Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

Abstract

Lysobacter enzymogenes is a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown in L. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time from L. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis in L. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology of L. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions in L. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis of L. enzymogenes.

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“…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).…”

“…Some initial insights into HSAF regulation have been obtained; however, the regulatory picture is far from complete. We and our collaborators have shown that HSAF levels are increased when L. enzymogenes is grown in poorer medium, e.g., 0.1ϫ tryptic soy broth (TSB), compared to regular 1ϫ TSB (8,11,12). This observation suggests that HSAF synthesis depends on extracellular stimuli.…”

“…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).…”

“…Because of its agricultural applications, L. enzymogenes is emerging as an important model for studying the regulation of HSAF biosynthesis. Previous studies identified several regulators of HSAF production (12,14,25,32). To expand the range of potential factors affecting HSAF synthesis and to gain insights into the mechanisms of such regulation, we systematically deleted genes encoding RRs of TCSs in L. enzymogenes.…”

“…In-frame gene deletions were verified by PCR using appropriate primers (Table 2). Complementation constructs for each mutant were generated as described previously (12). In brief, the DNA fragments containing full-length genes along with their upstream promoter regions were amplified by PCR and cloned into the broad-host-range vectors pBBR1-MCS5 and pUFR047 (Table 1).…”