pigment production. These
results are summarized in Table 3. Only glucose and sucrose
repressed helical cell formation, and these two, in addition to
maltose, mannose and
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816 D. A. HODCSON, P. SHAW AND L. SHAPIRO
TABLE 3
Effect of carbon source on pigment and helical cell
formation
Pigment Helical cell Additions to PYE production” formationb
None + + 0.5% Glucose - - 30 mM Sucrose 0.5% Acetate + + 0.5%
Arabinose + + 0.5% Fructose + +
+ 0.5% Maltose - + 0.5% Mannose - +
PYE + BM salts‘ + +
- -
0.5% Glycerol + +
0.5% Xylose -
(+) = production of pigment. * (+) = formation of helical cells
as determined under the electron
microscope. ‘ The salt and buffer components of buffered minimal
media
(CONTRERAS, SHAPIRO and HENRY 1978) were added to PYE in the
absence of glucose or other potential carbon sources.
xylose, repressed pigment formation. However, when nonrepressing
sugars, e.g., arabinose, were used as carbon source in minimal
media neither helical cells nor pigment were formed, indicating
that glucose was not the only cause of the suppression of these
phenotypes in B M G . The addition of the compo- nents of BMG
(other than glucose) to PYE media did not lead to repression of the
phenotypes (Table 3); hence, expression of both the helical cell
shape and pigment formation required some component(s) in PYE broth
that was not present in BM salt media. T o determine the genetic
lesion(s) responsible for the glucose-repressed helical cell and
pigment formation and the partial fatty acid dependence in AE6002,
the phenotypes were scored independently in the same crosses.
Mapping of mutant alleles in AE6002: a rapid method of
preliminary mapping genes in C. crescentus CB15 has recently been
devised (BARRETT et al. 198213). This method involves the
RP4-mediated conjugation of a series of strains that contain T n 5
inserted into genes of known chromosomal location and the strain
containing an allele of the gene to be mapped (Table 4). The
Tn5-marked gene was selected by virtue of its kanamycin resistance,
and its frequency of cotransfer with the wild-type allele of the
gene being mapped indicates the relative closeness of the two
genes. When crosses were performed with drug- resistant derivatives
of AE6002, the initial striking result was that the helical cell
formation (Hcl) and fatty acid bradytrophy (Fat) phenotypes
segregated from the pigment formation (Pig) phenotype (Table
4).
It appeared from the preliminary mapping with Tn5-derived
auxotrophs that hcl-501 and fat-506 cosegregated, but later crosses
showed they were close but separable; hence, only those crosses
involving genes close to cysD were per- formed when mapping fat-506
and hcl-501 precisely. It was known that thrA,
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MEMBRANE MUTANTS IN CAULOBACTER 817
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818 D. A. HODGSON, P. SHAW AND L. SHAPIRO
theflaE,F,G cluster (JOHNSON and ELY 1979; PURUCKER et al. 1982)
and hexB mapped close to cysD and the latter two were on opposite
sides (B. ELY, personal communication). Therefore, these genes had
to be accurately mapped to allow the constrtxtion of a fine
structure map of hcl-501 andfat-506. T o do this we performed
three-factor crosses (Table 5). If the genes were close enough we
used 4Cr3O-mediated transduction; if not we used RP4-mediated
conjugation. In cross I (Table 5) a cysD flaF strain was crossed
with a hexB strain by transduction. The relative cotransfer
frequencies show that cysD is closer to hexB than JaF. Therefore,
hexB could be ordered as indicated in column A or column B. It
would appear from examining the genotype distri- bution that hexB
segregates independently of j laF because the frequency of flaF
toflaF+ is the same in both hexB and hexB+ recombinants. This
implies that hexB is in the position indicated in column A. The
position indicated by the broken arrow would necessitate a
quadruple crossover event to generate hexBflaF recombinants, an
event known to be rare in C. crescentus (BARRETT et al. 198213).
