Flagellin Synthesis Research Articles

Pseudomonas aeruginosa regulates flagellin expression as part of a global response to airway fluid from cystic fibrosis patients.

Cystic fibrosis (CF) patients are highly susceptible to chronic lung infections by the environmental bacterium Pseudomonas aeruginosa. The overproduction and accumulation of dehydrated viscous respiratory mucus and excessive inflammation represents a defining feature of CF and constitutes the major environment encountered by P. aeruginosa during chronic infections. We applied whole-genome microarray technology to investigate the ability of P. aeruginosa to respond to signals found in muco-purulent airway liquids collected from chronically infected CF patients. Particularly notable was the activation of the Rhl-dependent quorum-sensing (QS) network and repression of fliC, which encodes flagellin. Activation of the Rhl branch of the QS network supports the observation that QS molecules are produced in the chronically infected CF lung. The shut-off of flagellin synthesis in response to CF airway liquids was rapid and independent of QS and the known regulatory networks controlling the hierarchical expression of flagellar genes. As flagellin is highly immunogenic and subject to detection by host pattern recognition receptors, its repression may represent an adaptive response that allows P. aeruginosa to avoid detection by host defense mechanisms and phagocytosis during the chronic phase of CF lung infections.

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus.

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.

Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the Delta tatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB(1) genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA(1) and reduced verotoxicity were detected in the Delta tatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

Use of two-dimensional electrophoresis To study differential protein expression in divercin V41-resistant and wild-type strains of Listeria monocytogenes.

The use of bacteriocins from food-grade lactic acid bacteria to fight against the food-borne pathogen Listeria monocytogenes has been gaining interest. However, the emergence of resistant cells is frequently reported when Listeria is exposed to such antibacterials. A two-dimensional electrophoresis study of whole-cell protein expression of Listeria monocytogenes variants sensitive or resistant to the action of a bacteriocin produced by Carnobacterium divergens V41, divercin V41, is reported in this paper. The resistant variant obtained from the sensitive strain of L. monocytogenes P was also resistant to piscicocins V1 and SF668, but remained sensitive to nisin. Its growth rate was 50% less than the sensitive strain, and the MIC for it was 10(4) times higher. No reversion of the resistance was observed after 20 successive cultures in the absence of divercin V41. Comparison of the protein patterns by two-dimensional gel electrophoresis analysis showed clear differences. In the resistant variant pattern, at least nine spots had disappeared and eight new ones were observed. One of the newly synthesized proteins was identified as a flagellin of L. monocytogenes. Direct interaction between flagellin and divercin V41 was not evidenced. Intracellular synthesis of flagellin is probably an indirect effect of a modification in transcriptional regulation with widespread effects through a sigma factor. An intense protein, only present in the sensitive strain, was identified as a non-heme iron-binding ferritin displaying strong similarities to Dps proteins. Common modifications in the transcriptional regulation for these two proteins are discussed.

FlbT, the post-transcriptional regulator of flagellin synthesis in Caulobacter crescentus, interacts with the 5' untranslated region of flagellin mRNA.

Flagellar gene expression' in Caulobacter crescentus is regulated by a complex trans-acting hierarchy, in which the assembly of early structural proteins is required for the expression of later structural proteins. The flagellins that comprise the filament are regulated at both the transcriptional and the post-transcriptional levels. Post-transcriptional regulation is sensitive to the assembly of the flagellar basal body and hook structures. In mutant strains lacking these structures, flagellin genes are transcribed, but not translated. Mutations in the flagellar regulatory gene, flbT, restore flagellin translation in the absence of flagellar assembly. In this report, we investigate the mechanism of FlbT-mediated post-transcriptional regulation. We show that FlbT is associated with the 5' untranslated region (UTR) of fljK (25 kDa flagellin) mRNA and that this association requires a predicted loop structure in the transcript. Mutations within this loop abolished FlbT association and resulted in increased mRNA stability, indicating that FlbT promotes the degradation of flagellin mRNA by associating with the 5' UTR. We also assayed the effects on gene expression using mutant transcripts fused to lacZ. Interestingly, the mutant transcript that failed to associate with FlbT in vitro was still repressed in mutants defective in flagellum assembly, suggesting that other factors in addition to FlbT couple assembly to translation.

Internalization of Listeria monocytogenes by nonprofessional and professional phagocytes.

