Several of these strains are +CbK resistant and produce a ... of the 25-kDa flagellins; (iv)flaF is required for filament assembly; and (v) +CbK resistance results.
JOURNAL OF BACTERIOLOGY, Oct. 1992, p. 6046-6053
Vol. 174, No. 19
0021-9193/92/196046-08$02.00/0 Copyright X) 1992, American Society for Microbiology
The Caulobacter crescentus flaFG Region Regulates Synthesis and Assembly of Flagellin Proteins Encoded by Two Genetically Unlinked Gene Clusters PATRICIA V. SCHOENLEIN,t JUDY LUI, LILLY GALLMAN,4 AND BERT ELY* Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208 Received 23 March 1992/Accepted 28 July 1992
At a specific time in the Caulobacter crescentus cell cycle, a single flagellar ifiament and multiple receptor sites for the swarmer-specific phage +Cbk are assembled at one pole of the predivisional cell. One cluster of genes required for this morphogenesis, theflaYG region, includes theflgJKL genes, which encode structural proteins of the flageliar filament. These flageilin genes are flanked by genes required for filament assembly, theflaYE genes at one end and theflaF-flbT-flbA-flaG genes at the other. In this study, we characterized mutants carrying large chromosomal deletions within this region. Several of these strains are +CbK resistant and produce a novel 22-kDa flagellin that is not assembled into flagella. Merodiploid strains containing either the entireJfaFG region or individual Jfi transcription units from this region were constructed. These strains were used to correlate the presence or absence of specific gene products to changes in flagellin synthesis, filament assembly, or phage sensitivity. As a result of these studies, we were able to conclude that (i) the production of the 22-kDa flageilin results from the absence of the flbA and faG gene products, which appear to be components of a flageilin-processing pathway common to the 25-, 27-, and 29-kDa flageilins; (ii) lbT negatively modulates the synthesis of the 27- and 25-kDa flagellins from two genetically unlinked gene clusters; (iii) flgL is the only flagellin gene able to encode the 27-kDa flagellin, and this flagellin appears to be required for the efficient assembly of the 25-kDa flagellins; (iv)flaF is required for filament assembly; and (v) +CbK resistance results from the deletion of at least two genes in theflaFG region. The life cycle of Caulobacter crescentus provides a model system for the analysis of programmed developmental events. During each DNA replication cycle, a predivisional cell divides asymmetrically to produce two cell types with distinct morphologies, a motile swarmer cell and a sessile stalked cell. Prior to cell division, flagellum biogenesis is initiated in the predivisional cell. At least 40 genes are required for the synthesis and assembly of a functional polar flagellar organelle (6). This complex organelle consists of a basal complex and a hook and rod structure, onto which flagellin proteins are assembled into a filament. A 29-kDa flagellin is initially assembled at the hook-proximal portion of the filament (3), after which the 27- and the 25-kDa proteins are successively assembled (16, 19, 34). The 25-kDa flagellin constitutes the distal two-thirds of the filament. The basal complex itself is embedded in the cell envelope by a series of protein rings. Six flagellin genes (fig) constitute a multigene family localized to two genetically unlinked loci, a and 13 (Fig. 1) (11). The flgJ (29-kDa flagellin), flgK (25-kDa flagellin), and flgL (27-kDa flagellin) genes comprise the a cluster of flagellin genes and are located within the flaYG gene cluster. This gene cluster also contains the flaY, flaE, flaF, flbT, and flbA-flaG transcription units. Previous genetic and physical studies (28, 29) and sequence analysis defined the gene order in this region as flaY-flaE-flgJ-flgK-flgL-flaF-flbT-flbA-flaG (Fig. 2). A second set of flagellin genes, the beta cluster, is *
located approximately 1,000 kb from the alpha cluster (Fig. 1) (6). No other flagellar genes have been found at this locus. During flagellum biogenesis, 4CbK phage receptor sites are also synthesized at the flagellar pole. C. crescentus cells are sensitive to the 4CbK phage during the period of motility and lose phage sensitivity concomitantly with the loss of the flagellum. The CbK phage receptor has not been identified, and its association with the flagellar organelle, if any, is not known. However, an intact flagellar filament is not required for infection, since most fla mutants are sensitive to the phage even though plaque-forming efficiency is reduced (2, 9). Screening 4CbK-resistant mutants for the concomitant loss of the ability to swim has resulted in the isolation of pleiotropic (ple) mutations (5, 10, 14). The ple mutations were localized to three specific regions of the C. crescentus chromosome, with the pleB mutations in strains SC603, SC604, SC613, and SC533 located in the vicinity of theflaYG gene cluster (5). Further studies demonstrated that thepleB mutations are deletions which affect the flaYG gene cluster (8). Several other deletions in the flaYG gene cluster which also result in the loss of motility have been identified in previous studies (14, 15, 26). Two of these strains, SC507 (fla-152) and SC514 (fla-158) produce large amounts of a 22-kDa flagellin species that is not assembled into filaments. In this study, Southern analyses and complementation studies have been used to define the extent of each of the deletions described above. In addition, the analysis of merodiploid strains demonstrated that the 22-kDa flagellin arises from the absence of either the flbT or the flbA-flaG gene products. Similarly, flgL was shown to code for the 27-kDa flagellin, and the flaF product was shown to be required for assembly of the flagellar filament. In addition,
Corresponding author.
t Present address: Medical College of Georgia, Department of
Cellular Biology and Anatomy, Augusta, GA 30912. t Present address: South Carolina Law Enforcement Division, Columbia, SC 29210.
