Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin

Abstract

This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.