ELY and coworkers had previously shown that thrA was to the left of
cysD as drawn (BARRETT et al. 1982a). Therefore, cross I1 of Table
5 was performed. The cotransfer frequencies indicate that cysD is
closer to flaF than thrA, hence, the two possible positions of thrA
shown in Table 5. Exam- ination of the genotype distribution
indicates dependent segregation; namely, there is a severe shortage
of theflaF+ thrA+ recombinant class. This is precisely the class
that could only be generated by a quadruple crossover event if thrA
is in the position indicated in column A. When the results from
these two crosses are combined, the gene order is thrA,flaF, cysD,
hexB (Figure 2) .
We were able to use this gene order to determine the position of
hclA. We used the same reasoning outlined for cross I, and cross
I11 (Table 5) shows that hclA is to the right of cysD as indicated
in the upper line of column A. Cross IV shows clear dependent
segregation (like cross 11) of hclA and hexB. Therefore, hclA is
also to the right of hexB (column A). It had previously been
demonstrated that cysE is to the right of cysD (B. ELY, personal
communica- tion). Therefore, cross V was performed (Table 5). The
lack of the hclA+ hexB+ class of recombinants allows us to conclude
that hclA lies between hexB and cysE (Table 5 , column A).
We next determined the map position offat-506. Cross I11 (lower
line) shows that fat-506 maps to the right of hclA (there is
dependent segregation of fat and hclA and independent segregation
offlaF and fat). Unfortunately, we could not use cysE to further
map fat-506, because this Tn5-marked allele obscures the Fat
phenotype. This may occur because strains containing this allele
grow very poorly on BMG media even if supplemented with cysteine or
thiosulfate. Therefore, lacA was used to position fat-506. Crosses
by ELY and coworkers (personal communication) indicate that lacA
maps between hclA and cysE. Therefore, the gene was used for
further mapping. Unfortunately, fa tA was so close to hclA that it
was difficult to separate the two by conjugation (data not shown)
and the lacA to hclA transduction frequency was so low as to make
analysis difficult (cross VI, Table 5). However, the absence of
thefat-506 hclA+ recombinant class is indicative of the order shown
in column A which, taken
-
MEMBRANE MUTANTS IN CAULOBACTER 819
together with cross 111, indicates thatfat-506 maps within the
lacA to hclA gap. This is the map position previously determined
for thefatA gene (HODGSON et al. 1982b). The map position offut-506
in addition to its fatty acid and biotin requirements suggests that
it is an allele offutA.
The data presented in Table 4 indicate that pheA is the closest
Tn5-marked gene to pigA, the gene responsible for pigment
formation. It had been shown previously (B. ELY, personal
communication) that serA maps to the right of pheA and that JaZ
maps close to PheA, but the position was uncertain. There- fore,
cross VI1 was performed to map both pigA and JuZ (Table 5). From
analysis of the cotransfer frequencies, JaZ appears closest to
pheA, followed by PigA and then serA. Inspection of the pigA/serA
segregation data indicates that PigA is to the left of pheA,
because segregation was independent (column A, upper line).
Segregation of serA andJaZ was dependent and could be ex- plained
ifJaZ is between serA and pheA (column A, lower line). Examination
of the pigA/JaZ segregation data confirmed this position.
Therefore, the final order is pigA pheA J u Z serA (Figure 2).
Further crosses (data not shown) in- volving uroC, which has been
mapped between serA andflu2 (BARRETT et al. 1982b), confirmed that
pigA is to the left to pheA as drawn.
Mapping of fatB: The mutant strain AE6000 is a fatty acid
auxotroph whose supplemented requirements differ from AE6000
(fatA50l) and AE6002 (fatA506) (Table 2; HODGSON et al. 1984a). The
data presented in Table 4 indicate that thefatB gene, mutated in
AE6001 to give the Fat- phenotype, is close toJaF. It should be
noted that these cotransfer frequencies are probably overestimates
because the futB503 allele was so detrimental to cell growth, even
in the presence of supplement, that during the overnight incubation
of the cross mixture on PYE plus Brij and oleic acid thefutB+ cells
overgrew the fatB cells. This was only a problem when conjugation
was used.