6046
C. CRESCENTUS flaFG REGION
VOL. 174, 1992
6047
TABLE 1. C. crescentus strains used in this study Relevant genotype
Reference or source
Wild type flbT650 flaF132 flaG133 flaF134 fla-152 fla-156 fla-158 fla-1S9 fla-672 (formerly pleB109) flaF673 (formerly pleB132) fla-674 (formerly pleB133) fla-676 (formerly pleB142) flaG617::TnS proA103 str-140 flbA604::TnS proA103 str-140
25 14 14 14 14 14 14 14 14 Spontaneous in CB15 Spontaneous in CB15 Spontaneous in CB15
Strain
CB15
SC276 SC279 SC280 SC282
SC507 SC512 SC514
f lgM-- a iOOO motA
rrnB
3000
SC515 SC533 SC603 SC604 SC613
Mato f laRZ
SC1047 SC1065
metF pheA
Spontaneous in CB15 4 4
cheR i5cO R500
2500
serA
hun6 f 1bQ
f laA leuA
trpB
mM leucine, 0.05 mM thiamine, 0.3 mM threonine, and 1.0 mM proline when necessary. Complex motility medium consisted of half-strength PYE and 0.3% agar (14). The following antibiotic concentrations were used to select for recombinant plasmids: ampicillin, 100 mg/liter; streptomycin, 50 mg/liter in PYE solid medium and 200 mg/liter in LB; kanamycin, 50 mg/liter; sulfanilamide, 300 mg/liter; and tetracycline, 1 mg/liter in PYE or 5 mg/liter in LB. As reported previously, optimal growth of C. crescentus in liquid medium requires a reduction in the concentrations of streptomycin and kanamycin to 10 mg/liter each (28). The pleB mutants were more sensitive to sulfanilamide and were grown with sulfanilamide concentrations of 75 mg/liter. Analysis of merodiploid strains. The mutants originally designated pleB mutants do not grow well on defined (phosphate-imidazole-glucose) medium, especially when they harbor the flaFG region on a recombinant cloning vector. Therefore, bacterial matings with these mutants as recipients were performed on complex nonselective medium by streaking a pipette tip dipped in an overnight culture of the E. coli donor onto a PYE plate. The pleB recipient was then
his 2000
rrnA
FIG. 1. A genetic map of the C. crescentus chromosome, showing the locations of the flagellar genes. BB, basal body gene cluster; hook, hook gene cluster.
we demonstrated that 4CbK resistance results from the deletion of at least two genes in the flaFG region.
MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Tables 1 and 2, respectively. C crescentus wild-type strain CB15 and mutant strains were grown at 30°C in liquid peptone-yeast extract (PYE) medium or in a phosphateimidazole-glucose medium (29). Escherichia coli strains were grown at 37°C in either complex LB medium (22) or defined E medium (33), which was supplemented with 0.2
pPVS 149
m flaE
flaY Sa Ba
Rla
II
,, I
Ha
fIgJ
fIgK
f9L
29kd
25kd
27kd
II Pb Aa
I
Pa
SCRb II
Sb
Pc
SdI RC II
PdTa
-1 pPVS 162 -1 pPVS 150,151 pPVS 152,153
bflaF Rd I Se La BbI ILb I' I I l'IIPe I C HbAbAc Pe Pf HCPg
BcSf Re
L
11
PhTb
1 Kb
FIG. 2. Physical map of the flaYG gene cluster. The locations of deletion mutations are shown below the physical map, and plasmids containing cloned fragments are depicted above the map. Deletionsfla-152 (SC507),fla-156 (SC512),fla-158 (SC514), andfla-159 (SC515) were isolated as flagellar mutations, while deletions fla-672 (SC533), fla-673 (SC603), fla-674 (SC6054), and fla-676 (SC613) were originally designated pleB mutations. Abbreviations: A, HpaI; B, BamHI; C, ClaI; H, HindIII; L, BglII; P, PstI; R, EcoRI; S, Sail; T, SstI.
6048
Plasmid
pPVS149 pPVS150
pPVS151 pPVS152 pPVS153 pPVS162 pPVS163 pPVS164
pPVSi9O
SCHOENLEIN ET AL.
