Identification of a small molecule signaling factor that regulates the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes

Abstract

Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic HSAF (heat-stable antifungal factor), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of L. enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained gfp gene under the control of a DSF (diffusible signaling factor)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 up-regulated the expression of the global regulator Clp (cAMP receptor-like protein). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway

“…Why elevated c-di-GMP levels are inhibitory for HSAF production also remains unknown. Consistent with the notion that c-di-GMP signaling plays an important role in HSAF production is our earlier observation that HSAF production was lower in the rpfG mutant (13). The RpfG protein is an RR containing an HD-GYP domain, which is predicted, based on its similarity to RpfG from Xanthomonas (34), to have c-di-GMP phosphodiesterase activity.…”

“…Two RR proteins (RpfG and PilG) were known to control HSAF levels, based on our earlier work (13,14). In the present work, we used the rpfG deletion mutant (ΔrpfG) as a control and confirmed that HSAF levels were significantly decreased in the mutant.…”

“…In support of this hypothesis, two two-component systems (TCSs) that affect HSAF biosynthesis in L. enzymogenes have been identified (12)(13)(14). One of these TCSs, i.e., RpfC-RpfG, activates HSAF production in response to extracellular levels of the fatty acid signaling molecule diffusible signaling factor 3 (DSF3) (12,13). Another member of a TCS family, PilG, which is an orphan response regulator (RR) protein, was found to negatively regulate HSAF biosynthesis in response to as yet unknown stimuli (14).…”

“…This observation suggests that HSAF synthesis depends on extracellular stimuli. In support of this hypothesis, two two-component systems (TCSs) that affect HSAF biosynthesis in L. enzymogenes have been identified (12)(13)(14). One of these TCSs, i.e., RpfC-RpfG, activates HSAF production in response to extracellular levels of the fatty acid signaling molecule diffusible signaling factor 3 (DSF3) (12,13).…”

Marine Microbiological Enzymes: Studies with Multiple Strategies and Prospects

“…This strategy is also relatively newly developed for marine microorganisms compared with soil studies. Recently, with Lysobacter , researchers identified a small molecule metabolite ( Le DSF3) that regulates the biosynthesis of HSAF [40]. The addition of Le DSF3 in L. enzymogenes cultures increases HSAF biosynthetic gene transcription and HSAF yield.…”