T o order the fatB gene in the vicinity ofJaE, i.e., hexB, thrA
and cysD, three: factor crosses were performed using these markers
(Table 5). Transduction cross VI11 (Table 5) demonstrates that fatB
is to the left of JaE (column A), because independent segregation
had occurred. Furthermore, cross IX indi- cates that fate is
between cysD and thrA (column A) because dependent seg- regation
had occurred and the predicted recombinant class (fatB hexB) was
reduced. From cross VI11 we can infer that futB is positioned
between thrA and J a F , and a transduction between AE5525
(JuE::Tn5, thrA) and AE6001 (futB) confirmed this prediction (data
not shown). Therefore, the two fatty acid auxotrophs have been
shown to map to different positions on the genome.
Mapping of gpsA: Lesions in the gpsA gene result in
glycerol-3-phosphate auxotrophy and the inhibition of phospholipid
synthesis when growth is at- tempted in the absence of supplement
(CONTRERAS et al. 1979). It was dem- onstrated in vitro that
mutations in gpsA resulted in a loss of sn-glycerol-3- phosphate
dehygrogenase activity. The map position of this gene involved in
membrane lipid synthesis was determined.
The initial mapping of gpsA505 with Tn5-derived auxotrophs
indicated that gpsA was closest to leuA and metF (Table 4).
However, when cotransduction was attempted no linkage of gpsA with
either marker could be detected. ELY
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D. A. HODGSON, P. SHAW AND L. SHAPIRO
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822 D. A. HODGSON, P. SHAW AND L. SHAPIRO
, f/u F
FIGURE 2.-A portion of the C. crescentus CBI5 genome showing the
relative map positions of marker genes on the right (BARRETT et al.
1982a,b) and genes mapped in this and a previous paper (HODGSON et
al. 1984b) on the left. 4, Genes with Tn5 insertions; and
parentheses, ambi- guity in gene order. The map distances are not
necessarily drawn to scale.
and coworkers (personal communication) had also found an
abnormally low ratio of frequency of cotransduction to frequency of
coconjugation in this same area of the map. Therefore, we had to
construct the donor strains AE5555 (me@ gpsA) and AE5556 (ZeuA
gpsA) by conjugation (see MATERIALS AND METH- ODS). Some of the
crosses performed with these strains are illustrated in Table 5.
When AE5555 (metF gpsA) was crossed with recipient strains
containing proA, argC, m u R (data not shown) or purA (cross X) the
same result was obtained: dependent segregation with the gpsA purA
(or equivalent) recombi- nant class was underrepresented. This
indicated that gpsA was to the left (as shown in column A) of all
of these genes, the order of which (see Figure 2) had been
previously determined (B. ELY, personal communication; BARRETT
-
MEMBRANE MUTANTS IN CAULOBACTER a23
et al. 1982a,b). However, when a proC recipient was used (Cross
XI) it ap- peared that proC and gpsA segregated independently,
suggesting that they were on opposite sides of metF (column A).
This was in contradiction of the pub- lished position of proC
(BARRETT et al. 1982a,b). The method used to construct the double
donor AE5555 (see MATERIALS AND METHODS) indicated that rzf" was
also on the opposite side of metF in relation to gpsA. If rzf" was
on the same side as gpsA, AE5555 could only have arisen by a
quadruple crossover event. However, the frequency of gpsA+ rif" to
gpsA rzf" was 1 : 1. Crosses with AE5556 (lacA gpsA) confirmed
these results; namely, gpsA was to the left of nov", nov" aroG and
purA were to the left of leuA and ProA and proC were to its right
(Figure 2).
Crosses between either AE5555 or AE5556 and AE5660 (aroB nov")
gave conflicting results. The probable reason for this ambiguity is
that the linkage frequencies between aroB or gpsA and each of the
Tn5-marked genes were very nearly the same. Therefore, as no other
Tn5-marked genes had been isolated that were closer to aroB, an
alternative cross was performed (Table 5; cross XII). The cross was
plated out onto BMG plus glycerol and glycerol-3- phosphate.
