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

The biosynthesis of mupirocin, a clinically significant antibiotic produced by <i>Pseudomonas</i> sp. NCIMB 10586, is activated by the <i>N</i>-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 <i>gacS</i> inactivation, <i>gacA</i> inactivation, or the double-mutant of the sRNAs ( RsmY and RsmZ). Similarly, the expressions of <i>mupR</i> and <i>mupI</i>, and AHL synthesis significantly decreased in <i>gacS, gacA,</i> or <i>rsmY/Z</i> 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 <i>rsmA/E/I/F/N</i> 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 <i>mupR</i> 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 <i>Pseudomonas</i> 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 <i>Pseudomonas</i> species, which significantly influences antibiotic production. Our study presented that the expressions of QS genes and <i>mup</i> gene cluster were downregulated in <i>gacS, gacA,</i> or <i>rsmY/Z</i> mutants compared to the wild-type. The inactivation of <i>rsmA/E/I/F/N</i> 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 <i>mupR</i> mRNA. These findings provide the understanding of the regulatory role of Gac/Rsm on mupirocin biosynthesis and <i>mupR/I</i> QS system and lay foundations for further improving mupirocin production.