Section 10
Chapter 9,044

Molecular cloning of two Pseudomonas flagellin genes and basal body structural genes

Winstanley, C.; Morgan, J.A.; Pickup, R.W.; Saunders, J.R.

Microbiology 140: 2019-2031


ISSN/ISBN: 1350-0872
PMID: 7921252
DOI: 10.1099/13500872-140-8-2019
Accession: 009043681

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Pseudomonas putida strains PaW8 and PRS2000 produce flagellins with apparent molecular masses of 81 kDa and 50 kDa respectively. Two Tn5 insertion mutants of P. putida PaW8 lacking the ability to bind the flagellin-specific monoclonal antibody MLV1 were isolated. Mutant PaW8-flg2 contained a Tn5 insertion within a 2.6 kb EcoRI fragment of the P. putida chromosome carrying putative basal body genes. DNA and deduced protein sequences suggested the presence on this fragment of two complete genes homologous to flgH and flgI from Salmonella typhimurium. The insertion of Tn5 occurred in the flgI locus and appeared severely to reduce expression of the P. putida flagellin gene. A Tn5-containing fragment of DNA from a second mutant, PaW8-flg1, was cloned and found to contain sequences that hybridized strongly with the Pseudomonas aeruginosa flagellin gene. A 2.3 kb HindIII fragment containing all but 62 bp of the P. putida PaW8 flagellin gene was cloned and used as a probe to identify clones carrying the equivalent gene from P. putida PRS2000. Flagellin genes from both P. putida 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 P. putida PaW8 flagellin. The deduced molecular mass of P. putida 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 P. putida PRS2000, where the predicted and apparent molecular masses were similar.

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