J. BA=RIOL.
TABLE 2. Plasmids used in this study Construction 2.4-kb BamHI-SstI fragment containing flbT and flaF transcription units replacing BamHI-SstI region of pKT230; flbA is proximal to the amp promoter of pKT230 3.2-kb EcoRI flbA-flaG region cloned into EcoRI site of pKT230; flbA is proximal to the amp promoter of pKT230 3.2-kb EcoRI flbA-flaG region cloned into EcoRI site of pKT230; flaG is proximal to the amp promoter of pKT230 0.8-kb EcoRI flaF region cloned into EcoRI site of pKT230; HindIII site is proximal to the amp promoter of pKT230 0.8-kb EcoRI flaF region cloned into EcoRI site of pKT230; HindIII site is distal to the amp promoter of pKT230 3.0-kb HpaI-EcoRI flbA-flaG region replacing HpaI-EcoRI region of pKT230; flbA is proximal to the amp promoter of pKT230 4.0-kb SstI flaFG region cloned into SstI site of pKT230; flaG is proximal to the amp promoter of pKT230 4.0-kb Sstl flaFG region cloned into SstI site of pKT230; flaF is proximal to the amp promoter of pKT230 4.0-kb SstI flaFG region containing a deletion of 0.7 kb in flbT region between EcoRIb and BglII. sites; flaF is proximal to the amp promoter of pKT230
streaked directly over the E. coli donor. These mating mixtures were incubated at 30°C for 6 h or overnight. Bacteria from the area of growth were streaked for single colonies on a PYE plate containing kanamycin to select bacterial colonies that contained the pKT230 recombinant plasmids. C. crescentus colonies were identified by their distinctive colony morphology and streaked onto PYEkanamycin medium for further purification. After purification, C. crescentus colonies containing recombinant clones were transferred with sterile toothpicks into motility medium to test for the ability to form concentric swarms at 23°C. Because pseudoreversion, the generation of second-site mutations, has been observed in several fla mutant strains containing large deletion mutations in the flaYG gene cluster (27), we analyzed plasmid-free segregants to confirm that the swarming ability of all merodiploid strains was lost concomitantly with antibiotic resistance. The ability of recombinant clones to restore phage sensitivity to the pleB mutants was determined by the protocol described by Johnson and Ely (14) except that swarmer cell-specific +CbK phage were spotted on the indicator lawns at 107, 105, 10 , and 102 PFU/ml. Recombinant DNA techniques and Southern analyses. C. crescentus chromosomal DNA isolation procedures have been described previously (1). Procedures for Southern blots and hybridizations, agarose and acrylamide gel electrophoresis, nick translation, and restriction mapping were performed as described by Maniatis et al. (21). The locations and extents of the deletion mutations were determined by either agarose or polyacrylamide gel electrophoresis and Southern hybridization. DNA fragments fractionated on polyacrylamide gels were electrotransferred to nitrocellulose as described by Smith et al. (31). Characterization of intracellular and assembled flagellins. Assembled flagellar structures were characterized by electron microscopy (14) and phase contrast microscopy after flagellar staining (13). Released flagellar filaments were purified from culture fluids, and their flagellin components were characterized as described before (28). Flagellin proteins were analyzed by immunoprecipitation studies or Western (immunoblot) analyses of log-phase cultures as described previously (28). Immunoprecipitation studies could not be performed on the mutant strains SC533, SC603, SC604, and SC613 because of their inability to be
Reference
28 28 28 28 28
28 28 28 28
lysed with Triton X-100 when they contained plasmid pKT230 derivatives. Therefore, the merodiploid strains resulting from complementation analyses were analyzed by Western blot procedures. RESULTS Mapping deletion mutations within theflaYG gene cluster of the C. crescentus chromosome. We have recently cloned and characterized the 4.0-kb flaFG region, which contains the flaF, flbT, and flbA-flaG transcription units (29). As a follow-up to these studies, the integrity of each of these genes was investigated in all strains containing chromosomal deletions that previous studies had localized to this region (Table 1). Southern analyses with a variety of DNA fragments as probes were used to determine the endpoints of each of the deletion mutations in strains SC507, SC512, SC514, and SC515. These deletions were 3 to 6 kb in length and located entirely within the flaYG gene cluster (Fig. 2). All of these deletions had one end in the flaYE region, as shown in previous studies (26). The distal ends of the fla-156 (SC512)
and fla-159 (SC515) deletions were located in the flgK and flaF genes, respectively. The fla-152 deletion (SC507) extended into the flbT gene, and the fla-158 deletion (SC514) extended beyondflbT and into theflbA gene. In contrast, the pleB mutants contained larger deletions with only a single endpoint within the flaYG region. When SC533, SC603, SC604, and SC613 DNAs were cleaved with the restriction endonuclease BglII, Southern analysis demonstrated that each of these deletions resulted in the loss of at least 10 kb of chromosomal DNA. Subsequent analysis by pulsed-field gel electrophoresis demonstrated that the distal endpoints of these deletions were also unique and that the deletions ranged from 10 to 17 kb in size (8). Each of the deletions in these strains removes the flbA-flaG and flbT transcription units. In addition, the deletion in SC603 removes at least 0.4 kb of the flaF gene, while the deletions in SC613 and SC533 remove the entireflaF gene and part of theflgL gene (Fig. 2). Since each of these deletions spans at least two flagellar genes and could be complemented by the clones containing the flaYG region (see below), we redesignated the pleB mutations in SC533, SC603, SC604, and SC613 fla-672, fla-673, fla-674, and fla-676, respectively. Characterization of the flagellin patterns of mutant strains.
VOL. 174, 1992
C. CRESCENTUS flaFG REGION
6049
TABLE 3. Flagellin species detected in C. crescentus fla mutantsa Flagellin producedb
Relevant genotype
Strain
29 kDa
27 kDa
25 kDa
24 kDa
22 kDa
++
++
CB15 CB15 SC279 SC282 SC276 SC1065 SC1047 SC1062 SC288 SC512 SC515
Wild type Wild typec flaF flaF flbT
+ + ++ ++
+ + ++
+ +
++
++
++
++
++
flbA AflaG
-
R R R R R
-
+ +
SC603
A(flaG flbATflaF) A(fIaG flbA 7)
R R R -
flaE flaY
A(flaKTflaYE)
A(flaFflgLKJflaEY) A(flaGflbATflaFflgL)
SC533
SC604
-
+-
+ -
-
-
-
++ ++ ++ ++ ++ ++
A(flaGflbATflaFflgL) A(flbATflaFflgLKJflaE) ++ ++ A(flbTflaFflgLKTflaE) Immunoprecipitation studies or Western analyses with antiflagellum antibody were performed on each mutant as described in Materials and Methods. b Symbols: +, wild-type amount; -, nondetectable amount; +, trace (almost nondetectable) amount; + +, greater-than-wild-type amount or large amount of
SC613 SC514 SC507 a
a flageilin species (22 kDa) not detected in the wild-type CB15 control; R, lower-than-wild-type amount. c Wild type grown to stationary phase (refer to Materials and Methods).
