JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 627-634 0021-9193/79/01-0627/08$02.00/0
Vol. 137, No. 1
Analysis of Nonmotile Mutants of the Dimorphic Bacterium Caulobacter crescentus REID C. JOHNSON' AND BERT ELY".2* Department of Biology' and Department of Microbiology and Immunology,2 University of South Carolina, Columbia, South Carolina 29208
Received for publication 21 September 1978
A total of 69 spontaneous nonmotile mutants were isolated from the dimorphic bacterium Caulobacter crescentus. The majority of the mutants were unable to assemble a flagellar filament (Fla-), although eight were able to synthesize a short stub of a flagellum. A third mutant class assembled flagella of normal morphology but were nonmotile (Mot-). Genetic analysis by oCr30-mediated transduction revealed 27 linkage groups for the fla and stub-forming mutations, and three linkage groups for the mot mutations. Intracellular flagellin detected by immunodiffusion was at the limit of detectability in most of the Fla- and stub-forming mutants but normal in the Mot- mutants. The Fla- and stub-forming mutants also showed decreased sensitivity to the swarmer-specific phages oCbK and oCb5 while maintaining complete sensitivity to the randomly adsorbing phages oCr30 and 4Cr40. One additional strain was totally resistant to oCbK, and the mutation in this strain has been designated pleA. Each of the mutants containing mot mutations showed wild-type sensitivity to all of the phages tested.
Flagellum biogenesis in Caulobacter represents an attractive system for the study of organelle differentiation in procaryotes. A single flagellum is present on swarmer cells along with pili and phage receptors at one pole of the cell (20). As this swarmer cell differentiates into a stalk cell, the entire flagellar structure, including the filament, hook, and rod, is released into the medium (18, 21; R. C. Johnson, J. P. Walsh, B. Ely, and L. Shapiro, J. Bacteriol., submitted), and pili and phage receptors are lost (20). The stalk cell undergoes DNA replication and then elongates into a predivisional cell with a new flagellum at the opposite pole from the stalk (for a review, see reference 20). This cell subsequently divides to produce a new swarmer cell and a stalk cell which can immediately undergo DNA replication, whereas the swarmer cell must lose its flagellum and form a stalk before it can replicate its DNA. Not only is the formation of the flagellum structure, and thus cell motility, temporally regulated in the Caulobacter cell cycle, but flagellin, the primary component of flagella, is synthesized coincidentally with the appearance of the flagellum on the cell surface (21). Osley et al. (17) have recently shown by immunoprecipitation studies with antiflagellin antibody and synchronized C. crescentus cell populations that two species of flagellin (see below) are synthesized immediately after, and are dependent upon, the completion of DNA replication.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella resolves two protein components of flagella: a minor 27,500dalton protein and the major 25,000-dalton protein (13, 16, 17, 22). Lagenaur and Agabian (13) have shown that these two proteins are distinct with regard to their amino acid composition, but the proteins seem to be related because antibody made against the major 25,000-dalton flagellin does cross-react with both purified flagellins in immunodiffusion assays (13). To study the regulation of flagellum biogenesis in Caulobacter, we isolated a large number of mutants with altered motility. We present here a characterization of these mutants, particularly with regard to genetic analysis. MATERIALS AND METHODS
Bacteria and bacteriophage. C. crescentus wildtype strain CB15 was used as a parent for all strains described below. Swarmer-specific bacteriophage 0CbK, which adsorbs to the polar end of the cell near the base of the flagellum (1), and 4Cb5, which adsorbs to pili also found at the cell pole (19), were gifts of L. Shapiro and N. Agabian, respectively. The flagellotropic phage 0Cp34 was obtained from A. Fukuda (7). 4)Cr30 and OCr40, which adsorb randomly to all cell types, have been described previously (11). Growth conditions. Complete (PYE) and minimal (M2) liquid and solid media for growing Caulobacter and Caulobacter phage have been reported previously (10). Rich swarming medium was prepared by adding 0.25% agar (Difco Laboratories) to half-strength PYE. 627
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JOHNSON AND ELY
Minimal swarming medium was made by the addition of the appropriate amount of carbon source to M2 plus 0.25% agar (Difco). Isolation of motility mutants. Spontaneously occurring Caulobacter motility mutants were isolated by an enrichment procedure based on that used by Armstrong et al. for the isolation of Escherichia coli chemotaxis mutants (4). Colonies of wild-type CB15 were stabbed into minimal swarming medium plus 0.05% glucose or xylose and incubated at 33°C. The bacteria were allowed to swarm for 4 to 5 days, and then the center of the swarm was restabbed into the same medium and incubated again. After four to six serial enrichments, the center was removed and suspended in PYE, and 0.1 ml of the appropriate dilution was mixed with 20 ml of rich swarming medium and poured onto a sterile petri dish. After 2 days of incubation at 33°C, the nonswarming colonies were rediluted and plated on rich swarming medium as before to get wellseparated colonies. Examples of the colony morphologies of wild-type cells and three nonmotile mutants on minimal swarming medium are shown in Fig. 1. The purified colonies were streaked onto PYE plates for further study. Only one isolate was retained from each selection to insure independent clones. Electron microscopy. A 10-ml amount of a logphase culture grown in PYE broth with aeration was pelleted in a Sorvall table top centrifuge and gently resuspended in 0.5 ml of fresh PYE. Equal volumes of the cell suspension and 1 or 2% phosphotungstic acid (pH 7.0) were mixed and placed on a carbon-stabilized nitrocellulose-coated 400-mesh grid. After 20 s, the grid was blotted, air dried, and observed under a Seimans Elmiskop model IA or a Japan Electronic Optics Laboratory Ltd. model 100B electron microscope at 80 kV. Transductions. Transductions were performed with the generalized transducing phage 4Cr30 (6). Equal volumes of a stationary phase culture grown in PYE broth and a 10-2 or 10-3 dilution of a UV-irradiated phage lysate (6) were combined, and 0.025 ml of the mixture was streaked across a minimal swarming medium plate containing 0.1% glucose with a 0.2-ml pipette. After 5 days of incubation at 330C, recombinant flares emanating from the streak were counted. An example of a transduction plate is shown in Fig. 2. No evidence of abortive transductants or trails was ever observed. Assay for intracellular flagellin. A 100-ml amount of a culture grown to an optical density at 560 nm of 0.5 in PYE broth was harvested, washed once in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 8.0), and resuspended in 1 ml of the same buffer. Extracts were prepared by sonic treatment of the cell suspensions with six 30-s bursts, followed by centrifugation at 30,000 x g for 30 min; 1.5 ml of saturated ammonium sulfate was added to the extracts, and they were incubated at 4°C for 4 h. The precipitates were pelleted at 16,000 x g for 30 min and suspended in 100 ,ul of 0.05 M tris(hydroxymethyl)aminomethane (pH 8.0). A 20-,ul quantity of the ammonium sulfate precipitate was added to each of the outer wells, and 20 IL of C. crescentus flagellin-specific rabbit serum (16) (gift of L. Shapiro) was added to the inner well of immunodiffusion plates containing 0.01 M phosphate buffer
J. BACTERIOL.
FIG. 1. Minimal swarm plate containing 0.05% glucose inoculated with CB15 (wild type), SC229 (Fla-), SC274 (stub), and SC303 (Mot-).
