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 virulen ce determinant expressed by this organism during chronic respiratory i nfections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternat ive sigma factor which is 66% identical to and functionally interchang eable,vith sigma(E) from Escherichia coli and Salmonella typhimurium. sigma(E) ha's been implicated in the expression of systems enhancing b acterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. a eruginosa from multiple promoters (P-1 through P-5) that fall into thr ee categories: (i) the P-1 and P-3 promoters, which display strong sim ilarity to the -35 and -10 canonical sequences of sigma(E) promoters a nd were found to be absolutely dependent on AlgU; (ii) the P-2 promote r, which was less active in algU mutants, but transcription of which w as not completely abrogated in algU::Tc-r cells; and (iii) the transcr ipts corresponding to P-4 and P-5, which were not affected by inactiva tion 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; transcript ion and brought the P-2 signal back to the wild-type level. The AlgU-d ependent promoters P-1 and P-3 were inducible by heat shock in wild-ty pe nonmucoid P. aeruginosa PAO1. At the protein level, induction of Al gU synthesis under conditions of extreme heat shock was detected by me tabolic labeling of newly synthesized proteins, two-dimensional gel an alysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of al gR, was also found to be induced by heat shock Under conditions of hig h osmolarity, growth at elevated temperature induced alginate synthesi s in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively these re sults 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 a nd, 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.