A regulatory RNA (PrrB RNA) modulates expression of secondary metabolite genes in Pseudomonas fluorescens F113

The GacS-GacA two-component signal transduction system, which is highly con served in gram-negative bacteria, is required for the production of exoenzy mes and secondary metabolites in Pseudomonas spp. Screening of a Pseudomona s fluorescens F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacS and gacA mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious Pseudomonas protein coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically comple ment gacS and gacA mutants and is itself regulated by the GacS GacA two-com ponent signal transduction system. Primer extension analysis of the 132-bas e transcript identified the transcription start site located downstream of a sigma(70) promoter sequence from positions -10 to -35. Inactivation of th e prrB gene in F113 resulted in a significant reduction of 2,4-diacetylphlo roglucinol (Phl) and hydrogen cyanide (HCN) production, while increased met abolite production was observed when prrB was overexpressed. The prrB gene sequence contains a number of imperfect repeats of the consensus sequence 5 '-AGGA-3', and sequence analysis predicted a complex secondary structure fe aturing multiple putative stem-loops with the consensus sequences predomina ntly positioned at the single-stranded regions at the ends of the stem-loop s. This structure is similar to the CsrB and RsmB regulatory RNAs in Escher ichia coli and Erwinia carotovora, respectively. Results suggest that a reg ulatory RNA molecule is involved in GacA-GacS-mediated regulation of Phl an d HCN production in P. fluorescens F113.