FIG. 2. Transduction of SC239 (flaC108) with
4OCr30phage grown on SC507 (flaEF152). (pH 7.0), 0.15 M NaCl, 0.01% sodium azide, and 1% agarose. Plates were incubated at room temperature (23°C), and the resultant precipitin lines were observed after 24 and 48 h. Immunoprecipitations were performed essentially as described by Lagenaur and Agabian (14). Phage sensitivity. Phage sensitivity was assayed by spotting drops of phage lysates (109 to 1010 plaqueforming units per mi) onto PYE soft agar seeded with 0.2 ml of a culture of indicator bacteria grown to stationary phase in PYE broth. Lysis was observed after 40 h of incubation.
RESULTS Isolation of nonmotile mutants. A total of 93 motility mutants were obtained by selection
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C. CRESCENTUS MOTILITY MUTANTS
for those cells which were unable to form a swarm in soft agar as described above. Of these mutants, 24 had normal motility as observed under the light microscope. Because they were motile but did not form a swarm, we presumed that these mutants were defective in chemotaxis. Of the remaining nonmotile mutants, 54 had no flagella when mid-log-phase cultures were observed in the electron microscope (Fig. 3G), and 8 had a short fragment or stub of a flagellum as
_
A
629
shown in Fig. 3E and F. Since these latter mutants did not have a complete flagellar structure, they were designated Fla- along with those mutants which lacked the entire structure. Similar stub-forming mutants have been observed by Stove and Stanier (25). Seven additional mutants had normal flagella which were indistinguishable from those of wild-type cells (Fig. 3A, C, and D). Since these seven mutants were completely nonmotile, we presumed the flagella to
B
C
E
G
_19A
F
FIG. 3. Electron micrographs of C. crescentus wild-type and mutant cells stained with phosphotungstic acid. (A) CB15 (wild-type) swarmer cell. (B) CB15 stalk cell. (C) CB15 predivisional cell. (D) SC398 (Mot-) swarmer and predivisional cell. (E) SC244 (stub) swarmer cell. (F) SC288 (stub) swarmer cell and released hook and flagellar stub structure. (G) SC520 (Fla-) swarmer and predivisional cell. Bar, 1 tum for A, B, C, D, and G and 0.5 ,um for E and F.
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be paralyzed and therefore designated them closely linked to pleA102 in SC419, a nonrevertMot- mutants. All of the mutants appeared to ing pleiotropic mutation which is representative have normal morphology with respect to all of one of the predominant groups of nonmotile other characteristics. mutants selected as 4CbK resistant (R. C. JohnEach of the nonmotile mutants was examined son and B. Ely, unpublished data). On the other for temperature sensitivity by inoculation into hand, SC293 was sensitive to @CbK, and the two rich swarming medium plates and incuba- mutation for this strain was not linked to tion at 23 and 33°C. Four of the mutants (SC230 pleAl02. Thus, the mutations for strains SC293 [stub], SC231 [Mot-], SC266 [Fla-], SC274 and SC296 must map at different loci and rep[stub]) which were nonmotile at 330C were able resent two additional linkage groups. to swarm at 23°C and showed normal motility Among the stub-forming mutants, only SC255 when observed under a light microscope at this and SC274 were able to serve as recipients in temperaure. The remainder of the mutants were transduction experiments. The mutation in nonmotile at both temperatures. SC288 was linked to that in SC255, whereas the Genetic analysis. Generalized transducing rest of the stub-forming mutations were unphage 4Cr30 was grown on each of the mutants linked to those in either of the two recipients. In and used to transduce various nonmotile recipi- addition, each of the unlinked stub-forming muents as described above. The number of recom- tants, along with SC255 and SC274, was crossed binant flares resulting from a transduction with with a recipient from each of the fla linkage phage grown on a mutant was compared with groups. Four of the five unlinked mutations, the number of recombinants obtained when those in SC176, SC230, SC260, and SC285, were wild-type phage was used. If the, number was tightly linked to flaA104. Confrmnation of this less than 10% of the expected value with wild- linkage was obtained by the recovery of tempertype phage, the mutation was assumed to be ature-sensitive mutants among the transduclinked with that in the recipient. Typical results tants of SC299 (flaAl04) from crosses with are shown in Table 1. Reduced numbers of re- phage grown on SC230. These mutants were combinant flares were formed when strains con- shown to be motile at the permissive temperataining mutations designated flaC were crossed ture and to produce stubs at the restrictive temwith one another. Similar results were obtained perature and thus displayed the SC230 phenoin crosses with those containing mutations des- type. The mutation in the remaining stub-formignated flaD or flaH. From this analysis and ing mutant, SC175, appeared to be unlinked to from crosses with the other strains containing all of the linkage groups and probably represents fla mutations (data not shown), we were able to a separate locus. The mot mutations mapped in three linkage distinguish 22 linkage groups, each containing from 1 to 12 fla mutations; this data is summa- groups, with the mutations in each group relarized in Table 2. Mutations in strains SC293 and tively loosely linked to one another. RepresentSC296 were unlinked to representatives of the ative data are presented in Table 3. Mutations 22 linkage groups, but these strains could not be in SC231 and SC268 had the weakest linkage of crossed against each other since they both re- any group, but linkage was confirned by the verted at too high a frequency to be used as recovery of temperature-sensitive transductants recipients. SC296 was found to be resistant to of SC268 when phage grown on the temperature*CbK (see below) and contained a mutation sensitive mutant SC231 was used. Each of the Mot- mutants was crossed with a recipient from each of the fla and stub-forming linkage groups, TABLE 1. Mapping of fla mutations and no evidence of linkage was observed. No. of fares obtained with the following Evidence for linkage between separate cisrecipient:' trons was also obtained. Mutations in flaB, flaC, Donor and flaD formed one cluster, with 20 to 25% SC239 SC243 SC271 SC256 recombination between mutations in different (flaC) (ftaC) (flaD) (fla.H genes and less than 10% recombination between 0 2 SC239 (flaC) 33 74 mutations within a gene (Table 1 and data not SC306 (flaC) 16 3 3 81 shown). Similar results were obtained with flaE, 0 SC271 (flaD) 21 29 94 SC258 (flaD) 11 17 4 83 flaF, and flaG, with flaJ, flaK, flaL, and flaM, 0 SC256 (flaH) 44 64 ND and with flaH and flau In addition, two muta0 52 ND SC284 (flaH) 85 tions, flaEF152 and flaEF158, were found to be CB15 (wild type) 67 67 80 108 polycistronic deletions, since they would not rea Values are numbers of flares obtained in a trans- combine with mutations in flaE or in flaF. duction experiment. ND, Not determined. FlaEF152 recombined with mutations in flaG
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TABLE 2. C. crescentus motility mutants Linkage
gellinc
OCp34 sensitivityd
Fla
-
+
Stub
2± 2-
+
Fla-
_
+
FlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaStub Stub Stub
_
±
_
+
Mutantsa
Phenotypeb
G H I J K L M N 0 P Q R S T U V W X Y Z
SC229 (flaA104), SC237 (flaA107), SC240 (flaA109), SC244 (flaAlll), SC247 (flaAI12), SC249 (flaA114), SC257 ( flaA118), SC261 (flaA122), SC264 (flaA123), SC292 (flaA139), SC299 (flaA145), SC302 (flaA147) SC176 ( flaA103), SC230 (flaAlO5),* SC260 (flaA121), SC285 (flaA136), SC516 (flaB160) SC239 (flaC108), SC243 (flaCI10), SC306 (flaC149), SC506 ( flaC151), SC518 (flaCI62) SC252 (flaD15), SC258 (flaD119), SC271 (flaD127), SC510 (flaD154), SC513 (flaD157) SC512 ( flaEl56), SC519 ( flaE163), SC520 (flaE164) SC279( flaF132), SC282 (flaFI34) SC507 (flaEF152), SC514 (flaEF158) SC278( flaG131), SC280 (flaG133), SC515 (flaG159) SC256 (flaH117), SC284 (flaH135) SC270 (flaI126) SC269 (flaJ125) SC298 ( flaK144), SC511 (flaK155) SC300 (flaL146) SC266 (flaMl24)* SC272 (flaN128) SC290 (flaO138) SC295 (flaP141) SC307( flaQ150), SC235 (flaQ106) SC305 (flaR148) SC508 (flaS153) SC517(flaT161) SC548 (flaU165) SC293 (flaV140) SC259 (flaW120), SC297 (flaW143) SC255 ( flaX116), SC288 (flaX137) SC274 (flaY129)* SC175 (flaZ102)
A
SC296 (pleAlOl)
Ple-
group
A
B C
D E F
Intracellular fla-
FlaFla-
_
±
_
+
_
±
_
±
+
+
+
_
+
±
+
_ ±
i
+ +
+ +
-
+ +
+
+ +
+ +
_
+
+ + MotA SC231 (motAlOl),* SC268 (motA102) + + MotB SC286 ( motB103), SC301 (motB104) + + MotSC303( motC105), SC398 ( motC106), SC509 (motC107) C a The mutants are arranged according to their linkage relationship with one another as described in the text; an asterisk (*) indicates that the mutation is temperature sensitive. b Fla- indicates no flagella assembled, stub indicates presence of stubs of flagella, and Mot- indicates normal resistant, and 4Cb5 resistant. but paralyzed flagella. Ple- indicates that the strain is pleiotropically Fla-, ' Intracellular flagellin was detected by the presence of cross-reacting material against flagellin-specific antibody in cell extracts. +, High levels of cross-reacting material; ±, low levels of cross-reacting material; and -, no cross-reacting material as observed after 48 h. d +, Plaques formed on lawns of the appropriate strain; -, phage unable to form plaques; ±, spot lysis with no plaques.
