Global Gac/Rsm regulatory system activates the biosynthesis of mupirocin by controlling the MupR/I quorum sensing system in Pseudomonas sp. NCIMB 10586.

Abstract

The biosynthesis of mupirocin, a clinically significant antibiotic produced by Pseudomonas sp. NCIMB 10586, is activated by the N-acyl homoserine lactone (AHL) MupR/I quorum sensing (QS) system. However, to date, limited research has focused on the influence of global regulators such as the GacS/A two-component system (TCS) on the MupR/I QS system or mupirocin biosynthesis. In this study, we characterized the regulatory components of the Gac/Rsm transduction system in the mupirocin-producing model strain NCIMB 10586 and investigated their interconnection with the MupR/I QS circuit and subsequent mupirocin biosynthesis. The production of mupirocin was hampered by either gacS inactivation, gacA inactivation, or the double-mutant of the sRNAs ( RsmY and RsmZ). Similarly, the expressions of mupR and mupI, and AHL synthesis significantly decreased in gacS, gacA, or rsmY/Z mutants, indicating that the GacS/A system stimulates mupirocin biosynthesis via the MupR/I QS system. Five CsrA family proteins, RsmA/E/I/F/N, were found in strain NCIMB 10586, and the single and multiple mutants of rsmA/E/I/F/N showed different phenotypes with respect to mupirocin production. Our results revealed that mupirocin biosynthesis was likely to be negatively regulated by RsmA/E/I, but positively regulated by RsmF. Additionally, the RsmF protein was shown to interact with the 5' leader of mupR mRNA. In summary, the Gac/Rsm system positively regulates the biosynthesis of mupirocin mainly through the MupR/I QS system, and the model of the regulatory mechanism is proposed. The elucidation of the Gac/Rsm-MupR/I regulatory pathway could help devise ways for improving mupirocin production through genetic engineering.IMPORTANCEThe Gac/Rsm regulatory system plays a global regulatory role in bacterial physiology and metabolism, including secondary metabolism. Mupirocin is a clinically important antibiotic, produced by Pseudomonas sp. NCIMB 10586, whose biosynthesis is activated by the MupR/I quorum sensing system. Global regulators have important impacts on the gene expression of secondary metabolic gene clusters and QS genes, and the GacS/A two-component system is one of the main regulators across Pseudomonas species, which significantly influences antibiotic production. Our study presented that the expressions of QS genes and mup gene cluster were downregulated in gacS, gacA, or rsmY/Z mutants compared to the wild-type. The inactivation of rsmA/E/I/F/N in NCIMB 10586, encoding CsrA family proteins, showed different regulatory traits of mupirocin production, in which the RsmF protein could interact with the 5' UTR region of mupR mRNA. These findings provide the understanding of the regulatory role of Gac/Rsm on mupirocin biosynthesis and mupR/I QS system and lay foundations for further improving mupirocin production.