To determine whether the deletion of specific genes in the
flaFG region resulted in changed patterns of flagellin synthesis, Western analyses and immunoprecipitation studies were performed on all the deletion strains (Table 3). For comparative purposes, the wild-type strain CB15 and strains containing single mutations in the flaYG gene cluster were also analyzed. The 27- and 29-kDa flagellins were not detected in strains SC507, SC514, SC533, SC603, SC604, and SC613. Interestingly, each of these strains produced large amounts of the 22-kDa flagellin species (Table 3). The 22-kDa flagellin is not assembled into the flagellar filament and is not synthesized by wild-type cells in detectable amounts except during the stationary phase (Table 3 and Fig. 3). In addition, SC507 also overproduced the 25-kDa flagellin and a 24-kDa flagellin species, while all of the other strains produced only trace or nondetectable amounts of the 25-kDa flagellin. The overall rates of flagellin synthesis in these strains were higher than they were in either wild-type CB15 or the flaG mutant SC1047, which produces small amounts of the 22-kDa flagellin and trace amounts of the 25-kDa flagellin (Table 3). In contrast, deletion strains SC512 and SC515 did not produce detectable levels of the 22- or 24-kDa flagellins. SC512 produced low levels of the 27- and 25-kDa flagellins, while le t t et
0
1v1
en
1-
tn 1-l
(0(0(0eD10 a U) (D U) Cn 3
0
qt
(
29 kd
25
kd
24
kd
-22 kd
FIG. 3. Western analyses of the flagellin proteins produced by deletion strains containing the 4.0-kb SstI flaFG region (pPVS164). wt, wild type; wt 24 hr, wild type grown to stationary phase.
SC515 produced only trace levels of the 25-kDa flagellin (data not shown). In each strain, the trace amounts of the 25-kDa flagellin were consistently detected by immunoprecipitations studies but were not detected by Western analyses. The flagellin patterns we obtained with SC603, SC507, SC512, and SC514 are consistent with previous analyses of these strains (26). The altered flagellin patterns of each of these strains were correlated to the locations of their respective deletion mutations (Fig. 2 and Table 3), and the following observations were made. (i) The 22-kDa flagellin species was produced only in strains containing deletions that extend into the flbT or the flbA-flaG transcription unit; compare SC515 (fla-159) and SC512 (fla-156) with SC514 (fla-158), SC507 (fla-152), SC603 (fla-673), SC604 (fla-674), SC613 (fla-676), and SC533 (fla-672). (ii) The absence or presence of the a cluster of flagellin genes and of the flaY orflaE gene does not alter the production of the 22-kDa flagellin species; compare SC507 (fla-152) and SC514 (fla-158) with SC603 (fla-673), SC604 (fla-674), SC613 (fla-676), and SC533 (fla-672). (iii) The presence of functional flbA and flaG gene products appears to be essential for the production of the 25-kDa flagellin synthesized from the genetically unlinked 13 cluster of flagellins. For instance, both SC507 and SC514 contain deletions of the a cluster of flagellins. However, only SC507, which contains a functional flbA-flaG transcription unit, produces a 25-kDa flagellin from the 13 cluster. Complementation of deletion mutations by thejlaFG region and the role offlgL in flagellin assembly. Thefla-672 (SC533), fla-673 (SC603), fla-674 (SC604), and fla-676 (SC613) deletions extend at least 5.0 kb beyond the SstIb site of theflaFG region (Fig. 2). Therefore, the alterations in flagellin synthesis, the defects in filament assembly, and/or the 4CbK resistance of these strains could result from the deletion of unidentified genes beyond theflaG gene. To test this hypothesis, the 4.0-kb SstI fragment containing functional flaF, flbT, and flbA-flaG transcription units (pPVS164) was introduced into each of these deletion strains. In each case, plasmid pPVS164 restored motility, the ability to swarm, and 4)CbK sensitivity to the resulting merodiploid strains (Table
6050
SCHOENLEIN ET AL.
J. BAcTERIOL.
TABLE 4. Complementation analysis of deletion mutations Gene(s) provided in trans
Complementationa SC533 SC603 SC604 SC613
(fla-672) (fla-673) (fla-674) (fla-676) ++ flaF, flbT, flbA, flaG (pPVS164) ++ ++ ++ flbA, flaG (pPVS150) S S S + flbA, flaG (pPVS151) S S S + flbA, flaG (pPVS162) S S + S flbT, flbA (pPVS149) flaF (pPVS152) flaF (pPVS153) a Symbols: + +, merodiploid strains were +CbK sensitive and motile and could swarm in motility medium; +, merodiploid strains were motile and 4CbK sensitive but could not swarm in semisolid medium; S, merodiploid strains assembled a short, straight filament (stub) and were 4CbK resistant; -, merodiploid strains were 4CbK resistant, unable to swim, and unable to swarm in motility medium.
