Identification and Functional Characterization of flgM , a Gene Encoding the Anti-Sigma 28 Factor in Pseudomonas aeruginosa

Abstract

ABSTRACT We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa . FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus . A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the σ 28 binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The β-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC , was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor σ 28 was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus σ 54 recognition sites and one σ 28 site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either σ 28 or σ 54 . A transcript was detected by primer extension that was 8 bp downstream of the consensus σ 28 -binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both σ 28 -dependent and -independent mechanisms of transcription exist.