Characterization and functional analysis of seven flagellin genes in Rhizobium leguminosarum bv. viciae. Characterization of R. leguminosarum flagellins.
ABSTRACT: BACKGROUND: Rhizobium leguminosarum bv. viciae establishes symbiotic nitrogen fixing partnerships with plant species belonging to the Tribe Vicieae, which includes the genera Vicia, Lathyrus, Pisum and Lens. Motility and chemotaxis are important in the ecology of R. leguminosarum to provide a competitive advantage during the early steps of nodulation, but the mechanisms of motility and flagellar assembly remain poorly studied. This paper addresses the role of the seven flagellin genes in producing a functional flagellum. RESULTS: R. leguminosarum strains 3841 and VF39SM have seven flagellin genes (flaA, flaB, flaC, flaD, flaE, flaH, and flaG), which are transcribed separately. The predicted flagellins of 3841 are highly similar or identical to the corresponding flagellins in VF39SM. flaA, flaB, flaC, and flaD are in tandem array and are located in the main flagellar gene cluster. flaH and flaG are located outside of the flagellar/motility region while flaE is plasmid-borne. Five flagellin subunits (FlaA, FlaB, FlaC, FlaE, and FlaG) are highly similar to each other, whereas FlaD and FlaH are more distantly related. All flagellins exhibit conserved amino acid residues at the N- and C-terminal ends and are variable in the central regions. Strain 3841 has 1-3 plain subpolar flagella while strain VF39SM exhibits 4-7 plain peritrichous flagella. Three flagellins (FlaA/B/C) and five flagellins (FlaA/B/C/E/G) were detected by mass spectrometry in the flagellar filaments of strains 3841 and VF39SM, respectively. Mutation of flaA resulted in non-motile VF39SM and extremely reduced motility in 3841. Individual mutations of flaB and flaC resulted in shorter flagellar filaments and consequently reduced swimming and swarming motility for both strains. Mutant VF39SM strains carrying individual mutations in flaD, flaE, flaH, and flaG were not significantly affected in motility and filament morphology. The flagellar filament and the motility of 3841 strains with mutations in flaD and flaG were not significantly affected while flaE and flaH mutants exhibited shortened filaments and reduced swimming motility. CONCLUSION: The results obtained from this study demonstrate that FlaA, FlaB, and FlaC are major components of the flagellar filament while FlaD and FlaG are minor components for R. leguminosarum strains 3841 and VF39SM. We also observed differences between the two strains, wherein FlaE and FlaH appear to be minor components of the flagellar filaments in VF39SM but these flagellin subunits may play more important roles in 3841. This paper also demonstrates that the flagellins of 3841 and VF39SM are possibly glycosylated.
Project description:Rotary flagella propel bacteria through liquid and across semisolid environments. Flagella are composed of the basal body that constitutes the motor for rotation, the curved hook that connects to the basal body, and the flagellar filament that propels the cell. Flagellar filaments can be composed of a single flagellin protein, such as in Escherichia coli, or made up of multiple flagellins, such as in Agrobacterium tumefaciens The four distinct flagellins FlaA, FlaB, FlaC, and FlaD produced by wild-type A. tumefaciens are not redundant in function but have specific properties. FlaA and FlaB are much more abundant than FlaC and FlaD and are readily observable in mature flagellar filaments, when either FlaA or FlaB is fluorescently labeled. Cells producing FlaA with any one of the other three flagellins can generate functional filaments and thus are motile, but FlaA alone cannot constitute a functional filament. In flaA mutants that manifest swimming deficiencies, there are multiple ways by which these mutations can be phenotypically suppressed. These suppressor mutations primarily occur within or upstream of the flaB flagellin gene or in the transcription factor sciP regulating flagellin expression. The helical conformation of the flagellar filament appears to require a key asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be spontaneously mutated to render helical flagella in the absence of FlaA, reflecting their overall similarity and perhaps the subtle differences in the specific functions they have evolved to fulfill.IMPORTANCE Flagellins are abundant bacterial proteins comprising the flagellar filaments that propel bacterial movement. Several members of the alphaproteobacterial group express multiple flagellins, in contrast to model systems, such as with Escherichia coli, which has one type of flagellin. The plant pathogen Agrobacterium tumefaciens has four flagellins, the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. Mutational analysis reveals that FlaA requires at least one of the other flagellins to function, as flaA mutants produce nonhelical flagella and cannot swim efficiently. Suppressor mutations can rescue this swimming defect through mutations in the remaining flagellins, including structural changes imparting helical shape to the flagella, and putative regulators. Our findings shed light on how multiple flagellins contribute to motility.
