Cell wall stress activates expression of a novel stress response facilitator (SrfA) under σ22 (AlgT/U) control in Pseudomonas aeruginosa

The ECF (extracytoplasmic function) alternative sigma factor, σ22 (AlgT/U), is required for expression of the algD promoter of the operon for alginate biosynthesis in Pseudomonas aeruginosa. Alginate production promotes chronic pulmonary infections by this opportunistic pathogen in patients with cystic fibrosis and chronic obstructive pulmonary disease. σ22 is normally sequestered, but its deregulation and activation occur either by mutation in mucA (encoding an anti-sigma factor) or in response to envelope stress, such as inhibition of peptidoglycan synthesis. The σ22 stress response system includes many genes in addition to those for alginate. In the present study, we characterized an intergenic region between ORFs PA2559 and PA2560 in PAO1 for a σ22-dependent, stress-responsive transcript, described here as PA2559.1. Northern analysis and transcript end-mapping indicated the PA2559.1 transcript was ~310 nt in length. Examination of the DNA sequence upstream of +1 revealed a σ22 core promoter motif, GAATTT-N16-TCTGT, and site-directed mutagenesis confirmed this to be a σ22-dependent promoter that was highly activated during cell wall stress. PA2559.1 also contained an ORF that demonstrated increased translational activity upon cell wall stress. As determined by mutant analysis, the protein encoded by PA2559.1 was shown to play a positive role in the σ22-dependent activation of the algD promoter under stress in both sessile (i.e. biofilm) and planktonic conditions. Thus, it appeared to act as a stress response facilitator and so was named SrfA. The sequence of SrfA was found to be novel in nature and extremely well conserved only in P. aeruginosa, suggesting that it is of high evolutionary importance in this species.