Cloning and comparison of fliC genes and identification of glycosylation in the flagellin of Pseudomonas aeruginosa a-type strains

Pseudomonas aeruginosa a-type strains have flagellin proteins which vary in molecular weight between strains. To compare the properties of a-type flagellins, the flagellin gene of several Pseudomonas aeruginosa a-type strains, as determined by interaction with specific anti-a monoclonal antibody, were cloned and sequenced. Specifically, strains 170018, 5933, and 5939 were chosen for cloning of the fliC gene. These strains were selected because their flagellins varied in molecular weight in SDS PAGE analysis. PCR amplification of a portion of the a-type flagellin gene from five strains produced a 1 kb product. This indicated that the gene size is not likely to be responsible for the observed molecular weight differences among the a-type strains. The amino acid seqence of several a-type strains and PAOl, a b-type strain, were compared. Comparisons revealed that a-type strains have a high percent similarity in amino acid sequence while comparison between the a-types and PAOl reveal higher divergence. Post-translational modification was explored for its contribution to the observed differences in molecular weight among the a-type strains. A biotin-hydrazide glycosylation assay was performed on the flagellin of three a-type strains (170018, 5933, and 5939) and one b-tjqje strain (M2) revealing a positive glycosylation reaction for strains 5933 and 5939 and a negative reaction for 170018 and M2. Deglycosylation of the flagellin proteins with trifluoromethanesulfonic acid (TPMS) confirmed the glycosylation results. A molecular weight shift was observed for the TPMS treated flagellins of 5933 and 5939. These results indicated that the molecular weight discrepancies observed for the a-type flagellins can be attributed to, at least in part, glycosylation of the protein. Anti-a flagellin monoclonal antibody reacted with the TFMS treated flagellins which suggested that the glycosyl groups are not a necessary component of the epitope for the human anti-a monoclonal antibody, Preliminary sequence comparison and hydrophilicity results indicated two possible monoclonal antibody binding sites. Comparisons between a-type and b-type sequences will allow the epitope for this monoclonal antibody to examined more closely.