Flagellin Structural Gene Research Articles

Aeromonas salmonicida isolated from wild and farmed fish and invertebrates in Oman

Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit fish and tilapia, and an invertebrate (abalone) in Oman during December 2011–May 2012. The cytotoxic enterotoxin ast gene was found widely distributed among the isolates; aerolysin-like protein (act) and the flagellin structural gene lafA less so; and the nuclease gene (nuc) not at all. However, there was not any evidence of pathogenicity among the isolates when tested in laboratory-based experiments using rainbow trout and Nile tilapia. Therefore, the risk of the pathogen to fish in Oman is unclear.

The Small RNA DsrA Influences the Acid Tolerance Response and Virulence of Salmonella enterica Serovar Typhimurium

The Gram-negative, enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) is exposed to various stress conditions during pathogenesis, of which acid stress serves as a major defense mechanism in the host. Such environments are encountered in the stomach and Salmonella containing vacuole of phagocytic and non-phagocytic cells. It is only recently that small RNAs (sRNAs) have come to the forefront as major regulators of stress response networks. Consequently, the sRNA DsrA which regulates acid resistance in Escherichia coli, has not been characterized in the acid tolerance response (ATR) of Salmonella. In this study, we show dsrA to be induced two and threefold under adaptation and challenge phases of the ATR, respectively. Additionally, an isogenic mutant lacking dsrA (ΔDsrA) displayed lower viability under the ATR along with reduced motility, feeble adhesion and defective invasion efficacy in vitro. Expression analysis revealed down regulation of several Salmonella pathogenicity island-1 (SPI-1) effectors in ΔDsrA compared to the wild-type, under SPI-1 inducing conditions. Additionally, our in vivo data revealed ΔDsrA to be unable to cause gut inflammation in C57BL/6 mice at 72 h post infection, although intracellular survival and systemic dissemination remained unaffected. A possible explanation may be the significantly reduced expression of flagellin structural genes fliC and fljB in ΔDsrA, which have been implicated as major proinflammatory determinants. This study serves to highlight the role of sRNAs such as DsrA in both acid tolerance and virulence of S. Typhimurium. Additionally the robust phenotype of non-invasiveness could be exploited in developing SPI-I attenuated S. Typhimurium strains without disrupting SPI-I genes.

A Novel Linear Plasmid Mediates Flagellar Variation in Salmonella Typhi

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljBz66) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage–related linear plasmid to be described in the Enterobacteriaceae.

σ 28 -Dependent Transcription in Salmonella enterica Is Independent of Flagellar Shearing

The FlgM anti-sigma28 factor is secreted in response to flagellar hook-basal body completion to allow sigma28-dependent transcription of genes needed late in flagellar assembly, such as the flagellin structural gene, fliC. A long-standing hypothesis was that one role of FlgM secretion was to allow rapid expression of flagellin in response to shearing. We tested this hypothesis by following FlgM secretion and fliC transcription in response to flagellar shearing. Experiments showed that the level of FlgM inside the cell was unchanged after shearing whereas the extracellular FlgM levels increased in the growth medium as time passed. Identical results were obtained with cells that were not exposed to shear forces: internal FlgM levels remained constant while external FlgM levels rose with time at rates similar to those for the sheared culture. Consistent with this find, FlgM/sigma28-dependent class 3 gene expression was unaffected by flagellar shearing but was affected by the growth phase of the cell. Regardless of exposure to shear forces, flagellar class 3 transcription rose sharply and then declined. These results demonstrate that flagellar regrowth following shearing is independent of FlgM secretion.

Quorum sensing in Yersinia enterocolitica controls swimming and swarming motility.

The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26 degrees C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) or fliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.

Structural, genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori.

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno -n o n-ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.

Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction.

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin. Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.

Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene.

