Cloning and characterization of Pseudomonas aeruginosa fliF, necessary for flagellar assembly and bacterial adherence to mucin.

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RESUMO

Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs. This process appears to be mediated by nonpilus adhesins which bind to mucin. To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P. aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin. All of these mutants were also found to be defective in motility. One such mutant, PAK-RR20, is characterized here. The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG. Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans. Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette. This mutant was also nonmotile and nonadhesive. However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon. The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P. aeruginosa were determined. Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P. aeruginosa with mutations in several regulatory genes. Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors. The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.

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