4). Thus, no genes affecting motility or 4)CbK sensitivity are located in the 5-kb region adjacent to the flaG gene. As expected, in control experiments, plasmid pPVS164 did not restore motility to SC507 (fla-152), SC514 (fla-158), or SC515 (fla-159), since the deletions in these strains extend beyond the flaFG region into the flaE gene (Fig. 2). The function of the flgL gene in both flagellin synthesis and filament assembly was examined directly in two of the merodiploid strains, SC533(pPVS164) and SC613(pPVS164). These strains do not contain a functional flgL gene because they contain chromosomal deletions that remove part of the flgL gene, and only the 5' end of the flgL gene is located within the 4.0-kb SstI fragment contained in pPVS164 (Fig. 2). Since flgL encodes the 27-kDa flagellin protein (23, 30), these motile strains are either assembling a flagellar filament without the 27-kDa flagellin protein or producing a 27-kDa flagellin originating from one of the other flg genes in the flagellin multigene family. Western analysis did not detect the 27-kDa flagellin protein in these merodiploid strains, but the 29- and 25-kDa flagellins were present in normal amounts compared with the intracellular flagellin profile of wild-type CB15 containing pPVS164 (Fig. 3, lanes 1 and 4). When similar analyses were performed on the merodiploid strains SC603(pPVS164) and SC604(pPVS164) (Fig. 3, lanes 2 and 3), which contain chromosomal deletions that do not extend into the flgL gene, the 27-kDa flagellin protein was detected in amounts comparable to those in CB15(pPVS164) (Fig. 3, lane 5). Thus, the absence of the 27-kDa flagellin in the merodiploids SC533(pPVS164) and SC613(pPVS164) is due to the loss of the flgL gene and not to the deleted region beyond the SstIb site. Furthermore, these data provide strong evidence that flgL is the only flagellin gene that encodes the 27-kDa flagellin and demonstrate that swarmer cells can assemble a flagellar filament in the absence of this flagellin. In addition, the absence of the 27-kDa flagellin did not affect synthesis of the 25- or 29-kDa flagellin. To confirm the absence of the 27-kDa flagellin in the assembled flagella of the merodiploid strain SC613 (pPVS164), flagellar filaments were isolated from this strain and analyzed for flagellin composition. The 27-kDa flagellin was not detected in these filaments, and there appeared to be a 20-fold reduction in the amount of assembled 25-kDa flagellin relative to the amount of the hook protein (Fig. 4). Similar results were obtained for SC533(pPVS164) (data not shown). Electron microscopy of logarithmically growing broth cultures determined that many of the swarmer cells
ID
~a
cn
CO, U-
U1) ...I
--hook
27 kd flagellin 25kd flagellin
FIG. 4. Flagellin proteins isolated from released flagellar filaments of SC613(pPVS164) compared with those from wild-type (wt) cells.
assembled short filaments (two to five sine waves) or did not assemble detectable filaments. However, some normallength flagella (seven to eight sine waves) were observed. Strains SC603(pPVS164), SC604(pPVS164), and CB15 (pPVS164), which synthesize reduced amounts of the 27kDa flagellin relative to strain CB15 containing no plasmid (Fig. 3), also assembled abbreviated filaments. Thus, although a filament can be assembled when 27-kDa flagellin synthesis is reduced or absent, these data demonstrate that inefficient assembly of the 25-kDa flagellin occurs. Regulation of the a and 13 clusters of flagellin genes through the trans-acting effects of thepfbA-JpaG andflbT gene products. In each of the merodiploids described above, the flaFG region (pPVS164) restored normal flagellin synthesis patterns, with concomitant loss of 22-kDa flagellin synthesis. To test how theflbA andflaG gene products affected this change in flagellin synthesis patterns, the flbA-flaG transcription unit (pPVS150, pPVS151, and/or pPVS162) was provided to each of these strains, and Western analyses were performed on the resulting merodiploids. None of these recombinant plasmids contained a functional flaF or flbT gene (Fig. 2). Merodiploid strains of SC603 and SC604 containing the flbA-flaG transcription unit in trans showed a dramatic reduction in the amount of the 22-kDa flagellin produced and the concomitant synthesis of the 25-, 27-, and 29-kDa flagellins (Fig. 5, lanes 1, 2, 3, and 6, and data not shown). Thus, the flbA-flaG transcription unit is required for the synthesis of the assembled flagellins, and loss of these gene products results in the production of the 22-kDa flagellin. Neither the flaF gene by itself (pPVS153) nor theflaF and flbTgenes together (pPVS149) restored the production of the wild-type flagellins to SC603, SC604, SC533, or SC613 (Fig. 6 and data not shown). However, providing an intact flbT gene (pPVS149) to each of these strains resulted in a significant reduction in the 22-kDa flagellin level without the appearance of new flagellin species. Similarly, providing an extra copy of the flbT gene to wild-type cells resulted in a reduction in the overall level of flagellin synthesis (Fig. 3, lane 5, and data not shown). Therefore, FlbT regulates the actual level of flagellin synthesis, but the species of flagellin proteins produced depends on whether the flbA and flaG gene products are functional. In their absence, only the 22-kDa flagellin is synthesized. The results described above demonstrate that FlbT regu-
VOL. 174, 1992 LO
v: 0
U)
0
U)
In v) 0.
U)
_j;W
(D u)
0
(0
U)
_ to
C. CRESCENTUS flaFG REGION
u
't
3
4
>
CL
CL
_
LOCO
U) UO CD
>> X X3 XL 0. CL0ew
>
L
Uqr v)4
U UCOt)U)COCl)
_
-
29kd
27kd
24kd
FIG. 5. Western analysis of strain SC604 containing the following transcription units in trans: the fibA-flaG transcription unit (pPVS150, pPVS151, and pPVS162); and the flaF, flbT, and flbA-flaG transcription units (pPVS164). SC604 contains a deletion spanning the flbT, flbA, and flaG genes. SC279 contains a point mutation in flaF. Extracts from log-phase and 24-h cultures of the wild-type (wt) strain were included as controls. lates the expression offlgL, the a cluster flagellin gene which
encodes the 27-kDa flagellin. However, the 25-kDa flagellin is encoded by both the a cluster flgK gene and the ,B cluster flagellin genes (7). To demonstrate that FlbT regulates the expression of the ,B cluster flagellin genes, similar analyses of the merodiploid strain SC514 containing the flbA-flaG transcription unit in trans (pPVS150, pPVS151, or pPVS162) were performed. Providing theflbA-flaG transcription unit to SC514 significantly reduced the level of the 22-kDa flagellin species and restored synthesis of the 25-kDa flagellin (Fig. 7, lanes 2, 3, and 4). Small amounts of a 24-kDa flagellin were also produced by these strains, in contrast to SC514 (pPVS164), which contained a functional flbT gene and therefore had reduced levels of flagellin synthesis (Fig. 7, lane 1). These flagellins must be produced from the ,B cluster of flagellin genes because all of the a cluster flagellin genes are deleted in SC514. Thus, the flbA and flaG gene products regulate the species of flagellin synthesized and theflbT gene product regulates the amount of flagellin synthesis from the ,B cluster as well as from the a cluster. In contrast, when the flbA-flaG transcription unit (pPVS150 and pPVS162) was present in trans in strain SC507, the flagellin species produced were not altered (Fig. 7, lanes 6 and 7). This result was expected because the chromosomal deletion in SC507 (fla152) does not enter the flbA-flaG transcription unit. However, when the entire flaFG region was provided in trans, the overall level of flagellin synthesis was reduced and the 22and 24-kDa flagellins were no longer detectable because a functional flbT gene was present along with extra copies of the flbA and flaG genes (Fig. 7, lane 8).
