The two-component GacS-GacA system activates lipA translation by RsmE but not RsmA in Pseudomonas protegens Pf-5

Abstract

In Pseudomonas spp., the Gac-Rsm signal transduction system is required for the production of lipases. The current model assumes that the system induces lipase gene transcription mediated through the quorum-sensing (QS) system. However, there are no reports of a QS system based upon N-acyl homoserine lactones or the regulation of lipase gene expression in Pseudomonas protegens. In this study, we investigated the regulatory mechanism acting on lipA expression activated by the Gac-Rsm system in P. protegens Pf-5 through deletion and overexpression of gacA, overexpression of rsmA or rsmE, expression of various lacZ fusions, reverse transcription-PCR analysis, and determination of whole-cell lipase activity. The results demonstrated that the GacS-GacA (GacS/A) system activates lipA expression at both the transcriptional and the translational levels but that the translational level is the key regulatory pathway. Further results showed that the activation of lipA translation by the GacS/A system is mediated through RsmE, which inhibits lipA translation by binding to the ACAAGGAUGU sequence overlapping the Shine-Dalgarno (SD) sequence of lipA mRNA to hinder the access of the 30S ribosomal subunit to the SD sequence. Moreover, the GacS/A system promotes lipA transcription through the mediation of RsmA inhibiting lipA transcription via an unknown pathway. Besides the transcriptional repression, RsmA mainly activates lipA translation by negatively regulating rsmE translation. In summary, in P. protegens Pf-5, the Gac-RsmE system mainly and directly activates lipA translation and the Gac-RsmA system indirectly enhances lipA transcription.