Therefore, aroB was selected against. It should be noted that pre-
vious studies had shown that the efficiency of plating of gpsA
strains was the same as gpsA+ strains on BMG plus supplement. Cross
XI1 indicated that gpsA mapped between aroB and novR because
segregation of gpsA was not inde- pendent of that of nov" and the
expected rare recombinant class, gpsA+ novS, occurred at a very low
frequency.
A summary of the map positions determined in this section is
presented in Figure 2.
DISCUSSION
In this paper we have presented evidence that the mutation
resulting in fatty acid bradytrophy of AE6002 (fat-506) is an
allele offatA. It maps to the same region, i.e., between hclA and
lacA, and can be supplemented by the same fatty acids and biotin.
There was one difference, however. fatA506 does not cause cell
death in the absence of supplement. This might be explained
iffatA506 caused only partial inactivation of the futA gene. We
have also identified a second Fat gene (futB) in C. crescentus.
Although fatB mapped close to fatA (HODGSON et al. 1982b) it was
separated by several unrelated genes (Figure 2).
The AE6002 strain was shown to carry a mutation that maps to a
single locus and affects cell shape. The biochemical basis of the
Hcl phenotype is unknown. However, the mutant phenotype was shown
to require some com- ponent(s) of PYE broth for its expression.
Helical cell mutations have been identified by TILBY (1981) in
Bacillus subtilis and by KURIKI (1981) in E. coli. In the former
case it was postulated that misalignment of cell wall filaments
composed of techoic acid and peptidoglycan were responsible for the
pheno- type (MENDELSON 1982; MENDELSON and KEENER 1982; TILBY
1981). In the latter case, Kuriki reported an unsaturated fatty
acid auxotroph that formed corkscrew filaments in the presence of
linoleate or linolenate at stationary phase and that had changes in
its membrane protein composition. No differ-
-
824 D. A. HODGSON, P. SHAW AND L. SHAPIRO
ence in membrane protein composition was observed in AE55 14
(cysD: :Tn5 hclA5Ol fa tA+ pigA+) when compared with AE55 13, an
isogenic hclAi strain (data not presented). Although hclA strains
formed long filaments in stationary phase, numerous individual
swarmers were still seen, indicating that cell divi- sion occurred
in some instances. Giemsa staining of the filaments indicated
uniform distribution of stained areas along the length of the
filament, implying that the cells were capable of normal nuclear
division.
The glucose and sucrose repression of the Hcl phenotype and the
repression of pigment formation by glucose in addition to several
other carbon sources was of interest. It was previously shown that
lactose utilization is repressed by glucose in C. crescentus
(SHAPIRO et al. 1972). However, it is not known whether the same
mechanism of repression affects all three systems. One method of
investigation would be the isolation of pigmented pigA cells on PYE
plus glucose. These mutants could then be tested for any
pleiotropic effect on the other two glucose repression phenomena.
Glucose-derepressed mutants have been isolated by equivalent
methods in another bacterium (HODGSON 1982). The pigment formed by
AE6002 has not been physically characterized. However, the absolute
requirement for some component(s) of PYE suggests that it may be
the product of a blocked metabolic pathway(s).
The isolation of three independent mutations in AE6002 during
NTG mu- tagenesis reinforces the warning of JOHNSON and ELY (1977)
that chemical mutagenesis could lead to multiple mutations in C.
crescentus. The presence of two closely linked mutations, fatA506
and hclA501, could be an indication of the process of comutation, a
phenomenon previously observed in E. coli (GUER- OLA, INGRAHAM and
CERDA-OLMEDO 197 1) and Streptomyces coelicolor A3(2) (RANDAZZO et
al. 1973), wherein the DNA replication forks have been identi- fied
as hot spots for mutations. The genetic studies of AE6002 have
demon- strated that, if NTG mutagenesis was used and multiple
mutations generated, the present genetic techniques available for
C. crescentus CBI 5 allow separation of these mutations. The gene
pigA has helped bridge a gap in the conjugation map and has been
useful in the mapping offlaZ, and hclA, fa tA and fatB have filled
in the thrA to cysE region of the map, a region of particular
interest as many genes involved in motility @le, che, mot andfla),
a cell cycle-dependent phenomenon in Caulobacter, have been mapped
on this area (BRYAN et al. 1984; JOHNSON and ELY 1979; PURUCKER et
al. 1982; B. ELY, personal com- munication). The mapping of gpsA
led to the discovery that proC and rifare below metF (as drawn in
Figure 2) and not above, as previously published. Finally, the
mapping of fa tA, fa tB and gpsA has allowed us to construct double
mutants in which studies of the epistatic effects of the genes have
been possible (HODGSON et al. 1984a).