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n>
CL
>
CL CL m _n
cL
.0 .0
0
U
U
OU)
U(D
:
-- t
.
...... :
-
~~~~~~29kd ~~~~27kd ~~~25kd
t___2kd .....
FIG. 7. Flagellin profiles by Western analysis of strains SC507 (fla-152) and SC514 (fla-158) containing the following transcription units in trans: the flbA-flaG transcription unit (pPVS150, pPVS151, and pPVS162), the flaF and flbT genes (pPVS152), and the 4.0-kb SstI flaF-flbT-flbA-flaG region (pPVS164).
Role ofJlaF in filament assembly and flagellin gene expression. The flbA-flaG transcription unit restored the full complement of wild-type flagellins to SC603 and SC604 (see above). Therefore, we characterized filament assembly and function and 4CbK sensitivity patterns in these strains to determine how the absence of the flaF and/or flbT gene affected these phenotypes. Strain SC604, containing the flbA-flaG transcription unit (pPVS150, pPVS151, or pPVS 162), was motile and 4CbK sensitive (Table 4). However, these strains were less sensitive to 4CbK than when the full transcription unit was present, and they were unable to swarm in motility medium, indicating a chemotaxis defect. This phenotype is exactly that observed for an flbT mutant and was expected, since all three of these merodiploid strains lack a functional flbT gene. In addition, flagellar stains showed that many filaments were three to five sine waves in length, in contrast to filaments seven and eight sine waves in length produced by cultures of CB15 containing any of these recombinant plasmids (data not shown). A similar flagellar proffle has been seen in cultures of the flbT mutant SC276 (28). In contrast, when SC533, SC603, SC604, or SC613 harbored the flbA-flaG transcription unit in trans, the resulting merodiploid strains were not motile but did assemble a very short, straight filament (stub) (Table 4). Western analyses of these merodiploid strains indicated that they synthesize similar levels of the 25-kDa flagellins and that SC603 and SC604, which have an intact flgL gene, synthesize similar levels of 27-kDa flagellin (Fig. 8). However, only the SC604 merodiploid strains assembled a functional flagellum, indicating that the absence of the flaF gene in SC603 must prevent the assembly of the flagellins into filaments.
Re
CvI) CII)
LO
It
CVO
=
4t
U) U) 29
kd"\
27 kd -_ 25 kd22 kd/
FIG. 6. Western analysis of the pleB mutant strains SC533 and SC603, containing the flaF and flbT genes on plasmid pPVS149.
N
3:
_._
_
U) U)
U)
o X X n 0.0. 0.0. CO) It e# t X O (O - 0 Xl (9 0
0000U (0 0(0(
00'-X
X
V-
U)
U)
)
U)
L.
a0. 29 kd
..
_
__-
SI
27 kd -25 kd
222 kd
FIG. 8. Western analysis of strains SC603, SC604, SC613, and SC533 in the presence and absence of the flbA-flaG region cloned into pPVS150. Fla prep, flagellins obtained from isolated flagellar
filaments.
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J. BACTERIOL.
SCHOENLEIN ET AL. 29 Kd
27 Kd
FlbA FlaG FlaA FlaR
FlbT
24 Kd 22 Kd
25 Kd
FIG. 9. Proposed relationship between the flagellins found in the flagellar filament and the 22- and 24-kDa flagellins. It is not clear whether the 22- and 24-kDa flagellins are precursors to or breakdown products of the other flagellins. However, overproduction of flagellins resulting from loss of theflbT gene or loss of the fbA-fiaG, flaA, or flaR transcription unit results in the appearance of the 22and 24-kDa flagellins.