4CbK
at 20% of the wild-type level, whereas flaEF158 recombined at 9% of the wild-type level; these data are summarized in Fig. 4. Synthesis of flagellin. Extracts were made from cultures of each of the nonmotile mutants, and immunodiffusions were performed with C. crescentus flagellin-specific antibody (Fig. 5). Only extracts from the stub-forming mutants
with mutations in flaX and flaY gave prominent precipitin bands which were comparable to the single precipitin band produced with the wildtype extract. Light precipitin bands were obtained with SC272 (flaN128) and SC235 (flaQ106) and with two additional stub-forming mutants (SC230, [flaA105] and SC260 [flaA121]). In addition, faint precipitin bands
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formed in many of the remaining mutants after prolonged incubation and probably reflect a low level of synthesis in these strains. In contrast, each of the Mot- mutants gave a reaction indistinguishable from that of the wild type, suggesting that the synthesis of flagellin is probably not affected in these mutants. A summary of the data obtained from the immunodiffusion analysis is presented in Table 2. To confirm the results, immunoprecipitates obtained from several of the mutants were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. All of the mutants tested, including those with mutations in cistrons X and Y, had greatly reduced levels of both flagellin proteins (data not shown). A detailed analysis of immunoprecipitates from the Fla- mutants will be presented in a subsequent publication. Phage sensitivity. Because mutations caus-
ing resistance to Caulobacter polar-adsorbing phage have been shown to pleiotropically affect motility (8, 12, 15), we spotted drops of OCb5
TABLE 3. Mapping of mot mutations No. of flares with the following recipient: Donor
SC268 (motA)
SC398 (motC)
SC286 (motB)
SC256
(flai)
FIG. 5. Immunodiffusion assay of flagellin. The center well contained anti-flagellin antibody. Portions (20,ul) of 1,000-fold-concentrated cell extracts were placed in the outer wells, except well A, which contained approximately 15 pg ofpurified flagellin. Well B contained SC274 (flaY129) extract, well C contained SC176 (flaA103) extract, well D contained SC507 (flaEF152), and well E contained CB15 (wildtype) extract.
38 SC231 (motA) 23 30 59 0 SC268 (motA) 57 23 99 SC286 (motB) 95 0 57 87 85 19 52 107 SC301 (motB) 80 60 0 99 SC398 (motC) 62 55 64 CB15 (wild type) 60 a Values are numbers of flares obtained in a transduction experiment.
flaB160
25%
flaCi10 flaCl62 flaC1O8 flaC149
20%
flaCi51
flaE156
35% --
flaE163
ftaF132
30%
flaF134
flaE164
flaD19 flaDl27 flaD154 flaD157
flaG131 flaG133
flaG159 flaEF152
20% ..........
9%
flaEF158
flaJ125 flaH117 flaH135
30%
flaK144
flaK155 504%
30-50%
25%
flaL146
flaM124
flaI126
FIG. 4. Linkage between C. crescentus fla genes. Linkage values reflect the number of Fla' recombinants obtained from transduction of recipient bacteria with phage grown on a mutant, as compared with phage grown on the wild-type strain. The probable order of the mutations is given where possible.
VOL. 137, 1979
and 4sCbK onto PYE plates spread with 0.1 ml of a saturated culture of each of the motility mutants. All of the Fla- and all of the stubforming mutants showed a decreased amount of lysis with either qCb5 or 4CbK as compared with the wild-type controls. On the other hand, all of the Mot- mutants maintained total sensitivity to these phages. Upon titering of oCbK, all of the mutants except SC296 gave plaques of varying turbidity and no more than a twofold decrease in plating efficiency. As expected, 4CbK was unable to form plaques on SC296 (pleA101). Strains sensitive to the pilus-adsorbing phage 4Cb5 gave only barely visible plaques, with a plating efficiency significantly lower than that of wild type. Pili were observed on many of the Fla- mutants, but a correlation could not be made since it is very difficult to determine the presence or absence of Caulobacter pili by electron microscopy. No differences in sensitivity were noted with the randomly adsorbing phages 4sCr3O or oCr40, indicating that this altered pattern of sensitivity is unique to the polar-adsorbing phage. Drops of the reported flagellotropic phage 0Cp34 (7) were spotted onto PYE plates spread with 0.1 ml of a saturated culture of at least one representative of each linkage group. Spot lysis was observed on mutants from all of the linkage groups except those containing the mutations flaO, flaW, and pleA. In addition, qCp34 could not form plaques on cells containing flaE, H, I, 0, S, W, and pleA mutations (Table 2). These mutants also had the greatest reduction in sensitivity with 4CbK (data not shown). Thus, mutations in only 7 of the 30 genes involved in motility cause resistance to 4Cp34.