Project description:Vibrio cholerae is a Gram-negative bacterium with a monotrichous flagellum that causes the human disease cholera. Flagellum-mediated motility is an integral part of the bacterial life cycle inside the host and in the aquatic environment. The V. cholerae flagellar filament is composed of five flagellin subunits (FlaA, FlaB, FlaC, FlaD, and FlaE); however, only FlaA is necessary and sufficient for filament synthesis. flaA is transcribed from a class III flagellar promoter, whereas the other four flagellins are transcribed from class IV promoters. However, expressing flaA from a class IV promoter still facilitated motility in a strain that was otherwise lacking all five flagellins (?flaA-E). Furthermore, FlaA from V. parahaemolyticus (FlaAVP; 77% identity) supported motility of the V. cholerae ?flaA-E strain, whereas FlaA from V. vulnificus (FlaAVV; 75% identity) did not, indicating that FlaA amino acid sequence is responsible for its critical role in flagellar synthesis. Chimeric proteins composed of different domains of FlaAVC and FlaD or FlaAVV revealed that the N-terminal D1 domain (D1N) contains an important region required for FlaA function. Further analyses of chimeric FlaAVC-FlaD proteins identified a lysine residue present at position 145 of the other flagellins but absent from FlaAVC that can prevent monofilament formation. Moreover, the D1N region of amino acids 87 to 153 of FlaAVV inserted into FlaAVC allows monofilament formation but not motility, apparently due to the lack of filament curvature. These results identify residues within the D1N domain that allow FlaAVC to fold into a functional filament structure and suggest that FlaAVC assists correct folding of the other flagellins.IMPORTANCEV. cholerae causes the severe diarrheal disease cholera. Its ability to swim is mediated by rotation of a polar flagellum, and this motility is integral to its ability to cause disease and persist in the environment. The current studies illuminate how one specific flagellin (FlaA) within a multiflagellin structure mediates formation of the flagellar filament, thus allowing V. cholerae to swim. This knowledge can lead to safer vaccines and potential therapeutics to inhibit cholera.
Project description:Aeromonas caviae is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of A. caviae to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the A. caviae polar flagellin locus and identified five polar flagellar genes, in the order flaA, flaB, flaG, flaH, and flaJ. Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing, mutant analysis, and Western blotting demonstrated that A. caviae has a complex flagellum filament composed of two flagellin subunits encoded by flaA and flaB. The predicted molecular mass of both flagellins was approximately 31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 35,500 Da. This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays. Single mutations in either flaA or flaB did not result in loss of flagella but did result in decreased motility and adherence by approximately 50%. Mutation of flaH, flaJ, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence. However, mutation of flaG did not affect motility but did significantly reduce the level of adherence. Centrifugation of the flagellate mutants (flaA, flaB, and flaG) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (flaH, flaJ, and flaA flaB) increased adherence slightly. We conclude that maximum adherence of A. caviae to human epithelial cells in vitro requires motility and optimal flagellar function.
Project description:Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, which prevents a switch of handedness. The complex filaments of Rhizobium lupini H13-3 and those of Sinorhizobium meliloti are composed of three and four flagellin (Fla) subunits, respectively. The Fla-encoding genes, named flaA through flaD, are separately transcribed from sigma(28)-specific promoters. Mutational analysis of the fla genes revealed that, in both species, FlaA is the principal flagellin and that FlaB, FlaC, and FlaD are secondary. FlaA and at least one secondary Fla protein are required for assembling a functional flagellar filament. Western analysis revealed a ratio close to 1 of FlaA to the secondary Fla proteins (= FlaX) present in wild-type extracts, suggesting that the complex filament is assembled from FlaA-FlaX heterodimers. Whenever a given mutant combination of Fla prevented the assemblage of an intact filament, the biosynthesis of flagellin decreased dramatically. As shown in S. meliloti by reporter gene analysis, it is the transcription of flaA, but not of flaB, flaC, or flaD, that was down-regulated by such abortive combinations of Fla proteins. This autoregulation of flaA is unusual. We propose that any combination of Fla subunits incapable of assembling an intact filament jams the flagellar export channel and thus prevents the escape of an (as yet unidentified) anti-sigma(28) factor that antagonizes the sigma(28)-dependent transcription of flaA.