DISCUSSION In this study, the roles of theflgL,flaF,flbT, andflbA-flaG transcription units in flagellum biogenesis and the origin of the novel 22- and 24-kDa flagellins were investigated. Deletion mutations within the flaYG gene cluster resulted in altered patterns and levels of flagellin synthesis (Table 3) and either partial or complete resistance to the polar-specific phage +CbK. For the four deletion strains originally identified as pleB mutants, providing the 4.0-kb SstI flaFG region in trans restored wild-type flagellin patterns, motility, ability to swarm in motility medium, and sensitivity to 4CbK phage (Table 4). Strains deleted for the flgL gene did not produce detectable levels of the 27-kDa flagellin (Fig. 3) but were able to assemble flagellar filaments if all of the other flagellar genes were functional. Thus, flagellum assembly can occur in the absence offlgL expression. However, further analysis of the assembled filaments demonstrated that the 25-kDa flagellin produced in these strains was not assembled efficiently (Fig. 4), resulting in shorter filaments. Similar abbreviated filaments have been observed in otherfla mutants that have reduced levels of the 27-kDa flagellin (19, 34). Therefore, 25-kDa flagellin assembly is facilitated by the prior assembly of the 27-kDa flagellin. Strains that lacked a functionalflbA-flaG transcription unit produced only the 22-kDa flagellin in significant quantities. When these strains did not contain a functional flbT gene as well, large amounts of this flagellin were detected. In contrast, merodiploid strains lacking only a functional flbT gene produced the full complement of wild-type flagellins, in addition to a 22- and a 24-kDa flagellin. In these strains, the 25-kDa flagellin, and the 27-kDa flagellin if theflgL gene was present, was overproduced. This flbT phenotype is consistent with that described for the flbT mutant SC276 in a previous study (28). In some of the strains analyzed, the entire at cluster of flagellins was deleted. Thus, the 22-, 24-, and 25-kDa flagellin species present in these strains must have been produced by a gene(s) of the cluster. Nevertheless, theflbA-flaG andflbT transcription units regulated their synthesis in the same manner as they regulated that of the 29- and 27-kDa flagellins from the a cluster. Taken together, these results demonstrate that the flbA-flaG transcription unit encodes proteins that are required for either the production or maintenance of the 29-, 27-, and 25-kDa flagellins, while FlbT regulates the level of synthesis of the 25- and 27-kDa flagellins (Fig. 9). The production of the 24- and 22-kDa flagellins in strains lacking a functional flbT gene product or in wild-type cells in stationary phase suggests that the FlbA or FlaG proteins become limiting under these conditions. We confirmed this hypothesis by providing the flbA-flaG transcription unit in trans to the flbT mutant SC276. When a plasmid containing the flbA-flaG transcription unit was present in addition to the chromosomal copy of
the operon, the 22- and 24-kDa flagellins were not present and increased amounts of the 27-kDa flagellin were observed (data not shown). In addition to the flbA, flaG, and flbT mutations, other mutations that are not linked to the flaYG gene cluster result in the production of the 22-kDa flagellin. The flaA and flaR mutations result in flagellin patterns that are similar to those resulting from flbA and flaG mutations (15). Since there appear to be only six flagellin genes and three of these appear to encode the 25-kDa flagellin (7, 11), it is unlikely that unique genes exist for all of the different flagellin species that have been observed. A more plausible explanation for the production of the different flagellin species is the existence of a common "processing" pathway for the flagellin gene products (Fig. 9). In this pathway, the 22- and 24-kDa flagellin species may be either precursors to or breakdown products of the structural flagellins. However, even during short pulse-chase experiments, a product or precursor relationship cannot be seen between the 22-kDa flagellin and the structural flagellins (15, 19). Additional experiments will be required to resolve this apparent paradox. The flaF gene product appears to be required for normal levels of flagellin gene expression from both the a and 13 clusters of flagellins. Thus, point mutations in the flaF gene (SC282 and SC279) result in reduced synthesis of the 25- and 27-kDa flagellins, and the deletion fla-159 (SC515), which is missing the a cluster of flagellins along with the flaF gene, results in reduced synthesis of the P cluster 25-kDa flagellin. However, the requirement for a functionalflaF gene product is abrogated when both the flaF and flbT genes are deleted. In all mutants and merodiploid strains carrying bothflaF and flbT mutations, the flbT phenotype is always dominant and flagellin proteins are overproduced. It is possible that the lowered production of the 25- and 27-kDa flagellin proteins in flaF mutants is due to a feedback mechanism resulting from the requirement for the flaF gene product for filament assembly. In this model, the FlbT protein would be required for feedback to occur in response to the absence of the FlaF protein. Therefore, elimination of FlbT would cause 25- and 27-kDa flagellin production to be unregulated. However, the interactions are probably more complex, since the introduction of a functional flaF gene in SC515 results in increased flagellin synthesis without any demonstrable assembly. It is not clear why deletions occur at a high frequency in the flaYG region. Analyses of spontaneous flagellar mutations at other locations on the chromosome have failed to identify deletions among the mutations at any of these loci (12, 17, 18, 24). Furthermore, analyses of spontaneous ilv and op mutations resulted in the identification of only a single deletion that occurred in the trpFBA region (32, 35). Therefore, spontaneously occurring deletions do not occur at a high frequency in C. crescentus. In contrast, six deletions have been identified among 14 mutations in the flaYG region, and five additional deletions were obtained by screening (CbK-resistant mutants for loss of motility. Thus, there is an unusual propensity for deletion formation in the flaYG region of the chromosome. Initially, we had hypothesized that these deletions might arise from a common endpoint. However, Southern analyses and pulsed-field gel electrophoresis experiments demonstrated that each deletion has a unique set of endpoints. Therefore, some other mechanism must be responsible for deletion formation in this region. The cause of oCbK resistance in the deletion mutants SC507, SC514, SC603, SC604, SC613, and SC533 is somewhat unclear. The only gene common to the deletions