This investigation was supported by Public Health Service grants
GM 11301 and GM 32506- 0 2 from the National Institutes of Health
and the Core Cancer Center grant NIH/NCI P30 CA13330.
LITERATURE CITED
BARRETT, J. T., R. H. C R O ~ ; D. M. FERBER, C. J. GERARDOT, P.
V. SCHOENLEIN and B. ELY, 1982a Genetic mapping with TnSderived
auxotrophs of Caulobacter crescentus. J. Bacteriol. 151:
889-898.
-
MEMBRANE MUTANTS IN CAULOBACTER 825
BARRETT, J. T., C. S. RHODES, D. M. FERBER, B. JENKINS, S. A.
KUHL and B. ELY, 1982b Construction of a genetic map for
Caulobacter crescentus. J. Bacteriol. 149 889-896.
BRYAN, R., M. PURUCKER, S. LOPES-COMES, W. ALEXANDER and L.
SHAPIRO, 1984 Analysis of the pleiotropic regulation of flagella
and chemotaxis gene expression in Caulobacter crescentus using
plasmid complementation. Proc. Natl. Acad. Sci. USA 81:
1341-1345.
CONTRERAS, I., R. BENDER, J. MANSOUR, S. HENRY and L. SHAPIRO,
1979 Caulobacter crescentus mutant defective in membrane
phospholipid synthesis. J. Bacteriol. 140 612-619.
CONTRERAS, I., L. SHAPIRO and S. HENRY, 1978 Membrane
phospholipid composition of Caulo- bacter crescentus. J. Bacteriol.
135 1130-1 136.
CONTRERAS, I., A. WEISSBORN, K. AMEMIYA, J. MANSOWR, S. HENRY,
R. BENDER and L. SHAPIRO, 1980 The effect of termination of
membrane phospholipid synthesis on cell cycledependent events in
Caulobacter. J. Mol. Biol. 138 401-409.
Generalized transduction in Caulobacter crescentus. Genetics
ELY, B. and R. C. JOHNSON, 1977
GUEROLA, N., J. L. INGRAHAM and E. CERDA-OLMEDO, 1971 Induction
of closely-linked multiple mutations by nitrosoguanidine. Nature
(New Biol.) 230 122-125.
HODGSON, D., P. SHAW, V. LETIS, S. HENRY and L. SHAPIRO, 1984a
Genetic analysis and char- acterization of a mutant of Caulobacter
crescentus defective in membrane biogenesis. J. Bacte- rial. 158
430-440.
HODCSON, D., P. SHAW, M. O’CONNELL, S. HENRY and L. SHAPIRO,
1984b A fatty aciddepend- ent cell cycle mutant of Caulobacter
crescentus. J. Bacteriol. 158 156-162.
HODGSON, D. A., 1982 Glucose repression of carbon source uptake
and metabolism in Streptomyces coelicolor A3(2) and its
perturbation in mutants resistant to 2deoxyglucose. J. Gen.
Microbiol.
JOHNSON, R. C. and B. ELY, 1977 Isolation of
spontaneouslyderived mutants of Caulobacter
JOHNSON, R. C., D. M. FERBER and B. ELY, 1983 Synthesis and
assembly of flagellar components by Caulobacter motility mutants.
J. Bacteriol. 154: 1137-1 144.
JOHNSON, R. C., M. P. WALSH, E. ELY and L. SHAPIRO, 1979
Flagellar hook and basal body complex of Caulobacter crescentus. J.
Bacteriol. 138 984-989.
