Regulation of cyclic lipopeptide orfamide A biosynthesis in Pseudomonas protegens by the Gac–Rsm–LuxR Cascade

Orfamide A, a lipopeptide produced by Pseudomonas protegens Pf-5, is a key determinant of its biocontrol properties. In this study, we investigated the regulatory interactions among the GacS/A two-component system, small RNAs (sRNAs), repressor proteins, and two LuxR-type transcription factors in orfamide A biosynthesis. We found that GacS/A indirectly regulates orfamide A production by enhancing transcription of three sRNAs (RsmX, RsmY, and RsmZ). RsmY and RsmZ synergistically relieve repression by RsmA and RsmE, while RsmX plays a lesser role, likely counteracting only one repressor. LuxR-type transcription factors, LuxR1 and LuxR2, which positively regulate orfamide A synthesis, are directly repressed by RsmA and RsmE via binding to their 5′ untranslated regions, linking them to the Gac–Rsm signaling cascade. We further demonstrated that LuxR2 activates luxR1 expression, which in turn facilitates orfamide A production by binding to the promoter of the orfamide A biosynthetic gene cluster. Importantly, we showed that this entire regulatory cascade operates in the rhizosphere and directly influence biocontrol efficacy. These findings provide a comprehensive understanding of the Gac–Rsm–LuxR pathway in orfamide A biosynthesis and offer valuable insights for the development of biocontrol agents based on Pseudomonas strains.