4‐Hydroxybenzoic acid is a diffusible factor that connects metabolic shikimate pathway to the biosynthesis of a unique antifungal metabolite in Lysobacter enzymogenes

Abstract

Heat-stable antifungal factor (HSAF) produced by Lysobacter enzymogenes is a potential lead compound for developing new antibiotics. Yet, how L. enzymogenes regulates the HSAF biosynthesis remains largely unknown. Here, we show that 4-hydroxybenzoic acid (4-HBA) serves as a diffusible factor for regulating HSAF biosynthesis. The biosynthesis of 4-HBA involved an oxygenase, LenB2, and mutation of lenB2 almost completely abolished 4-HBA production, leading to significantly impaired HSAF production. Introduction of a heterologous gene coding for 4-HBA biosynthetic enzyme into the lenB2 mutant restored the production of 4-HBA and HSAF to their corresponding wild-type levels. Exogenous addition of 0.5-1 μM 4-HBA was sufficient to restore HSAF production in the lenB2 mutant. Furthermore, the shikimate pathway was found to regulate the biosynthesis of HSAF via 4-HBA. Finally, we identified a LysR-family transcription factor (LysR ) with activity directed to HSAF production. LysR could bind to the HSAF promoter and, as a result, regulates expression of HSAF biosynthesis genes. The 4-HBA could bind to LysR and appeared to partly enhance formation of the LysR -DNA complex Collectively, our findings suggest that L. enzymogenes produces 4-HBA to serve as an adaptor molecule to link the shikimate pathway to the biosynthesis of a unique antifungal metabolite (HSAF).

Signaling specificity in the c-di-GMP-dependent network regulating antibiotic synthesis in Lysobacter

“…Binding of the Clp-GST fusion protein to the HSAF operon promoter ( 39 ) was determined by MST using Monolith NT.115 (NanoTemper Technologies, Germany), according to the recently described protocol ( 16 ). Briefly, the 50-bp DNA fragments were labeled with 5-carboxy-fluorescein (FAM).…”

“…Lysobacter enzymogenes is a nonpathogenic, plant-associated gammaproteobacterium known for synthesis of the antifungal antibiotic Heat-Stable Antifungal Factor, HSAF ( 11–14 ), which makes it an attractive biological control agent for fungal diseases of agriculturally important plants ( 11 , 12 , 15 ). Therefore, identifying genes and growth conditions that regulate HSAF production has been of significant interest ( 16 , 17 ). Our earlier work revealed that elevated intracellular c-di-GMP levels are detrimental for HSAF production due to lower HSAF biosynthesis gene expression ( 18 ).…”

The impact of the omics era on the knowledge and use ofLysobacterspecies to control phytopathogenic micro-organisms

“…; Su et al . ). The two signalling molecules have a distinct role in the physiology of L. enzymogenes OH11, with 3‐HBA involved in the biosynthesis of the xanthomonadin‐like pigment (Qian et al .…”

“…) and 4‐HBA directly involved in HSAF biosynthesis through the activity of the regulatory protein LysR Le , belonging to the LysR family (Su et al . ).…”

“…These signalling systems are shared by members of Xanthomonadaceae family and are mediated by the diffusible signal factor (DSF) and the diffusible factor (DF; Qian et al 2013). The DF signalling system relies on the signalling molecules 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic acid (4-HBA), synthesized by a bifunctional chorismatase encoded by lenB2 Su et al 2017). The two signalling molecules have a distinct role in the physiology of L. enzymogenes OH11, with 3-HBA involved in the biosynthesis of the xanthomonadinlike pigment ) and 4-HBA directly involved in HSAF biosynthesis through the activity of the regulatory protein LysR Le , belonging to the LysR family (Su et al 2017).…”