Mechanism Of C-di-GMP Signaling Pathway In Regulating Antibiotic Biosynthesis In Lysobacter Enzymogenes OH11

The Gram-negative genus Lysobacter comprises a group of ubiquitous environmental bacteria of more than fifty species.This genus is gaining increasing interests because it has emerged as a rich repertoire for discovering new antibiotics.Lysobacter enzymogenes strain OH 11 is a soil proteobacterium isolated from the rhizosphere soil of pepper.It secretes HSAF(Heat Stable Anti-fungal Factor),comprising a polycyclic tetramate macrolactam skeleton and exhibits potent broad-spectrum anti-fungal activities via a new mode of targeting on the sphingolipid biosynthesis pathway of filamentous pathogens.In addition to the new mode of action,the biosynthetic mechanism of HSAF is also unique,of which one hybrid polyketide synthase and nonribosomal peptide synthetase,encoded by lafB,has been found to catalyze the reactions linking ornithine to two polyketide chains during HSAF biosynthesis.c-di-GMP is a bacterial second messenger identified in 1987.It is synthesized by diguanylate cyclases(DGCs)containing the GGDEF/GGEEF domain,and degraded by phosphodiesterases(PDEs)comprising the EAL or HD-GYP domain.In many bacteria,c-di-GMP has been found to control the planktonic and biofilm lifestyle switch,bacterial pathogenicity,as well as cell cycle and/or chromosome replication.These findings undoubtedly unveil the significance of this small molecule in bacteria physiology.However,the role and/or mechanistic detail of c-di-GMP in antibiotic biosynthesis,such as HSAF,remains unclear.In this study,heterologous expression of known DGC and PDE in L.enzymogenes OH11 ga-ve us a clue that c-di-GMP maybe participate in HSAF regulation.By scanning the whole genome of OH11,26 c-di-GMP metabolizing proteins containing the GGDEF,EAL or HD-GYP domains were identified.We generated libraries of individual deletion mutants(24 in total)and overexpression strains and by quantifying HSAF levels in each of these strains,LchP(a dual GGDEF-EAL protein)and LchD(a single GGDEF protein)were identified to control HSAF biosynthesis.LchP serves as a PDE to modulate HSAF production,and controls transcription of few genes,and most of them are highly related to HSAF biosynthesis gene cluster.LchD is an active DGC that cooperatively works with LchP by physical interaction.Clp,a transcriptional factor,acts as a specific c-di-GMP receptor and controls HSAF biosynthesis by directly binding to lafB promoter.Moreover,Clp both physically interacts with LchP and LchD,and functions as a genetic downstream regulator of the LchP-dependent c-di-GMP signaling pathway.Clp could promote the PDE activity of LchP by physical interaction.We also found Clp is in the downstream of quorum-sensing signal DSF-RpfC/RpfG pathway.Our results not only identify new regulators controlling HSAF production,but also suggest a specific c-di-GMP signaling system consisting of LchP(PDE),LchD(DGC)and Clp(receptor),is likely specific for the antibiotic HSAF biosynthesis,which provides a unique example showing how the ubiquitous second messenger,c-di-GMP,operates its signaling specificity in an antibiotic-producing bacterium.