C. CRESCENTUS flaFG REGION
VOL. 174, 1992
causing 4OCbK resistance is flbT (Fig. 2). However, an insertion in the flbT gene (SC276) does not result in (CbK resistance (data not shown). Bender et al. (2) have shown that strains lacking a flagellar filament have reduced sensitivity to this phage. However, our studies have shown that the deletion strains that lack both flaF and flbT (SC507) or flbA-flaG and flbT (SC514, SC603, SC604, SC613, and SC533) are significantly more resistant to the phage than strains containing flagellar mutations affecting a single flagellar gene. Thus, loss of either set of genes has a greater effect than mutations in any one of the individual genes. ACKNOWLEDGMENTS This work was supported by grant DMB-8704112 from the National Science Foundation and Public Health Service grant GM33580 from the National Institutes of Health. REFERENCES 1. Barrett, J. T., R. H. Croft, D. M. Ferber, C. J. Gerardot, P. V. Schoenlein, and B. Ely. 1982. Genetic mapping with Tn5-derived auxotrophs of Caulobacter crescentus. J. Bacteriol. 149:888898. 2. Bender, R. A., C. M. Refson, and E. A. O'Neill. 1989. Role of the flagellum in cell-cycle-dependent expression of bacteriophage receptor activity in Caulobacter crescentus. J. Bacteriol. 171: 1035-1040. 3. Driks, A., R. Bryan, L. Shapiro, and D. DeRosier. 1989. The organization of the Caulobacter crescentus flagellar filament. J. Mol. Biol. 206:627-636. 4. Ely, B., and R. H. Croft. 1982. Transposon mutagenesis in Caulobacter crescentus. J. Bacteriol. 149:620-625. 5. Ely, B., R. H. Croft, and C. J. Gerardot. 1984. Genetic mapping of genes required for motility in Caulobacter crescentus. Genetics 108:523-532. 6. Ely, B., and T. Ely. 1989. Use of pulsed field gel electrophoresis and transposon mutagenesis to estimate the minimal number of genes required for motility in Caulobacter crescentus. Genetics 123:649-654. 7. Ely, B., and T. Ely. 1992. Unpublished data. 8. Ely, B., and C. J. Gerardot. 1988. Use of pulsed field gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome. Gene 68:323-330. 9. Ely, B., and R. C. Johnson. 1977. Generalized transduction in Caulobacter crescentus. Genetics 87:391-399. 10. Fukuda, A., M. Asada, S. Koyasu, H. Yoshida, K. Yaginuma, and Y. Okada. 1981. Regulation of polar morphogenesis in Caulobacter crescentus. J. Bacteriol. 145:559-572. 11. Gill, P. R., and N. Agabian. 1983. The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus. J. Biol. Chem. 258:7395-7401. 12. Hahnenberger, K. M., and L. Shapiro. 1987. Identification of a gene cluster involved in flagellar basal body biogenesis in Caulobacter crescentus. J. Mol. Biol. 194:91-103. 13. Heimbrook, M. E., W. L. Wang, and G. Campbell. 1989. Staining bacterial flagella easily. J. Clin. Microbiol. 27:26122615. 14. Johnson, R. C., and B. Ely. 1979. Analysis of non-motile mutants of the dimorphic bacterium Caulobacter crescentus. J. Bacteriol. 137:627-634. 15. Johnson, R. C., D. M. Ferber, and B. Ely. 1983. Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants. J. Bacteriol. 154:1137-1144.
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16. Johnson, R. C., M. P. Walsh, B. Ely, and L. Shapiro. 1979. Flagellar hook and basal complex of Caulobacter crescentus. J. Bacteriol. 138:984-989. 17. Khambaty, F. M., and B. Ely. 1992. Molecular genetics of the flgI region and its role in flagellum biosynthesis in Caulobacter crescentus. J. Bacteriol. 172:4101-4109. 18. Kidwai, A., and B. Ely. Unpublished data. 19. Koyasu, S. 1984. On flagellar formation in Caulobacter crescentus: novel flagellin synthesis in stub-forming non-motile mutants of C. crescentus. J. Biochem. 96:1351-1364. 20. Koyasu, S., M. Asada, A. Fukada, and Y. Okada. 1981. Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella. J. Mol. Biol. 153:471-475. 21. Maniatis, T., E. F. Fritsch, and J. Sambrook 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 22. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Minnich, S. A., and A. Newton. 1987. Promoter mapping and cell cycle regulation of flagellin gene transcription in Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 84:1142-1146. 24. Ohta, N., E. Swanson, B. Ely, and A. Newton. 1984. Physical mapping and complementation analysis of transposon TnS mutations in Caulobacter crescentus: organization of transcriptional units in the hook gene cluster. J. Bacteriol. 158:897-904. 25. Poindexter, J. S. 1964. Biological properties and classification of the Caulobacter group. Bacteriol. Rev. 28:231-295. 26. Purucker, M., R. Bryan, K. Amemiya, B. Ely, and L. Shapiro. 1982. Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile TnS insertion mutants. Proc. Natl. Acad. Sci. USA 79:6797-6801. 27. Schoenlein, P. V. 1989. The structure of the flaFG region and its role in controlling the temporally-expressed flagellar genes of Caulobacter crescentus. Ph.D. thesis. University of South Carolina, Columbia, S.C. 28. Schoenlein, P. V., and B. Ely. 1989. Characterization of strains containing mutations in the contiguous flaF, flbT, or flbA-flaG transcription units and the identification of a novel Fla phenotype in Caulobacter crescentus. J. Bacteriol. 171:1554-1561. 29. Schoenlein, P. V., L. M. Gallman, and B. Ely. 1989. Organization of the flaFG gene cluster and the identification of two additional genes involved in flagellum biogenesis in Caulobacter crescentus. J. Bacteriol. 171:1544-1553. 30. Schoenlein, P. V., L. S. Gailman, M. E. Winkler, and B. Ely. 1990. Nucleotide sequence analysis of the Caulobacter crescentus flaF and flbT genes and an analysis of codon usage in organisms with GC-rich genomes. Gene 92:17-25. 31. Smith, M. R., C. S. Devine, S. M. Cohn, and M. W. Lieberman. 1984. Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose. Anal. Biochem.
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