strains PaW8 and PRS2000 produce flagellins with apparent molecular masses of 81 kDa and 50 kDa respectively. Two Tn5 insertion mutants of PaW8 lacking the ability to bind the flagellin-specific monoclonal antibody MLV1 were isolated. Mutant PaW8- contained a Tn5 insertion within a 2.6 kb RI fragment of the chromosome carrying putative basal body genes. DNA and deduced protein sequences suggested the presence on this fragment of two complete genes homologous to and from The insertion of Tn5 occurred in the locus and appeared severely to reduce expression of the flagellin gene. A Tn5-containing fragment of DNA from a second mutant, PaW8-, was cloned and found to contain sequences that hybridized strongly with the flagellin gene. A 2.3 kb HindIII fragment containing all but 62 bp of the PaW8 flagellin gene was cloned and used as a probe to identify clones carrying the equivalent gene from PRS2000. Flagellin genes from both strains were sequenced and their amino acid sequences deduced. Both flagellins were found to contain conserved amino- and carboxy-terminal regions when compared to other flagellins, with the central region being more variable. The epitope for MLV1 is likely to lie within this central region of PaW8 flagellin. The deduced molecular mass of PaW8 flagellin (68 kDa) differed significantly from its apparent molecular mass estimated by PAGE, possibly as a consequence of post-translational modification. This was not the case with flagellin from PRS2000, where the predicted and apparent molecular masses were similar.


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