DISCUSSION The morphogenesis of flagella in Caulobacter appears to be rather complex, as judged by the large number of genes involved. Mutations in 27 loci (including pleA) resulted in total loss of flagella, whereas mutations in four loci gave partially assembled flagella. Since most loci were represented by a single mutation, additional loci will probably be found as more Caulobacter motility mutants are isolated. Also, some of the linkage groups could contain more than one gene since the mutants were grouped on the basis of low transduction frequencies. Further genetic mapping, along with the isolation of additional mutants and the development of a system of complementation, will provide a more accurate estimate of the number of genes involved in flagellum morphogenesis. Extensive analysis by recombination and com-
C. CRESCENTUS MOTILITY MUTANTS
633
plementation of E. coli Fla- mutants has revealed up to 20 genes involved in flagellum synthesis (9, 24). The greater number of genes in Caulobacter is not too surprising since the biogenesis of flagella is temporally and spatially regulated in Caulobacter. Furthermore, there is the additional step of the release of the flagella as the swarmer cell differentiates into the nonflagellated stalk. Some of the mutations might cause changes in the cell envelope which would affect flagellum assembly only indirectly (2). Three loci giving a Mot- phenotype were found in C. crescentus, in contrast to the two found in E. coli (9, 24). In Salmonella, mot mutations occur at a third locus (motC), but. these mutations are part of the flaAII cistron and do not represent a unique gene (5, 9). Since none of the C. crescentus mot mutations were found to be linked to any of the fla genes, we conclude that Caulobacter may have an additional mot locus, as compared with E. coli and Salmonella. Another interesting difference between the genetics of C. crescentus motility mutants and those analyzed in other bacteria is that the Caulobacter fla genes are not clustered. In both E. coli and Salmonella, motility genes are clustered in three main regions (9, 24), with the majority of the E. coli genes being transcribed in polycistronic operons (23, 24). Furthermore, both Proteus mirabilis (3) and Pseudomonas aeruginosa (9) fla genes have been found to map in a cluster. Relatively weak linkage has been established between several C. crescentus fla genes (Fig. 4), but linkage between some genes is to be expected due to the large number of genes involved and the large phage used for mapping (6). Thus, Caulobacter represents the first example where a bacterium does not have its fla genes tightly clustered. Clustering of biosynthetic genes also has not been observed when similar transductional analyses were performed on groups of C. crescentus auxotrophs (Ely et al., unpublished data). Only one mutant, SC296, was resistant to both 4CbK and 4Cb5, as has been found with the pleiotropic nonmotile mutants isolated previously (8, 12, 15; Johnson and Ely, unpublished data). The remainder of the mutants described had only a slightly decreased sensitivity to 4CbK, even though they had a marked decrease in sensitivity to 4Cb5. In contrast, each of the Mot- mutants was totally sensitive to both of these phages. We conclude, therefore, that the presence but not the activity of the flagella must enhance infection by the polar-adsorbing phage. It is important to note that, although many swarmer-stage polar characteristics were affected in these mutants, stalk cell polar charac-
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teristics such as stalk formation and rosette formation were normal. ¢OCp34 was reported by Fukuda et al. to require actively rotating flagella to support successful infection (7). However, we found that, although some Fla- mutants were resistant to OCp34, both the Mot- and many of the Flamutants were sensitive to the phage. Thus, OCp34 can infect cells which have either a paralyzed flagellum or no flagellum at all. The mutants isolated in this study will be invaluable for studying polar assembly and temporal regulation, as well as general flagellum morphogenesis in a nonenteric bacterium. Studies are currently underway to determine which cistron is responsible for a particular function or structure in the synthesis of the flagellum. The C. crescentus basal body was recently isolated and found to contain an additional structure compared with those observed on E. coli and Salmonella basal bodies (Johnson et al., submitted). Also, a flagellum composed of two flagellin proteins is unique to Caulobacter. These characteristics, along with the temporal expression of the organelle, make the C. crescentus flagellar system distinct from other procaryotic flagellar systems studied. Lagenaur and Agabian (14) have recently shown that the onset of hook protein biosynthesis occurs immediately before the biosynthesis of the two flagellins. The two flagellins are first detected coincidentally with one another (14, 17), suggesting a common temporal control of both flagellins which is separate from the control of hook synthesis. Studies are underway to determine whether synthesis of the hook and rod structure occurs normally in all mutants lacking a flagellum. Preliminary results indicate that hook protein synthesis is unaffected and that hook structures are assembled in most of the mutants (Johnson et al., submitted; Johnson and Ely, unpublished data). These data support the idea that there are separate control mechanisms for hook and flagellin syntheses. ACKNOWVLEDGMEENTS We thank L. Shapiro for the gift of the C. crescentus flagellin-specific antibody. This work was supported in part by grant PCM76-82742 from the National Science Foundation.
LITERATURE CrIED 1. Agabian-Keshishian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage 4CbK. J. Virol. 5:795-800. 2. Ames, G. F.-L., E. N. Spudich, and H. Nikaido. 1974. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406-416.
J. BACTERIOL. 3. Appelbaum, P. C., and 0. W. Prozesky. 1973. Transductional analysis of non-motile mutants in Proteus mirabilis. J. Gen. Microbiol. 77:89-97. 4. Armstrong, J. B., J. Adler, and M. M. Dahl. 1967. Nonchemotactic mutants of Escherichia coli. J. Bacteriol. 93:390-398. 5. Collins, A. L. T., and B. A. D. Stocker. 1976. Salmonella typhimurium mutants generally defective in chemotaxis. J. Bacteriol. 128:754-765. 6. Ely, B., and R. C. Johnson. 1977. Generalized transduction in Caulobacter crescentus. Genetics 87:391-399. 7. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. A flagellotropic bacteriophage and flagella formation in Caulobacter. Virology 71:583-592. 8. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. Regulation of polar surface structures in Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors. Mol. Gen. Genet. 149:167-173. 9. Iino, T. 1977. Genetics of structure and function of bacterial flagella. Annu. Rev. Genet. 11:161-182. 10. Johnson, R. C., and B. Ely. 1977. Isolation of spontaneous derived mutants of Caulobacter crescentus. Genetics 86:25-32. 11. Johnson, R. C., N. B. Wood, and B. Ely. 1977. Isolation and characterization of bacteriophages for Caulobacter crescentus. J. Gen. Virol. 37:323-335. 12. Kurn, N., S. Ammer, and L. Shapiro. 1974. A pleiotropic mutation affecting expression of polar development events in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 71:3157-3161. 13. Lagenaur, C., and N. Agabian. 1976. Physical characterization of Caulobacter crescentus flagella. J. Bacteriol. 128:435-444. 14. Lagenaur, C., and N. Agabian. 1978. Caulobacter flagellar organelle: synthesis, compartmentation, and assembly. J. Bacteriol. 135:1062-1069. 15. Lagenaur, C., S. Farmer, and N. Agabian. 1977. Adsorption properties of stage-specific Caulobacter phage 4CbK. Virology 77:401-407. 16. Marino, W., S. Ammer, and L. Shapiro. 1976. Conditional surface structure mutants of Caulobacter crescentus: temperature-sensitive flagella formation due to an altered flagellin monomer. J. Mol. Biol. 107:115-130. 17. Osley, M. A., M. Sheffery, and A. Newvton. 1977. Regulation of flagellin synthesis in the cell cycle of Caulobacter: dependence on DNA replication. Cell 12: 393-400. 18. Poindexter, J. S., P. R. Hornack, and P. A. Armstrong. 1967. Intracellular development of a large DNA bacteriophage lytic for Caulobacter crescentus. Arch. Mikrobiol. 59:237-246. 19. Schmidt, J. M. 1966. Observations on the adsorption of Caulobacter bacteriophages containing ribonucleic acid. J. Gen. Microbiol. 45:347-353. 20. Shapiro, L 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377407. 21. Shapiro, L, and J. V. Maizel, Jr. 1973. Synthesis and structure of Caulobacter crescentus flagella. J. Bacteriol. 113:478-485. 22. Sheffery, M., and A. Newton. 1977. Reconstitution and purification of flagellar filaments from Caulobacter crescentus. J. Bacteriol. 132:1027-1030. 23. Silverman, M., and M. Simon. 1973. Genetic analysis of bacteriophage Mu-induced flagellar mutants in Escherichia coli. J. Bacteriol. 116:114-122. 24. Silverman, M., and M. Simon. 1977. Bacterial flagella. Annu. Rev. Microbiol. 31:397419. 25. Stove, J. L., and R. Y. Stanier. 1962. Cellular differentiation in stalked bacteria. Nature (London) 196: 1189-1192.
