Zhengqing Fu, Zhenhe Su, Sen Han, Guoliang Qian, Fengquan Liu
Lysobacter enzymogenes is a Gram-negative, environmental ubiquitous bacterium that produces a secondary metabolite, called HSAF (Heat-stable antifungal factor), as an antifungal factor against plant and animal fungal pathogens. 4-hydroxybenzoic acid (4-HBA) is a newly identified diffusible factor that regulates HSAF synthesis via LysRLe, a LysR-type transcription factor (TF). Here, to identify additional TFs within the 4-HBA regulatory pathway that control HSAF production, we re-analyzed the LenB2-based transcriptomic data, in which LenB2 is the enzyme responsible for 4-HBA production. This survey led to identification of three TFs (Le4806, Le4969, and Le3904). Of them, LarR (Le4806), a member of the MarR-family proteins, was identified as a new TF that participated in the 4-HBA-dependent regulation of HSAF production. Our data show that: (i) LarR is a downstream component of the 4-HBA regulatory pathway controlling the HSAF level, while LysRLe is the receptor of 4-HBA; (ii) 4-HBA and LysRLe play an opposite regulatory effect on larR transcription, where larR transcript was negatively modulated by 4-HBA, while LysRLe, in contrast, exerted a positive transcriptional regulation by directly binding to the larR promoter without being affected by 4-HBA in vitro; (iii) LarR, similar to LysRLe, could bind to promoter of the HSAF biosynthetic gene operon, leading to a positive regulation of HSAF production; (iv) LarR and LysRLe could not interact, and instead control HSAF biosynthesis independently. These results outline a previously uncharacterized mechanism by which the antibiotic HSAF biosynthesis in L. enzymogenes is modulated by the interplay of 4-HBA, a diffusible molecule, and two different TFs.IMPORTANCE Bacteria use diverse chemical signaling molecules to regulate a wide range of physiological and cellular processes. 4-HBA is an "old" chemical molecule that was produced by diverse bacterial species, but its regulatory function and working mechanism remain largely unknown. We previously found that 4-HBA in L. enzymogenes could serve as a diffusible factor regulating HSAF synthesis via LysRLe Here, we further identified LarR, a MarR-family protein as a second TF that participates in the 4-HBA-dependent regulation of HSAF biosynthesis. Our results dissected how LarR acts as a protein linker to connect 4-HBA and HSAF synthesis, where LarR also has a cross-talk with LysRLe. Thus, our findings not only provide fundamental insight regarding how a diffusible molecule (4-HBA) adopts two different types of TFs for coordinating HSAF biosynthesis, but also present applied microbiology knowledge for increasing the antibiotic HSAF yield by modification of the 4-HBA regulatory pathway in L. enzymogenes.