Project description:Flagella-driven motility enables bacteria to reach their favorable niche within the host. The human foodborne pathogen Campylobacter jejuni produces two heavily glycosylated structural flagellins (FlaA and FlaB) that form the flagellar filament. It also encodes the non-structural FlaC flagellin which is secreted through the flagellum and has been implicated in host cell invasion. The mechanisms that regulate C. jejuni flagellin biogenesis and guide the proteins to the export apparatus are different from those in most other enteropathogens and are not fully understood. This work demonstrates the importance of the putative flagellar protein FliS in C. jejuni flagella assembly. A constructed fliS knockout strain was non-motile, displayed reduced levels of FlaA/B and FlaC flagellin, and carried severely truncated flagella. Pull-down and Far Western blot assays showed direct interaction of FliS with all three C. jejuni flagellins (FlaA, FlaB, and FlaC). This is in contrast to, the sensor and regulator of intracellular flagellin levels, FliW, which bound to FlaA and FlaB but not to FlaC. The FliS protein but not FliW preferred binding to glycosylated C. jejuni flagellins rather than to their non-glycosylated recombinant counterparts. Mapping of the binding region of FliS and FliW using a set of flagellin fragments showed that the C-terminal subdomain of the flagellin was required for FliS binding, whereas the N-terminal subdomain was essential for FliW binding. The separate binding subdomains required for FliS and FliW, the different substrate specificity, and the differential preference for binding of glycosylated flagellins ensure optimal processing and assembly of the C. jejuni flagellins.
Project description:Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.
Project description:Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.
Project description:Vibrio vulnificus is a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum. V. vulnificus possesses a total of six flagellin genes organized into two loci (flaFBA and flaCDE). We proved that all six of the flagellin genes were transcribed, whereas only five (FlaA, -B, -C, -D, and -F) of the six flagellin proteins were detected. To understand roles of the six V. vulnificus flagellins in motility and virulence, mutants with single and multiple flagellin deletions were constructed. Mutations in flaB or flaC or the flaCDE locus resulted in a significant decrease in motility, adhesion, and cytotoxicity, whereas single mutations in the other flagellin genes or the flaFBA locus showed little or no effect. The motility was completely abolished only in the mutant lacking all six flagellin genes (flaFBA flaCDE). Surprisingly, a double mutation of flaB and flaD, a gene sharing 99% identity with the flaB at the amino acid level, resulted in the largest decrease in motility, adhesion, and cytotoxicity except for the mutant in which all six genes were deleted (the hexa mutant). Additionally, the 50% lethal doses (LD50s) of the flaB flaD and the flaFBA flaCDE mutants increased 23- and 91-fold in a mouse model, respectively, and the in vitro and in vivo invasiveness of the mutants was significantly decreased compared to that of the wild type. Taken together, the multiple flagellin subunits differentially contribute to the flagellum biogenesis and the pathogenesis of V. vulnificus, and among the six flagellin genes, flaB, flaD, and flaC were the most influential components.
Project description:Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.
Project description:Vibrio cholerae, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum. Motility has been implicated as a virulence determinant in some animal models of cholera, but the relationship between motility and virulence has not yet been clearly defined. We have begun to define the regulatory circuitry controlling motility. We have identified five V. cholerae flagellin genes, arranged in two chromosomal loci, flaAC and flaEDB; all five genes have their own promoters. The predicted gene products have a high degree of homology to each other. A strain containing a single mutation in flaA is nonmotile and lacks a flagellum, while strains containing multiple mutations in the other four flagellin genes, including a flaCEDB strain, remain motile. Measurement of fla promoter-lacZ fusions reveals that all five flagellin promoters are transcribed at high levels in both wild-type and flaA strains. Measurement of the flagellin promoter-lacZ fusions in Salmonella typhimurium indicates that the promoter for flaA is transcribed by the sigma54 holoenzyme form of RNA polymerase while the flaE, flaD, and flaB promoters are transcribed by the sigma28 holoenzyme. These results reveal that the V. cholerae flagellum is a complex structure with multiple flagellin subunits including FlaA, which is essential for flagellar synthesis and is differentially regulated from the other flagellins.