Vol. 137, No. 1
Analysis of Nonmotile Mutants of the Dimorphic Bacterium Caulobacter crescentus REID C. JOHNSON' AND BERT ELY".2* Department of Biology' and Department of Microbiology and Immunology,2 University of South Carolina, Columbia, South Carolina 29208
Received for publication 21 September 1978
A total of 69 spontaneous nonmotile mutants were isolated from the dimorphic bacterium Caulobacter crescentus. The majority of the mutants were unable to assemble a flagellar filament (Fla-), although eight were able to synthesize a short stub of a flagellum. A third mutant class assembled flagella of normal morphology but were nonmotile (Mot-). Genetic analysis by oCr30-mediated transduction revealed 27 linkage groups for the fla and stub-forming mutations, and three linkage groups for the mot mutations. Intracellular flagellin detected by immunodiffusion was at the limit of detectability in most of the Fla- and stub-forming mutants but normal in the Mot- mutants. The Fla- and stub-forming mutants also showed decreased sensitivity to the swarmer-specific phages oCbK and oCb5 while maintaining complete sensitivity to the randomly adsorbing phages oCr30 and 4Cr40. One additional strain was totally resistant to oCbK, and the mutation in this strain has been designated pleA. Each of the mutants containing mot mutations showed wild-type sensitivity to all of the phages tested.
Flagellum biogenesis in Caulobacter represents an attractive system for the study of organelle differentiation in procaryotes. A single flagellum is present on swarmer cells along with pili and phage receptors at one pole of the cell (20). As this swarmer cell differentiates into a stalk cell, the entire flagellar structure, including the filament, hook, and rod, is released into the medium (18, 21; R. C. Johnson, J. P. Walsh, B. Ely, and L. Shapiro, J. Bacteriol., submitted), and pili and phage receptors are lost (20). The stalk cell undergoes DNA replication and then elongates into a predivisional cell with a new flagellum at the opposite pole from the stalk (for a review, see reference 20). This cell subsequently divides to produce a new swarmer cell and a stalk cell which can immediately undergo DNA replication, whereas the swarmer cell must lose its flagellum and form a stalk before it can replicate its DNA. Not only is the formation of the flagellum structure, and thus cell motility, temporally regulated in the Caulobacter cell cycle, but flagellin, the primary component of flagella, is synthesized coincidentally with the appearance of the flagellum on the cell surface (21). Osley et al. (17) have recently shown by immunoprecipitation studies with antiflagellin antibody and synchronized C. crescentus cell populations that two species of flagellin (see below) are synthesized immediately after, and are dependent upon, the completion of DNA replication.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella resolves two protein components of flagella: a minor 27,500dalton protein and the major 25,000-dalton protein (13, 16, 17, 22). Lagenaur and Agabian (13) have shown that these two proteins are distinct with regard to their amino acid composition, but the proteins seem to be related because antibody made against the major 25,000-dalton flagellin does cross-react with both purified flagellins in immunodiffusion assays (13). To study the regulation of flagellum biogenesis in Caulobacter, we isolated a large number of mutants with altered motility. We present here a characterization of these mutants, particularly with regard to genetic analysis. MATERIALS AND METHODS
Bacteria and bacteriophage. C. crescentus wildtype strain CB15 was used as a parent for all strains described below. Swarmer-specific bacteriophage 0CbK, which adsorbs to the polar end of the cell near the base of the flagellum (1), and 4Cb5, which adsorbs to pili also found at the cell pole (19), were gifts of L. Shapiro and N. Agabian, respectively. The flagellotropic phage 0Cp34 was obtained from A. Fukuda (7). 4)Cr30 and OCr40, which adsorb randomly to all cell types, have been described previously (11). Growth conditions. Complete (PYE) and minimal (M2) liquid and solid media for growing Caulobacter and Caulobacter phage have been reported previously (10). Rich swarming medium was prepared by adding 0.25% agar (Difco Laboratories) to half-strength PYE. 627
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JOHNSON AND ELY
Minimal swarming medium was made by the addition of the appropriate amount of carbon source to M2 plus 0.25% agar (Difco). Isolation of motility mutants. Spontaneously occurring Caulobacter motility mutants were isolated by an enrichment procedure based on that used by Armstrong et al. for the isolation of Escherichia coli chemotaxis mutants (4). Colonies of wild-type CB15 were stabbed into minimal swarming medium plus 0.05% glucose or xylose and incubated at 33°C. The bacteria were allowed to swarm for 4 to 5 days, and then the center of the swarm was restabbed into the same medium and incubated again. After four to six serial enrichments, the center was removed and suspended in PYE, and 0.1 ml of the appropriate dilution was mixed with 20 ml of rich swarming medium and poured onto a sterile petri dish. After 2 days of incubation at 33°C, the nonswarming colonies were rediluted and plated on rich swarming medium as before to get wellseparated colonies. Examples of the colony morphologies of wild-type cells and three nonmotile mutants on minimal swarming medium are shown in Fig. 1. The purified colonies were streaked onto PYE plates for further study. Only one isolate was retained from each selection to insure independent clones. Electron microscopy. A 10-ml amount of a logphase culture grown in PYE broth with aeration was pelleted in a Sorvall table top centrifuge and gently resuspended in 0.5 ml of fresh PYE. Equal volumes of the cell suspension and 1 or 2% phosphotungstic acid (pH 7.0) were mixed and placed on a carbon-stabilized nitrocellulose-coated 400-mesh grid. After 20 s, the grid was blotted, air dried, and observed under a Seimans Elmiskop model IA or a Japan Electronic Optics Laboratory Ltd. model 100B electron microscope at 80 kV. Transductions. Transductions were performed with the generalized transducing phage 4Cr30 (6). Equal volumes of a stationary phase culture grown in PYE broth and a 10-2 or 10-3 dilution of a UV-irradiated phage lysate (6) were combined, and 0.025 ml of the mixture was streaked across a minimal swarming medium plate containing 0.1% glucose with a 0.2-ml pipette. After 5 days of incubation at 330C, recombinant flares emanating from the streak were counted. An example of a transduction plate is shown in Fig. 2. No evidence of abortive transductants or trails was ever observed. Assay for intracellular flagellin. A 100-ml amount of a culture grown to an optical density at 560 nm of 0.5 in PYE broth was harvested, washed once in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 8.0), and resuspended in 1 ml of the same buffer. Extracts were prepared by sonic treatment of the cell suspensions with six 30-s bursts, followed by centrifugation at 30,000 x g for 30 min; 1.5 ml of saturated ammonium sulfate was added to the extracts, and they were incubated at 4°C for 4 h. The precipitates were pelleted at 16,000 x g for 30 min and suspended in 100 ,ul of 0.05 M tris(hydroxymethyl)aminomethane (pH 8.0). A 20-,ul quantity of the ammonium sulfate precipitate was added to each of the outer wells, and 20 IL of C. crescentus flagellin-specific rabbit serum (16) (gift of L. Shapiro) was added to the inner well of immunodiffusion plates containing 0.01 M phosphate buffer
J. BACTERIOL.
FIG. 1. Minimal swarm plate containing 0.05% glucose inoculated with CB15 (wild type), SC229 (Fla-), SC274 (stub), and SC303 (Mot-).
FIG. 2. Transduction of SC239 (flaC108) with
4OCr30phage grown on SC507 (flaEF152). (pH 7.0), 0.15 M NaCl, 0.01% sodium azide, and 1% agarose. Plates were incubated at room temperature (23°C), and the resultant precipitin lines were observed after 24 and 48 h. Immunoprecipitations were performed essentially as described by Lagenaur and Agabian (14). Phage sensitivity. Phage sensitivity was assayed by spotting drops of phage lysates (109 to 1010 plaqueforming units per mi) onto PYE soft agar seeded with 0.2 ml of a culture of indicator bacteria grown to stationary phase in PYE broth. Lysis was observed after 40 h of incubation.