Burkholderia pseudomallei is a human and animal pathogen in tropical regions, especially Southeast Asia and northern Australia. Currently little is known about the genetics and molecular biology of this organism. In this report, we describe the mutagenesis of B. pseudomallei with the transposon Tn5-OT182. B. pseudomallei 1026b transposon mutants were obtained at a frequency of 4.6 x 10(-4) per initial donor cell, and the transposon inserted randomly into the chromosome. We used Tn5-OT182 to identify the flagellin structural gene, fliC. We screened 3,500 transposon mutants and identified 28 motility mutants. Tn5-OT182 integrated into 19 unique genetic loci encoding proteins with homology to Escherichia coli and Salmonella typhimurium flagellar and chemotaxis proteins. Two mutants, MM35 and MM36, contained Tn5-OT182 integrations in fliC. We cloned and sequenced fliC and used it to complement MM35 and MM36 in trans. The fliC transcriptional start site and a sigmaF-like promoter were identified by primer extension analysis. We observed a significant difference in the expression of two distinct fliC-lacZ transcriptional fusions during bacterial growth, suggesting the presence of a latent intragenic transcriptional terminator in fliC. There was no significant difference in the virulence of 1026b compared to that of MM36 in diabetic rats or Syrian hamsters, suggesting that flagella and/or motility are probably not virulence determinants in these animal models of B. pseudomallei infection. A phylogenetic analysis based on the flagellins from a variety of bacterial species supported the recent transfer of B. pseudomallei from the genus Pseudomonas to Burkholderia.

The flk gene of Salmonella typhimurium couples flagellar P- and L-ring assembly to flagellar morphogenesis.

The flagellum of Salmonella typhimurium is assembled in stages, and the negative regulatory protein, FlgM, is able to sense the completion of an intermediate stage of assembly, the basal body-hook (BBH) structure. Mutations in steps leading to the formation of the BBH structure do not express the flagellar filament structural genes, fliC and fljB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regulatory gene, flk, which appears to sense the completion of another assembly stage in the flagellar morphogenic pathway just prior to BBH formation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural genes. Mutations by insertional inactivation in either the flk or flgM locus allow expression of the fljB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fljB gene in strains defective in all of the steps leading to BBH formation. The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk+ allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein, B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene product of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independent of the FlhDC and sigma28 flagellar transcription factors. The Flk protein observed in a T7 RNA polymerase-mediated expression system showed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly hydrophilic protein with a very C-terminal membrane-spanning segment preceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm.

Temperature-dependent regulation of Yersinia enterocolitica Class III flagellar genes.

Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30 degrees C and 37 degrees C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37 degrees C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (sigma F) and flgM (anti-sigma F), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific sigma-factor, sigma F, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-sigma F and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37 degrees C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM mutants, Y. enterocolitica flgM mutants are fully virulent.

Coupling of flagellin gene transcription to flagellar assembly in Bacillus subtilis.

The regulation of flagellin gene expression in Bacillus subtilis was examined in vivo by means of a lacZ translational fusion to the flagellin structural gene (hag). We have tested the effects of two known mutations (flaA4 and flaA15) in the major flagellar operon and of three deletions. One deletion was in frame in the fliI cistron, one was out of frame in the fliK cistron, and the last spanned about 21 kb of the flaA operon. In all instances, the expression of the flagellin gene was defective. Flagellin gene expression was restored in the strain with the 21-kb deletion by overexpression of the sigD gene under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac promoter. These results indicate that transcription of the flagellin gene is dependent on the formation of the flagellar basal body but that such a requirement can be bypassed by overexpression of sigD. Lack of expression of hag was observed in the presence of flaD1, flaD2, and delta sin mutations as well.

Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan.

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.

Comparative analysis of flagellin sequences from Escherichia coli strains possessing serologically distinct flagellar filaments with a shared complex surface pattern

