Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449.

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.     

Role of two flagellin genes in Campylobacter motility.

Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity. The flaA gene is transcribed from a o-28 promoter, and the flaB gene from a o-54 promoter. Gene replacement mutagenesis techniques were used to generate flaA+ flaB and flaA flaB+ mutants. Both gene products are capable of assembling independently into functional filaments. A flagellar filament composed exclusively of the flaA gene product is indistinguishable in length from that of the wild type and shows a slight reduction in motility. The flagellar filament composed exclusively of the flaB gene product is severely truncated in length and greatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products are required for a fully active flagellar filament. Although the wild-type flagellar filament is a heteropolymer of the flaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surface exposed along the length of the wild-type filament.     

Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae.

The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaB1, flaB2, and flaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaB1, flaB2, and flaB3 flagellins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between the flaB2 and flaB3 genes appears to increase the resistance of the flaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. The flaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the Mr values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flagellar filaments. Each flagellin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11- or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium.     

Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni.

The complex flagellar filaments of the LIO8 serogroup member Campylobacter coli VC167 are composed of two highly related subunit proteins encoded by the flaA and flaB genes which share 92% identity. Using oligonucleotide primers based on the known DNA sequence of both the flaA and flaB genes from C. coli VC167 in the polymerase chain reaction, we have shown conservation of both fla genes among isolates within the LIO8 heat-labile serogroup by digestion of the amplified product with PstI and EcoRI restriction endonucleases. Amplification and subsequent restriction analysis of the flaA flagellin gene from Campylobacter isolates belonging to 13 different LIO serogroups further identified 10 unique polymorphic groups. Within most of the serogroups examined, isolates appeared to contain flaA genes with conserved primary structures. Only in serogroups LIO11 and LIO29 did independent isolates possess flagellin genes with different primary structures. Furthermore, by employing primers specific for the flaB gene of C. coli VC167, all serogroups examined contained a second fla gene corresponding to flaB. In all serogroups except the LIO5 and LIO6 isolates which were identical to each other, the polymorphic pattern of this flaB gene was identical to that of the corresponding flaA gene. These data indicate that the presence of a second highly homologous flagellin gene is widespread throughout Campylobacter isolates and that in most instances, the primary structure of the two fla genes is conserved within isolates belonging to the same heat-labile LIO serogroup. This may represent the presence of clonal evolutionary groups in Campylobacter spp.     

Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification

Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.     

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.     

Genomic rearrangements in the flagellin genes of Proteus mirabilis

Molecular analyses have revealed that Proteus mirabilis possesses two genes, flaA and flaB, that are homologous to each other and to flagellin genes of many other species. Both swimmer and swarmer cells transcribe flaA, but not flaB. FlaA- mutants are non-motile and do not differentiate showing the essential role of flaA in swarmer cell differentiation and behaviour. At a low frequency, motile, differentiation-proficient revertants have been found in FlaA-populations. These revertants produce an antigenically and biochemically distinct flagellin protein. The revertant flagellin is the result of a genetic fusion between highly homologous regions of flaA and flaB that places the active flaA promoter and the 5' coding region of flaA adjacent to previously silent regions of flaB generating a hybrid flagellin protein. Analysis of the flaA-flaB region of two such revertants reveals that a portion of this locus has undergone a rearrangement and deletion event that is unique to each revertant. Using a polymerase chain reaction (PCR) to amplify the falA-flaB locus from wild-type swimmer cells, swarmer cells and cells obtained after urinary tract infection, we uncover at least six general classes of rearrangements between flaA and flaB. Each class of rearrangement occurs within one of nine domains of homology between flaA and flaB. Rearrangement of flaA and flaB results in a hybrid flagellin protein of nearly identical size and biochemical properties, suggesting a concerted mechanism may be involved in this process. The data also reveal that the frequency and distribution of flaAB rearrangements is predicted on environmental conditions. Thus, rearrangement between flaA and flaB may be a significant virulence component of P. mirabilis in urinary tract infections.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.     

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.     

Genomic organization and expression of Campylobacter flagellin genes.

Campylobacter coli VC167, which undergoes an antigenic flagellar variation, contains two full-length flagellin genes, flaA and flaB, that are located adjacent to one another in a tandem orientation and are 91.5% homologous. The gene product of flaB, which has an Mr of 58,946, has 93% sequence homology to the gene product of flaA, which has an Mr of 58,916 (S. M. Logan, T. J. Trust, and P. Guerry, J. Bacteriol. 171:3031-3038, 1989). Mutational analyses and primer extension experiments indicated that the two genes are transcribed under the control of distinct promoters but that they are expressed concomitantly in the same cell, regardless of the antigenic phase of flagella being produced. The flaA gene, which was expressed at higher levels than the flaB gene in both phases, was transcribed from a typical sigma 28-type promoter, whereas the flaB promoter was unusual. A mutant producing only the flaB gene product did not synthesize a flagellar filament and was nonmotile. Southern blot analysis indicated that flagellar antigenic variation involves a rearrangement of flagellin sequence information rather than the alternate expression of the two distinct genes.