RESULTS Isolation of nonmotile mutants. A total of 93 motility mutants were obtained by selection
VOL. 137, 1979
C. CRESCENTUS MOTILITY MUTANTS
for those cells which were unable to form a swarm in soft agar as described above. Of these mutants, 24 had normal motility as observed under the light microscope. Because they were motile but did not form a swarm, we presumed that these mutants were defective in chemotaxis. Of the remaining nonmotile mutants, 54 had no flagella when mid-log-phase cultures were observed in the electron microscope (Fig. 3G), and 8 had a short fragment or stub of a flagellum as
_
A
629
shown in Fig. 3E and F. Since these latter mutants did not have a complete flagellar structure, they were designated Fla- along with those mutants which lacked the entire structure. Similar stub-forming mutants have been observed by Stove and Stanier (25). Seven additional mutants had normal flagella which were indistinguishable from those of wild-type cells (Fig. 3A, C, and D). Since these seven mutants were completely nonmotile, we presumed the flagella to
B
C
E
G
_19A
F
FIG. 3. Electron micrographs of C. crescentus wild-type and mutant cells stained with phosphotungstic acid. (A) CB15 (wild-type) swarmer cell. (B) CB15 stalk cell. (C) CB15 predivisional cell. (D) SC398 (Mot-) swarmer and predivisional cell. (E) SC244 (stub) swarmer cell. (F) SC288 (stub) swarmer cell and released hook and flagellar stub structure. (G) SC520 (Fla-) swarmer and predivisional cell. Bar, 1 tum for A, B, C, D, and G and 0.5 ,um for E and F.
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JOHNSON AND ELY
J. BACTERIOL.
be paralyzed and therefore designated them closely linked to pleA102 in SC419, a nonrevertMot- mutants. All of the mutants appeared to ing pleiotropic mutation which is representative have normal morphology with respect to all of one of the predominant groups of nonmotile other characteristics. mutants selected as 4CbK resistant (R. C. JohnEach of the nonmotile mutants was examined son and B. Ely, unpublished data). On the other for temperature sensitivity by inoculation into hand, SC293 was sensitive to @CbK, and the two rich swarming medium plates and incuba- mutation for this strain was not linked to tion at 23 and 33°C. Four of the mutants (SC230 pleAl02. Thus, the mutations for strains SC293 [stub], SC231 [Mot-], SC266 [Fla-], SC274 and SC296 must map at different loci and rep[stub]) which were nonmotile at 330C were able resent two additional linkage groups. to swarm at 23°C and showed normal motility Among the stub-forming mutants, only SC255 when observed under a light microscope at this and SC274 were able to serve as recipients in temperaure. The remainder of the mutants were transduction experiments. The mutation in nonmotile at both temperatures. SC288 was linked to that in SC255, whereas the Genetic analysis. Generalized transducing rest of the stub-forming mutations were unphage 4Cr30 was grown on each of the mutants linked to those in either of the two recipients. In and used to transduce various nonmotile recipi- addition, each of the unlinked stub-forming muents as described above. The number of recom- tants, along with SC255 and SC274, was crossed binant flares resulting from a transduction with with a recipient from each of the fla linkage phage grown on a mutant was compared with groups. Four of the five unlinked mutations, the number of recombinants obtained when those in SC176, SC230, SC260, and SC285, were wild-type phage was used. If the, number was tightly linked to flaA104. Confrmnation of this less than 10% of the expected value with wild- linkage was obtained by the recovery of tempertype phage, the mutation was assumed to be ature-sensitive mutants among the transduclinked with that in the recipient. Typical results tants of SC299 (flaAl04) from crosses with are shown in Table 1. Reduced numbers of re- phage grown on SC230. These mutants were combinant flares were formed when strains con- shown to be motile at the permissive temperataining mutations designated flaC were crossed ture and to produce stubs at the restrictive temwith one another. Similar results were obtained perature and thus displayed the SC230 phenoin crosses with those containing mutations des- type. The mutation in the remaining stub-formignated flaD or flaH. From this analysis and ing mutant, SC175, appeared to be unlinked to from crosses with the other strains containing all of the linkage groups and probably represents fla mutations (data not shown), we were able to a separate locus. The mot mutations mapped in three linkage distinguish 22 linkage groups, each containing from 1 to 12 fla mutations; this data is summa- groups, with the mutations in each group relarized in Table 2. Mutations in strains SC293 and tively loosely linked to one another. RepresentSC296 were unlinked to representatives of the ative data are presented in Table 3. Mutations 22 linkage groups, but these strains could not be in SC231 and SC268 had the weakest linkage of crossed against each other since they both re- any group, but linkage was confirned by the verted at too high a frequency to be used as recovery of temperature-sensitive transductants recipients. SC296 was found to be resistant to of SC268 when phage grown on the temperature*CbK (see below) and contained a mutation sensitive mutant SC231 was used. Each of the Mot- mutants was crossed with a recipient from each of the fla and stub-forming linkage groups, TABLE 1. Mapping of fla mutations and no evidence of linkage was observed. No. of fares obtained with the following Evidence for linkage between separate cisrecipient:' trons was also obtained. Mutations in flaB, flaC, Donor and flaD formed one cluster, with 20 to 25% SC239 SC243 SC271 SC256 recombination between mutations in different (flaC) (ftaC) (flaD) (fla.H genes and less than 10% recombination between 0 2 SC239 (flaC) 33 74 mutations within a gene (Table 1 and data not SC306 (flaC) 16 3 3 81 shown). Similar results were obtained with flaE, 0 SC271 (flaD) 21 29 94 SC258 (flaD) 11 17 4 83 flaF, and flaG, with flaJ, flaK, flaL, and flaM, 0 SC256 (flaH) 44 64 ND and with flaH and flau In addition, two muta0 52 ND SC284 (flaH) 85 tions, flaEF152 and flaEF158, were found to be CB15 (wild type) 67 67 80 108 polycistronic deletions, since they would not rea Values are numbers of flares obtained in a trans- combine with mutations in flaE or in flaF. duction experiment. ND, Not determined. FlaEF152 recombined with mutations in flaG
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C. CRESCENTUS MOTILITY MUTANTS
VOL. 137, 1979
TABLE 2. C. crescentus motility mutants Linkage
gellinc
OCp34 sensitivityd
Fla
-
+
Stub
2± 2-
+
Fla-
_
+
FlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaFlaStub Stub Stub
_
±
_
+
Mutantsa
Phenotypeb
G H I J K L M N 0 P Q R S T U V W X Y Z
SC229 (flaA104), SC237 (flaA107), SC240 (flaA109), SC244 (flaAlll), SC247 (flaAI12), SC249 (flaA114), SC257 ( flaA118), SC261 (flaA122), SC264 (flaA123), SC292 (flaA139), SC299 (flaA145), SC302 (flaA147) SC176 ( flaA103), SC230 (flaAlO5),* SC260 (flaA121), SC285 (flaA136), SC516 (flaB160) SC239 (flaC108), SC243 (flaCI10), SC306 (flaC149), SC506 ( flaC151), SC518 (flaCI62) SC252 (flaD15), SC258 (flaD119), SC271 (flaD127), SC510 (flaD154), SC513 (flaD157) SC512 ( flaEl56), SC519 ( flaE163), SC520 (flaE164) SC279( flaF132), SC282 (flaFI34) SC507 (flaEF152), SC514 (flaEF158) SC278( flaG131), SC280 (flaG133), SC515 (flaG159) SC256 (flaH117), SC284 (flaH135) SC270 (flaI126) SC269 (flaJ125) SC298 ( flaK144), SC511 (flaK155) SC300 (flaL146) SC266 (flaMl24)* SC272 (flaN128) SC290 (flaO138) SC295 (flaP141) SC307( flaQ150), SC235 (flaQ106) SC305 (flaR148) SC508 (flaS153) SC517(flaT161) SC548 (flaU165) SC293 (flaV140) SC259 (flaW120), SC297 (flaW143) SC255 ( flaX116), SC288 (flaX137) SC274 (flaY129)* SC175 (flaZ102)
A
SC296 (pleAlOl)
Ple-
group
A
B C
D E F
Intracellular fla-
FlaFla-
_
±
_
+
_
±
_
±
+
+
+
_
+
±
+
_ ±
i
+ +
+ +
-
+ +
+
+ +
+ +
_
+
+ + MotA SC231 (motAlOl),* SC268 (motA102) + + MotB SC286 ( motB103), SC301 (motB104) + + MotSC303( motC105), SC398 ( motC106), SC509 (motC107) C a The mutants are arranged according to their linkage relationship with one another as described in the text; an asterisk (*) indicates that the mutation is temperature sensitive. b Fla- indicates no flagella assembled, stub indicates presence of stubs of flagella, and Mot- indicates normal resistant, and 4Cb5 resistant. but paralyzed flagella. Ple- indicates that the strain is pleiotropically Fla-, ' Intracellular flagellin was detected by the presence of cross-reacting material against flagellin-specific antibody in cell extracts. +, High levels of cross-reacting material; ±, low levels of cross-reacting material; and -, no cross-reacting material as observed after 48 h. d +, Plaques formed on lawns of the appropriate strain; -, phage unable to form plaques; ±, spot lysis with no plaques.
