Coordination of cyclic di-GMP and 4-hydroxybenzoic acid in regulating antifungal antibiotic biosynthesis in Lysobacter enzymogenes

Small molecules are able to regulate numerous cellular processes through binding to various bacterial receptor proteins, but the mechanisms and functions by which these chemicals coordinate and execute remain poorly understood. 4-hydroxybenzoic acid (4-HBA) and cyclic di-GMP (c-di-GMP) are two such molecules with distinct structures that are produced in Lysobacter enzymogenes to synergistically affect the secretion of an antifungal antibiotic, known as heat-stable antifungal factor (HSAF). In our earlier studies, we showed that CdgL, a YajQ-like protein without DNA-binding domain, was able to physically interact with LysR, a transcription factor, to enhance its binding affinity toward the upstream region of the HSAF biosynthesis operon promoter, hence increasing the HSAF biosynthesis. Interestingly, 4-HBA or c-di-GMP can bind to its cognate receptor of LysR or CdgL, respectively, to regulate the HSAF biosynthesis. Further, c-di-GMP acts by binding to CdgL to induce the dissociation of the CdgL-LysR complex, leading to decreased downstream expression. We now showed that CdgL controlled the transcription of lenB2, which encodes an oxygenase to convert chorismate to 4-HBA. Notably, overexpression of cdgL was found to stimulate lenB2 transcription, which likely increased the intracellular 4-HBA content. Also, 4-HBA could bind to LysR to interrupt the LysR-CdgL complex formation and release of CdgL, which caused a lower affinity of LysR toward DNA and hence decreased HSAF operon expression. These findings, along with our earlier report, allow us to propose a coordination mechanism demonstrating how the HSAF biosynthesis is co-regulated by 4-HBA and c-di-GMP through interactions with their cognate receptors. This new mechanism shall shed light on improving the HSAF yield for practical usage.