MULTIPLE PROMOTERS AND INDUCTION BY HEAT-SHOCK OF THE GENE ENCODING THE ALTERNATIVE SIGMA-FACTOR ALGU (SIGMA(E)) WHICH CONTROLS MUCOIDY IN CYSTIC-FIBROSIS ISOLATES OF PSEUDOMONAS-AERUGINOSA

Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable,vith sigma(E) from Escherichia coli and Salmonella typhimurium. sigma(E) ha's been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P-1 through P-5) that fall into three categories: (i) the P-1 and P-3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma(E) promoters and were found to be absolutely dependent on AlgU; (ii) the P-2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tc-r cells; and (iii) the transcripts corresponding to P-4 and P-5, which were not affected by inactivation of lgU. Introduction of E. coli rpoE (encoding sigma(E)) or algU into P. aeruginosa algU::Tc-r strains restored P-1 and P-3; transcription and brought the P-2 signal back to the wild-type level. The AlgU-dependent promoters P-1 and P-3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AIgU and, by extension, its homologs in other organisms (e.g., sigma(E) in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.