Regulation of rpoS Gene Expression in Pseudomonas : Involvement of a TetR Family Regulator

Abstract

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-densitydependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.The rpoS gene codes for sigma factor RpoS (also called s and 38 ) (12, 18), which was identified as a central regulator during stationary phase in Escherichia coli; this factor is involved in the survival of famine conditions and in cross-protection against osmotic, acidic, oxidative, and heat stresses (19,22). Since then, it has been identified in various gram-negative bacteria, including several species belonging to the fluorescent pseudomonads; these findings demonstrate that in these bacteria as well this factor has an important regulatory role, including adaptation to nutrient-limiting conditions, survival in the presence of several environmental stresses, and the production of virulence factors (16,17,34,37,42,43).RpoS is an alternative sigma factor, resulting in the alteration of RNA polymerase core specificity and thereby switching gene expression at stationary phase. The levels of RpoS within a bacterial cell are carefully controlled, since perturbations in the relative amounts can have severe consequences. Thus, the regulation of RpoS levels is of crucial importa...

Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391

“…It was previously shown for P. putida that PsrA regulates the expression of rpoS (Kojic & Venturi, 2001) by binding to its promoter (Kojic et al, 2002). Our results indicate a similar regulation in PCL1391.…”

“…A defect in the regulatory genes psrA or gacS results in a severe decrease of the amounts of RpoS. Similar observations were made in other strains (Kojic & Venturi, 2001;SchmidtEisenlohr et al, 2003;Whistler et al, 1998).…”

“…regulating the phz operon, according to results in other strains (Kojic & Venturi, 2001). Previous work in rich growth medium indicated that PsrA inhibits N-AHL and PCN production in PCL1391 (Chin-A-Woeng et al, 2005).…”

“…It was shown that psrA regulates rpoS in other Pseudomonas species (Kojic & Venturi 2001), probably via binding to the promoter of the rpoS gene at a PsrA-binding box. Therefore, an attempt was made to complement the psrA mutant PCL1111 with constitutively expressed rpoS.…”

“…The psrA gene of Pseudomonas putida was shown to regulate the transcription of the rpoS gene (Kojic & Venturi, 2001) by directly binding to the rpoS promoter (Kojic et al, 2002). rpoS encodes the stationary-phase alternative sigma factor s s , which is responsible for the switch in gene expression occurring upon exposure of cells to starvation and/ or various stresses (Lange & Hengge-Aronis, 1991).…”