Listeria monocytogenes was originally described by Murray et al. (1926), who named it Bacterium monocytogenes due to the characteristic monocytosis found in the blood of experimentally infected animals. In 1940 it was renamed into its present name. L. monocytogenes is a gram-positive, rod-shaped, non-spore forming, facultative anaerobic bacillus which grows in a wide range of temperatures. L. monocytogenes is catalase positive, oxidase negative, CAMP positive, and expresses a hemolysin producing characteristic zones of lysis on blood agar plates. L. monocytogenes is motile when grown between 20 and 25°C, but the synthesis of flagellin is repressed at 37°C and the bacteria are then nonmotile. The cell wall of L. monocytogenes contains peptidoglycan, teichoic acid, and lipoteichoic acid and shows all the charateristics of a typical gram-positive cell wall. While the teichoic acids are covalently linked to the peptidoglycan, the lipoteichoic acids are anchored to the cytoplasmic membrane via a glycolipid moiety. L. monocytogenes is wide spread in nature and is found on plants, in soil, and in freshwater, but also in silage, wastewater, and in human and animal feces (reviewed in Schuchat et al., 1991).

A phase variant of Azospirillum lipoferum lacks a polar flagellum and constitutively expresses mechanosensing lateral flagella.

Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing.

Development of a system for genetic manipulation of Bartonella bacilliformis.

Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.

A regulatory locus, pehSR, controls polygalacturonase production and other virulence functions in Ralstonia solanacearum.

We previously identified a locus that regulates production of polygalacturonase (PG), an extracellular plant cell wall-degrading enzyme important in bacterial wilt of plants caused by Ralstonia (Pseudomonas) solanacearum. The DNA sequence of this locus, called pehSR, was determined and two consecutive open reading frames (ORFs) of 1,905 and 1,680 bp were identified. The amino acid sequences predicted to be encoded by these ORFs are similar to those of regulators of pilin synthesis in Pseudomonas aeruginosa and Myxococcus xanthus and to a regulator of flagellin synthesis and adhesion in P. aeruginosa, as well as to other two-component regulators of the NtrB/C subfamily. pehSR mutants produced negligible levels of endo-PG activity, while exo-PG activity was reduced by 50%. Northern (RNA) blot analysis showed that PehSR regulates endo-PG expression at the transcriptional level. pehSR mutants grew normally in culture and in planta but were dramatically reduced in virulence; this loss of virulence was substantially greater than that observed for endo-PG structural gene mutants, suggesting that pehSR regulates additional factors important in virulence. Although pehSR mutants were essentially nonmotile, like the wild-type strain, multiple copies of pehSR conferred motility on the bacterium. Reporter gene studies indicated that pehSR expression increased when bacteria grew in plant tissue, and that the pehSR locus was itself negatively regulated by the global virulence gene regulator PhcA.

Decrease in Expression of the Master Operon of Flagellin Synthesis in a dnaA46 Mutant of Escherichia coli.

We examined the expression of the fliC, fliA and fihD genes in the dnaA46 mutant. In Northern blot analysis, the amounts of fliC, fliA, and flhD mRNA decreased in the dnaA46 mutant growing at 37 degrees C but not at 28 degrees C. The promoter activity of each gene also decreased in this mutant at 37 degrees C. The decrease in expression of the flh D gene was not related to the cAMP-catabolite activator protein (CAP) pathway. We tentatively conclude that DnaA protein is involved in the expression of the flhD gene by a mechanism independent of the cAMP-CAP pathway.

Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection.

To examine the role of flagella in pathogenesis of urinary tract infection caused by Proteus mirabilis, we constructed a nonmotile, nonswarming flagellum mutant of strain WPM111 (an hpmA hemolysin mutant of strain BA6163, chosen because of its lack of in vitro cytotoxicity in renal epithelial cell internalization studies). A nonpolar mutation was introduced into the flaD gene, which encodes the flagellar cap protein. This mutation does not affect the synthesis of flagellin but rather prevents the assembly of an intact flagellar filament. In in vitro assays, the genetically characterized nonmotile mutant was found to be internalized by cultured human renal proximal tubular epithelial cells in numbers less than 1% of those of the flagellated parent strain. Internalization of the nonmotile mutant was increased significantly (14- to 21-fold) by centrifugation onto the monolayer. To assess virulence in vivo, CBA mice were challenged transurethrally with 10(7) CFU of P. mirabilis BA6163 (wild type) (n = 16), WPM111 (hpmA mutant) (n = 46), or BB2401 (hmpA flaD mutant) (n = 46). Differences in quantitative cultures between the parent strain and the hemolysin-negative mutant were not significant. However, the hpmA flaD mutant was recovered in numbers approximately 100-fold lower than those of the hmpA mutant or the wild-type parent strain and thus was clearly attenuated. We conclude that while hemolysin does not significantly influence virulence, flagella contribute significantly to the ability of P. mirabilis to colonize the urinary tract and cause acute pyelonephritis in an experimental model of ascending urinary tract infection.