KURIKI, Y., 1981 Effects of unsaturated fatty acids on the
morphogenesis of an unsaturated fatty acid auxotroph of Escherichia
coli. J. Bacteriol. 147: 1 121-1 124.
KURN, N., L. SHAPIRO and N. AGABIAN, 1977 Effect of carbon
source and the role of cyclic adenosine 3’,5’-monophosphate on the
Caulobacter cell cycle. J. Bacteriol. 131: 951-959.
LE-, V., P. SHAW, L. SHAPIRO and S. HENRY, 1982 Synthesis and
utilization of fatty acids by wild-type and fatty acid auxotrophs
of Caulobacter crescentus. J. Bacteriol. 151: 1269-1278.
MANSOUR, J., S. HENRY and L. SHAPIRO, 1980 Differential membrane
synthesis during the cell cycle of Caulobacter crescentus. J.
Bacteriol. 144: 262-269.
MANSOUR, J., S. HENRY and L. SHAPIRO, 1981 Phospholipid
biosynthesis is required for stalk elongation in Caulobacter
crescentus. J. Bacteriol. 145 1404-1 409.
MENDELSON, H. H., 1982 Dynamics of Bacillus subtilis helical
macrofiber morphogenesis: wri- thing, folding, close packing, and
contraction. J. Bacteriol. 151: 438-449.
Clockwise and counter-clockwise spinwheel colony morphologies of
B. subtilis are correlated with the helix hand of the strain. J.
Bacteriol. 151: 455-457.
Physical mapping and complementation analysis of Tn5 mutations
in Caulobacter crescentus: organization of transcriptional units in
the hook cluster. J. Bacteriol. 158 897-904.
81: 391-399.
128 2417-2430.
crescentus. Genetics 86: 25-32.
MENDELSON, N. H. and S. L. KEENER, 1982
O I ~ T A , N., E. SWANSON, B. ELY and A. NEWTON, 1984
-
826
OSLEY, M. A. and A. NEWTON, 1977
D. A. HODGSON, P. SHAW AND L. SHAPIRO
Mutational analysis of developmental control of Caulobacter
Biological properties and classification of the Caulobacter
group. Bacte-
Isolation of a Caulobacter gene cluster specifying flagellum
production by using non-motile Tn5 insertion mutants. Proc. Natl.
Acad. Sci. USA 79: 6797-6801.
Comutation in Streptomyces. J.
Effect of dibutyryladen- osine 3’,5’-cyclic monophosphate on
growth and differentiation in Caulobacter crescentus. Proc. Natl.
Acad. Sci. USA 69: 1225-1229.
The synthesis of specific membrane proteins is a function of DNA
replication and phospholid synthesis in Caulobacter. J. Mol.
Biol.
Genetic analysis of the differentiating bacterium: Cau- lobacter
crescentus. pp. 317-339. In: Genetics as a Tool in Microbiology,
Edited by S. W. GLOVER and D. A. HOPWOOD. Cambridge University
Press, Cambridge.
Methylation involved in chemotaxis is regulated during
Caulobacter differentiation. Proc. Natl. Acad. Sci. USA 80:
crescentus. Proc. Natl. Acad. Sci. USA 74: 124-128.
POINDEXTER, J. S., I964
PURUCKER, M., R. BRYAN, K. AMEMIYA, B. ELY and L. SHAPIRO,
1982
rial. Rev 28: 231-295.
RANDAZZO, R., G. SERMONTI, A. CARERE and M. BIGNAMI, 1973
SHAPIRO, L., N. AGABIAN-KESHISHIAN, A. HIRSCH and 0. ROSEN,
1972
Bacteriol. 113: 500-501.
SHAPIRO, L., J. MANSOUR, P. SHAW and S. HENRY, 1982
159: 303-322.
SHAPIRO, L., P. NISEN and B. ELY, 1981
SHAW, P., S. L. COMES, K. SWEENEY, B. ELY and L. SHAPIRO,
1983
5261-5265.
TILBY, M. J., 1981 Helical shape and wall synthesis in a
bacterium. Nature 2 6 6 450-452.
Corresponding editor: G. MOSIG
