Comparative analysis of flagellin genes from wild type FLA⁺ and non-motile FLA⁻ P. aeruginosa strains

The major structural protein of the Pseudomonas aeruginosa flagellum is the protein, flagellin. Pseudomonas aeruginosa strains express two types of flagellin proteins: b-type that have a molecular weight (M_r) of 53kDa and a more heterologous a-type ranging in M_r from 45 to 52kDa. The purpose of this research is two fold. It was of interest firstly, to make a comparative study of b-type genes and secondly, to test whether a PCR technique could be used to extend this comparison to Fla^- strains. To facilitate these objectives, a PCR technique was modified and employed to amplify most of the N- and C-terminus and all of the central variable region of the Pseudomonas aeruginosa flagellin genes. A PCR gene fragment size of 1.02kb corresponds to the flagellin a-type gene whereas an amplified gene fragment of 1.25kb corresponds to the flagellin b-type gene (Winstanley et al., 1996). To confirm and extend these correlations, we performed monoclonal antibody colony immunoblots on a number of motile Fla⁺ and non-motile Fla^- strains and isolated and sequenced amplified gene fragments of 4 Fla^- isolates and the wild type M2. The Fla^- phenotype exhibited absence of reaction with type specific monoclonal antibodies, These results suggest that little or no flagellin is expressed in these non-motile mutants or that a mutation in the fliC gene is located in the Mab reactive epitope in the flagellin protein. The amino acid sequence results of strain M2 (Fla+, b-type) showed almost complete identity (99.7%) with the Fla⁺ b-type PA01, emphasizing the homologous nature of this group of flagellins. Variation in the b-type flagellins range from a single amino acid difference to a maximum of 5 amino acids in a Fla^- CF isolate. In analyzing the M2 protein sequence, we have found 3 potential sites for linear Mab epitopes in the b-type flagellin that are absent in the a-type flagellins. The conserved nature of the b-type genes enabled one to rapidly detect mutations that might occur in the Fla^- gene sequences. Only one of the fliC gene sequences provided evidence which linked mutations in the structural gene to the Fla^- phenotype. A stop codon was found in the N-terminal region of CF isolate 448bb. Mutations at the regulatory level could explain the other flagellin deficiencies. The a-type flagellin protein sequences exhibited less identity amongst P. aeruginosa strains. Results show evidence for protein sequence variation in the a-type flagellins. In strain PA103 (Fla^-, a-type), there were structural differences which involved two distinct deletion regions and three additional substitution regions, in comparison to the wild type a-type strain PAK. These sequence variations corresponded precisely to those of another GenBank a-type P. aeruginosa CF isolate K701 (Fla⁺) to which the PA103 fliC gene shares a high degree of homology. Location of mutations in a-type flagellins of Fla^- strains is more difficult because of their decreased sequence homology compared to the b-types. The PCR protocol described here, cannot only be used to identify and study the fliC gene and differentiate a- and b-type flagellins, but also it can be used to rapidly detect and type the fliC gene in Fla^- strains that do not react with monoclonal antibodies.