FlaD (sinR) mutations affect SigD-dependent functions at multiple points in Bacillus subtilis.

A flaD (sinR) null mutation depressed sigD-lacZ expression only two- to fourfold, whereas a flaD1 point mutation depressed it almost completely. Introduction of pHYSigD, a sigmaD-overproducing plasmid, corrected the filamentous phenotype common to both sinR mutants; autolysin synthesis was restored partially and completely in the flaD1 and flaD (sinR) null strains, respectively. Flagellin synthesis and motility were not restored at all in either strain.

The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin.

The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present. We previously showed that FlgM is also necessary for the virulence of S. typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence. In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S. typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S. typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S. typhimurium mutant resulted in a virulent phenotype. Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S. typhimurium. Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S. typhimurium.

Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus: phase variation leads to differential transcription of two flagellar genes ( fliCD)

The insect-pathogenic bacterium Xenorhabdus undergoes spontaneous phase variation involving a large number of phenotypes. Our previous study indicated that phase I variants were motile, whereas phase II variants of X. nematophilus F1 were nonflagellated cells which did not synthesize flagellin [Givaudan, A., Baghdiguian, S., Lanois, A. and Boemare, N. (1995) Appl. Environ. Microbiol. 61, 1408–1413]. In order to approach the study of the flagellar switching, a locus containing two ORFs from X. nematophilus F1 (phase I) was identified by using functional complementation of flagellin-negative E. coli. The sequence analysis revealed that the first ORF corresponds to the fliC gene coding for flagellin, and showed a high degree of homology between the N-terminal and C-terminal of Xenorhabdus FliC and flagellins from other bacteria. The second identified ORF in the opposite orientation encodes a homologue of the enterobacterial hook-associated protein 2, FliD. Both Xenorhabdus fliCD genes were required for the entire restoration of E. coli motility. A sequence highly homologous to the σ 28 consensus promoter was identified upstream from the coding sequences from both genes. The structure of the fliC gene and its surrounding region was shown to be the same in both phase variants, but Northern blot analysis revealed that fliC and fliD were, respectively, not and weakly transcribed in phase II variants. In addition, complementation experiments showed that motility and flagellin synthesis of phase II cannot be recovered by placing in trans fliCD genes from phase I. These latter results suggest that a gene(s) higher in the transcriptional hierarchy of the flagellar regulon is switched off in Xenorhabdus phase II variants.

Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin.

This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.

Control by phosphatidylglycerol of expression of the flhD gene in Escherichia coli

We reported elsewhere that mutation in the pgsA gene, responsible for the synthesis of phosphatidylglycerol, repressed the synthesis of flagellin and caused the loss of motility of Escherichia coli (Tomura et al., FEBS Letters 329, 287–290, 1993). We now describe evidence for a decrease in promoter activity of the flhD gene, a master gene for flagellum synthesis, in the pgsA3 mutant. We constructed a plasmid with a promoter region of the flhD gene connected with the structure region of the lacZ gene. The activity of β-galactosidase in the extract prepared from the pgsA3 mutant harboring the fusion plasmid was 30% of that in the wild type cells. This result means that phosphatidylglycerol is likely to be required for the initiation of transcription of the flhD gene. We also found that the motility-less phenotype of the mutant was partially suppressed by elevating incubation temperature. This suppression is caused by restoration of transcription of the flhD gene by high temperature. As the content of phosphatidylglycerol did not increase by elevating incubation temperature, we proposed that this suppression is caused by alternation of a physical structure of phospholipid bilayers in cytoplasmic membranes.