4CbK
at 20% of the wild-type level, whereas flaEF158 recombined at 9% of the wild-type level; these data are summarized in Fig. 4. Synthesis of flagellin. Extracts were made from cultures of each of the nonmotile mutants, and immunodiffusions were performed with C. crescentus flagellin-specific antibody (Fig. 5). Only extracts from the stub-forming mutants
with mutations in flaX and flaY gave prominent precipitin bands which were comparable to the single precipitin band produced with the wildtype extract. Light precipitin bands were obtained with SC272 (flaN128) and SC235 (flaQ106) and with two additional stub-forming mutants (SC230, [flaA105] and SC260 [flaA121]). In addition, faint precipitin bands
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JOHNSON AND ELY
J. BACTERIOL.
formed in many of the remaining mutants after prolonged incubation and probably reflect a low level of synthesis in these strains. In contrast, each of the Mot- mutants gave a reaction indistinguishable from that of the wild type, suggesting that the synthesis of flagellin is probably not affected in these mutants. A summary of the data obtained from the immunodiffusion analysis is presented in Table 2. To confirm the results, immunoprecipitates obtained from several of the mutants were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. All of the mutants tested, including those with mutations in cistrons X and Y, had greatly reduced levels of both flagellin proteins (data not shown). A detailed analysis of immunoprecipitates from the Fla- mutants will be presented in a subsequent publication. Phage sensitivity. Because mutations caus-
ing resistance to Caulobacter polar-adsorbing phage have been shown to pleiotropically affect motility (8, 12, 15), we spotted drops of OCb5
TABLE 3. Mapping of mot mutations No. of flares with the following recipient: Donor
SC268 (motA)
SC398 (motC)
SC286 (motB)
SC256
(flai)
FIG. 5. Immunodiffusion assay of flagellin. The center well contained anti-flagellin antibody. Portions (20,ul) of 1,000-fold-concentrated cell extracts were placed in the outer wells, except well A, which contained approximately 15 pg ofpurified flagellin. Well B contained SC274 (flaY129) extract, well C contained SC176 (flaA103) extract, well D contained SC507 (flaEF152), and well E contained CB15 (wildtype) extract.
38 SC231 (motA) 23 30 59 0 SC268 (motA) 57 23 99 SC286 (motB) 95 0 57 87 85 19 52 107 SC301 (motB) 80 60 0 99 SC398 (motC) 62 55 64 CB15 (wild type) 60 a Values are numbers of flares obtained in a transduction experiment.
flaB160
25%
flaCi10 flaCl62 flaC1O8 flaC149
20%
flaCi51
flaE156
35% --
flaE163
ftaF132
30%
flaF134
flaE164
flaD19 flaDl27 flaD154 flaD157
flaG131 flaG133
flaG159 flaEF152
20% ..........
9%
flaEF158
flaJ125 flaH117 flaH135
30%
flaK144
flaK155 504%
30-50%
25%
flaL146
flaM124
flaI126
FIG. 4. Linkage between C. crescentus fla genes. Linkage values reflect the number of Fla' recombinants obtained from transduction of recipient bacteria with phage grown on a mutant, as compared with phage grown on the wild-type strain. The probable order of the mutations is given where possible.
VOL. 137, 1979
and 4sCbK onto PYE plates spread with 0.1 ml of a saturated culture of each of the motility mutants. All of the Fla- and all of the stubforming mutants showed a decreased amount of lysis with either qCb5 or 4CbK as compared with the wild-type controls. On the other hand, all of the Mot- mutants maintained total sensitivity to these phages. Upon titering of oCbK, all of the mutants except SC296 gave plaques of varying turbidity and no more than a twofold decrease in plating efficiency. As expected, 4CbK was unable to form plaques on SC296 (pleA101). Strains sensitive to the pilus-adsorbing phage 4Cb5 gave only barely visible plaques, with a plating efficiency significantly lower than that of wild type. Pili were observed on many of the Fla- mutants, but a correlation could not be made since it is very difficult to determine the presence or absence of Caulobacter pili by electron microscopy. No differences in sensitivity were noted with the randomly adsorbing phages 4sCr3O or oCr40, indicating that this altered pattern of sensitivity is unique to the polar-adsorbing phage. Drops of the reported flagellotropic phage 0Cp34 (7) were spotted onto PYE plates spread with 0.1 ml of a saturated culture of at least one representative of each linkage group. Spot lysis was observed on mutants from all of the linkage groups except those containing the mutations flaO, flaW, and pleA. In addition, qCp34 could not form plaques on cells containing flaE, H, I, 0, S, W, and pleA mutations (Table 2). These mutants also had the greatest reduction in sensitivity with 4CbK (data not shown). Thus, mutations in only 7 of the 30 genes involved in motility cause resistance to 4Cp34.