Escherichia coli morphotype E flagellar filaments have a characteristic surface pattern of short-pitch loops when examined by electron microscopy. Seven of the 50 known E. coli H (flagellar antigen) serotypes (H1, H7, H12, H23, H45, H49, and H51) produce morphotype E filaments. Polymerase chain reaction was used to amplify flagellin structural (fliC) genes from E. coli strains producing morphotype E flagellar filaments and from strains with flagellar filaments representing other morphotypes. A single DNA fragment was obtained from each strain, and the size of the amplified DNA correlated with the molecular mass of the corresponding flagellin protein. This finding and hybridization data suggest that these bacteria are monophasic. fliC genes from three E. coli serotypes (H1, H7, and H12) possessing morphotype E flagellar filaments were sequenced in order to assess the contribution of conserved flagellin primary sequence to the characteristic filament architecture. The H1 and H12 fliC sequences were identical in length (1,788 bp), while the H7 fliC sequence was shorter (1,755 bp). The deduced molecular masses of the FliC proteins were 60,857 Da (H1), 59,722 Da (H7), and 60,978 Da (H12). The H1, H7, and H12 flagellins demonstrated 98 to 99% identity over the amino-terminal region (190 amino acid residues) and 89% (H7) to 99% (H1 and H12) identity in the carboxy-terminal region (100 amino acid residues). The complete primary amino acid sequences for H1 and H12 flagellins differed by only 10 amino acids, accounting for previously reported serological cross-reactions. However, the central region of H7 flagellin had only 38% identity with H1 and H12 flagellins. The characteristic morphology of morphotype E flagellar filaments is therefore not dependent on a highly conserved primary sequence within the exposed central region. Comparison of morphotype E E. coli flagellins with those from E. coli K-12, Serratia marcescens, and several Salmonella serovars supported the established concept of highly conserved terminal regions flanking a variable central region.

Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis.

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.

Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds.

Campylobacter coli VC167 has been shown to undergo a reversible flagellar antigenic variation between antigenic type 1 (T1) and antigenic type 2 (T2). VC167 contains two flagellin genes, and the products of both genes are incorporated into a complex flagellar filament in both antigenic types. Although there are only minor amino acid changes in the flagellins expressed by T1 and T2 cells, the two antigenic types of flagellins can be distinguished by differences in apparent M(r) on sodium dodecyl sulfate-polyacrylamide gels and by immunoreactivity with T1-specific (LAH1) or T2-specific (LAH2) antiserum. The isolation of stable variants of T1 and T2 has allowed for the transfer via natural transformation of the flagellin structural genes from the T1 background into the T2 background and from the T2 background into the T1 background. In addition, the flagellin genes from VC167 T1 and T2 have been transferred into strains of Campylobacter jejuni. The results indicate that the observed antigenic variations of VC167 flagellins are dependent on the host genetic background and independent of the primary amino acid sequence. These data provide evidence that posttranslational modifications are responsible for the antigenic variation seen in VC167 flagellins.

Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium

The complex regulation of flagellin gene expression in Salmonella typhimurium was characterized in vivo by using lac transcriptional fusions to the two flagellin structural genes (fliC [H1] and fljB [H2]). Phase variation was measured as the rate of switching of flagellin gene expression. Switching frequencies varied from 1/500 per cell per generation to 1/10,000 per cell per generation depending on the particular insertion and the direction of switching. There is a 4- to 20-fold bias in favor of switching from the fljB(On) to the fljB(Off) orientation. Random Tn10dTc insertions were isolated which failed to express flagellin. While most of these insertions mapped to loci known to be required for flagellin expression, several new loci were identified. The presence of functional copies of all of the genes responsible for complete flagellar assembly, except the hook-associated proteins (flgK, flgL, and fliD gene products), were required for expression of the fliC or fljB flagellin genes. Two novel loci involved in negative regulation of fliC and fljB in fla mutant backgrounds were identified. One of these loci, designated the flgR locus, mapped to the flg operon at 23 min on the Salmonella linkage map. An flgR insertion mutation resulted in relief of repression of the fliC and fljB genes in all fla mutant backgrounds except for mutants in the positive regulatory loci (flhC, flhD, and fliA genes).

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.

Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola.

The gene coding for the flagellin protein of Roseburia cecicola, an oxygen-intolerant, gram-negative, anaerobic bacterium indigenous to the murine cecum, has been cloned and sequenced. NH2-terminal amino acid sequence data from the flagellin protein were used as a basis for the synthesis of two mixed-sequence deoxyoligonucleotides. The oligonucleotides were used to identify and clone the flagellin structural gene. DNA sequence analysis of M13mp8 and mp9 subclones revealed a protein with a length of 293 amino acids and a molecular weight of 31,370. Comparisons with the sequences of flagellins of other species revealed conserved regions and suggested that although R. cecicola has structural characteristics of a gram-negative bacterium, it may be most closely related to the gram-positive bacteria.

Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation.

The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.

Isolation of a Caulobacter gene cluster specifying flagellum production by using nonmotile Tn5 insertion mutants

Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.