Effect of salicylate on expression of flagella by Escherichia coli and Proteus, Providencia, and Pseudomonas spp

Osmotic stress, salicylate, and Mar (multiple antibiotic resistance) mutation are known to block the expression of the OmpF porin. Since these conditions have also been shown to inhibit the expression of P and CFA fimbriae in Escherichia coli, we speculated that they might affect the expression of flagella as well. Hyperosmotic conditions have been shown to block the synthesis of flagellin and expression of flagella in E. coli (C. Li, C. J. Louise, W. Shi, and J. Adler, J. Bacteriol. 175:2229-2235, 1993). In the current study, sodium salicylate was found to inhibit the motility of E. coli, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii in a reversible, concentration-dependent manner. Swarming did not occur at 20 mM sodium salicylate. Salicylate also blocked the synthesis of flagellin in E. coli. Phenotypic Mar mutants of E. coli derived from motile strains were amotile. Flagella were markedly reduced as determined by scanning electron microscopy when P. mirabilis was grown in broth containing 20 mM salicylate. Salicylate had no apparent effect, however, on expression of a 40-kDa porin protein in P. mirabilis. This finding suggests that the noted effect of salicylate on Proteus spp. may be mediated through a mechanism other than porin production or that the Proteus porin may not be analogous to OmpF in E. coli. Salicylate decreased the motility of Pseudomonas cepacia but had no effect on Pseudomonas aeruginosa. The exact mechanism by which salicylate exerts its effect is not known, but it appears to be related to osmoregulation.

Swarming and Swimming Changes Concomitant with Phase Variation in Xenorhabdus nematophilus

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Phase I, the variant naturally isolated from the infective-stage nematode, provides better conditions than the phase II variant for nematode reproduction. This study has shown that Xenorhabdus phase I variants displayed a swarming motility when they were grown on a suitable solid medium (0.6 to 1.2% agar). Whereas most of the phase I variants from different Xenorhabdus spp. were able to undergo cycle of rapid and coordinately population migration over the surface, phase II variants were unable to swarm and even to swim in semisolid agar, particularly in X. nematophilus. Optical and electron microscopic observations showed nonmotile cells with phase II variants of X. nematophilus F1 which lost their flagella. Flagellar filaments from strain F1 phase I variants were purified, and the molecular mass of the flagellar structural subunit was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 36.5 kDa. Flagellin from cellular extracts or culture medium of phase II was undetectable with antiserum against the denatured flagellin by immunoblotting analysis. This suggests that the lack of flagella in phase II cells is due to a defect during flagellin synthesis. The importance of such a difference of motility between both phases is discussed in regard to adaptation of these bacteria to the insect prey and the nematode host.

Sequence, regulation, and functions of fis in Salmonella typhimurium

The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined. The overall fis operon organization in S. typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species. While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94. Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase. fis autoregulation was less effective in S. typhimurium than that in E. coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S. typhimurium fis promoter region. Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase. On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase. During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.

Expression of multiple flagellin-encoding genes of Proteus mirabilis.

The overproduction of flagella is a distinguishing characteristic of Proteus mirabilis swarmer cell differentiation. The synthesis of flagellin, the principal protein composing the flagellar filament, is coordinately regulated as part of a larger regulon of genes whose expression is a prerequisite in urinary pathogenesis. In this report, the regulation of expression of the flaA locus, comprising flaA and flaB, two tandemly linked and nearly identical copies of flagellin-encoding genes, is examined. Transcriptional expression studies reveal that flaA, but not flaB, is expressed by wild-type cells, and flaA transcription increases eightfold during differentiation. The flaA transcriptional start site for both swimmer and swarmer cells was determined to be located at a guanine, 8 bases downstream of the flaA sigma 28 promoter. FlaA- mutants are nonmotile and undifferentiated and do not synthesize flagellin, while FlaB- mutants are wild type, thus verifying that FlaA is the sole flagellin produced by wild-type cells and that flaB is silent. FlaA- mutants frequently revert to a Mot+ phenotype that is antigenically distinct from that of wild-type cells. Southern blot analysis of the flaA Mot+ revertants reveals a deletion of between 2 and 7kb in the flaA locus. Biochemical analyses of revertant flagellin indicate major changes in protein size and composition but conservation of the first 28 N-terminal residues. The result of this process is to produce an antigenically distinct flagellum that may be significant in ensuring the survival of P. mirabilis during pathogenesis.