DISCUSSION The morphogenesis of flagella in Caulobacter appears to be rather complex, as judged by the large number of genes involved. Mutations in 27 loci (including pleA) resulted in total loss of flagella, whereas mutations in four loci gave partially assembled flagella. Since most loci were represented by a single mutation, additional loci will probably be found as more Caulobacter motility mutants are isolated. Also, some of the linkage groups could contain more than one gene since the mutants were grouped on the basis of low transduction frequencies. Further genetic mapping, along with the isolation of additional mutants and the development of a system of complementation, will provide a more accurate estimate of the number of genes involved in flagellum morphogenesis. Extensive analysis by recombination and com-
C. CRESCENTUS MOTILITY MUTANTS
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plementation of E. coli Fla- mutants has revealed up to 20 genes involved in flagellum synthesis (9, 24). The greater number of genes in Caulobacter is not too surprising since the biogenesis of flagella is temporally and spatially regulated in Caulobacter. Furthermore, there is the additional step of the release of the flagella as the swarmer cell differentiates into the nonflagellated stalk. Some of the mutations might cause changes in the cell envelope which would affect flagellum assembly only indirectly (2). Three loci giving a Mot- phenotype were found in C. crescentus, in contrast to the two found in E. coli (9, 24). In Salmonella, mot mutations occur at a third locus (motC), but. these mutations are part of the flaAII cistron and do not represent a unique gene (5, 9). Since none of the C. crescentus mot mutations were found to be linked to any of the fla genes, we conclude that Caulobacter may have an additional mot locus, as compared with E. coli and Salmonella. Another interesting difference between the genetics of C. crescentus motility mutants and those analyzed in other bacteria is that the Caulobacter fla genes are not clustered. In both E. coli and Salmonella, motility genes are clustered in three main regions (9, 24), with the majority of the E. coli genes being transcribed in polycistronic operons (23, 24). Furthermore, both Proteus mirabilis (3) and Pseudomonas aeruginosa (9) fla genes have been found to map in a cluster. Relatively weak linkage has been established between several C. crescentus fla genes (Fig. 4), but linkage between some genes is to be expected due to the large number of genes involved and the large phage used for mapping (6). Thus, Caulobacter represents the first example where a bacterium does not have its fla genes tightly clustered. Clustering of biosynthetic genes also has not been observed when similar transductional analyses were performed on groups of C. crescentus auxotrophs (Ely et al., unpublished data). Only one mutant, SC296, was resistant to both 4CbK and 4Cb5, as has been found with the pleiotropic nonmotile mutants isolated previously (8, 12, 15; Johnson and Ely, unpublished data). The remainder of the mutants described had only a slightly decreased sensitivity to 4CbK, even though they had a marked decrease in sensitivity to 4Cb5. In contrast, each of the Mot- mutants was totally sensitive to both of these phages. We conclude, therefore, that the presence but not the activity of the flagella must enhance infection by the polar-adsorbing phage. It is important to note that, although many swarmer-stage polar characteristics were affected in these mutants, stalk cell polar charac-
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teristics such as stalk formation and rosette formation were normal. ¢OCp34 was reported by Fukuda et al. to require actively rotating flagella to support successful infection (7). However, we found that, although some Fla- mutants were resistant to OCp34, both the Mot- and many of the Flamutants were sensitive to the phage. Thus, OCp34 can infect cells which have either a paralyzed flagellum or no flagellum at all. The mutants isolated in this study will be invaluable for studying polar assembly and temporal regulation, as well as general flagellum morphogenesis in a nonenteric bacterium. Studies are currently underway to determine which cistron is responsible for a particular function or structure in the synthesis of the flagellum. The C. crescentus basal body was recently isolated and found to contain an additional structure compared with those observed on E. coli and Salmonella basal bodies (Johnson et al., submitted). Also, a flagellum composed of two flagellin proteins is unique to Caulobacter. These characteristics, along with the temporal expression of the organelle, make the C. crescentus flagellar system distinct from other procaryotic flagellar systems studied. Lagenaur and Agabian (14) have recently shown that the onset of hook protein biosynthesis occurs immediately before the biosynthesis of the two flagellins. The two flagellins are first detected coincidentally with one another (14, 17), suggesting a common temporal control of both flagellins which is separate from the control of hook synthesis. Studies are underway to determine whether synthesis of the hook and rod structure occurs normally in all mutants lacking a flagellum. Preliminary results indicate that hook protein synthesis is unaffected and that hook structures are assembled in most of the mutants (Johnson et al., submitted; Johnson and Ely, unpublished data). These data support the idea that there are separate control mechanisms for hook and flagellin syntheses. ACKNOWVLEDGMEENTS We thank L. Shapiro for the gift of the C. crescentus flagellin-specific antibody. This work was supported in part by grant PCM76-82742 from the National Science Foundation.
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J. BACTERIOL. 3. Appelbaum, P. C., and 0. W. Prozesky. 1973. Transductional analysis of non-motile mutants in Proteus mirabilis. J. Gen. Microbiol. 77:89-97. 4. Armstrong, J. B., J. Adler, and M. M. Dahl. 1967. Nonchemotactic mutants of Escherichia coli. J. Bacteriol. 93:390-398. 5. Collins, A. L. T., and B. A. D. Stocker. 1976. Salmonella typhimurium mutants generally defective in chemotaxis. J. Bacteriol. 128:754-765. 6. Ely, B., and R. C. Johnson. 1977. Generalized transduction in Caulobacter crescentus. Genetics 87:391-399. 7. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. A flagellotropic bacteriophage and flagella formation in Caulobacter. Virology 71:583-592. 8. Fukuda, A., K. Miyakawa, H. Iba, and Y. Okada. 1976. Regulation of polar surface structures in Caulobacter crescentus: pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors. Mol. Gen. Genet. 149:167-173. 9. Iino, T. 1977. Genetics of structure and function of bacterial flagella. Annu. Rev. Genet. 11:161-182. 10. Johnson, R. C., and B. Ely. 1977. Isolation of spontaneous derived mutants of Caulobacter crescentus. Genetics 86:25-32. 11. Johnson, R. C., N. B. Wood, and B. Ely. 1977. Isolation and characterization of bacteriophages for Caulobacter crescentus. J. Gen. Virol. 37:323-335. 12. Kurn, N., S. Ammer, and L. Shapiro. 1974. A pleiotropic mutation affecting expression of polar development events in Caulobacter crescentus. Proc. Natl. Acad. Sci. U.S.A. 71:3157-3161. 13. Lagenaur, C., and N. Agabian. 1976. Physical characterization of Caulobacter crescentus flagella. J. Bacteriol. 128:435-444. 14. Lagenaur, C., and N. Agabian. 1978. Caulobacter flagellar organelle: synthesis, compartmentation, and assembly. J. Bacteriol. 135:1062-1069. 15. Lagenaur, C., S. Farmer, and N. Agabian. 1977. Adsorption properties of stage-specific Caulobacter phage 4CbK. Virology 77:401-407. 16. Marino, W., S. Ammer, and L. Shapiro. 1976. Conditional surface structure mutants of Caulobacter crescentus: temperature-sensitive flagella formation due to an altered flagellin monomer. J. Mol. Biol. 107:115-130. 17. Osley, M. A., M. Sheffery, and A. Newvton. 1977. Regulation of flagellin synthesis in the cell cycle of Caulobacter: dependence on DNA replication. Cell 12: 393-400. 18. Poindexter, J. S., P. R. Hornack, and P. A. Armstrong. 1967. Intracellular development of a large DNA bacteriophage lytic for Caulobacter crescentus. Arch. Mikrobiol. 59:237-246. 19. Schmidt, J. M. 1966. Observations on the adsorption of Caulobacter bacteriophages containing ribonucleic acid. J. Gen. Microbiol. 45:347-353. 20. Shapiro, L 1976. Differentiation in the Caulobacter cell cycle. Annu. Rev. Microbiol. 30:377407. 21. Shapiro, L, and J. V. Maizel, Jr. 1973. Synthesis and structure of Caulobacter crescentus flagella. J. Bacteriol. 113:478-485. 22. Sheffery, M., and A. Newton. 1977. Reconstitution and purification of flagellar filaments from Caulobacter crescentus. J. Bacteriol. 132:1027-1030. 23. Silverman, M., and M. Simon. 1973. Genetic analysis of bacteriophage Mu-induced flagellar mutants in Escherichia coli. J. Bacteriol. 116:114-122. 24. Silverman, M., and M. Simon. 1977. Bacterial flagella. Annu. Rev. Microbiol. 31:397419. 25. Stove, J. L., and R. Y. Stanier. 1962. Cellular differentiation in stalked bacteria. Nature (London) 196: 1189-1192.
