Idetification And Funcational Characterization Of Lys385,a Second Messenger C-di-GMP Metabolising Protein In Lysobacter Enzymogenes

Lysobacter,belonging to Xanthomonadaceae family,is a genus of Gram-negative bacteria emerging as a new source of antibiotics.Of the genus,L.enzymogenes is the best characterized species.This species is known for its ability to produce a variety of extracellular lytic enzymes,including chitinase,cellulase and protease.These enzymes are able to destroy the cell wall of pathogenic fungi and oomycetes and are highly linked to biocontrol activity of L.enzymogenes against crop pathogens.In addition to producing abundant lytic enzymes,L.enzymogenes produces diverse bioactive natural products and is recently emerging as a new source of antibiotics,such as the antifungal factor HSAF(Heat-Stable Antifungal Factor)and the antibacterial compound WAP-8294A2.HSAF is broad-spectrum antifungal antibiotic with new structure and mode of action.WAP-8294A2 is a cyclic lipodepsipeptide with a strong activity against Gram-positive bacteria,including MRSA(methicillin-resistant Staphylococcus aureus).The biosynthesis of HSAF and WAP-8294A2 has been experimental documented or proposed in L.enzymogenes,however,the genetic regulatory mechanisms of both HSAF and WAP-8294A2 are beginning to be understood.C-di-GMP is one of the bacterial second messengers,and its intracellular synthesis and hydrolysis is dependent on two differential enzymes named diguanylate cyclases(DGCs)and phosphodiesterases(PDEs)respectively.C-di-GMP is synthesized by the condensation of 2 GTP molecules by DGCs that possess a GGDEF domain,while it is hydrolyzed respectively into pGpG or 2 GMP by PDEs that contain an EAL or HD-GYP domain.These three domains are widespread and highly conserved in a wide range of bacteria,but their numbers vary between different species.It is worthy to notice that GGDEF,EAL or HD-GYP domain is often found associated with sensor domains,arguing that DGC or PDE activity is controlled by direct signal input through these sensor domains.Alternation in intracellular concentration of c-di-GMP leads to a global effect on bacterial phenotypes(e.g.cell motility,biofilm formation and virulence)and multiple cellular behaviors(e.g.cell cycle and chromosome replication),implying the broad importance of this novel signaling molecule in pathogenic and non-pathogenic bacteria.Furthermore,c-di-GMP was found to regulate the biosynthesis of various exopolysaccharides and secondary metabolites,further expanding the regulatory effects of c-di-GMP in bacteria.Importantly,c-di-GMP was shown to regulate antibiotic biosynthesis in Streptomyces coelicolor,providing a strong evidence to link c-di-GMP signaling with antibiotic production in a Gram-positive bacterium.However,the regulation of c-di-GMP signaling on antibiotic biosynthesis in Gram-negative bacteria is lacking of a systematic study so far.Here,we use the Gram-negative L.enzymogenes as a subject,and aim to explore the potential regulation of c-di-GMP signaling on biosynthesis of the antifungal antibiotic HSAF and the antibacterial compound WAP-8294A2.In the present study,we identified a putative DGCs,named Lys385 containing an N-terminal cNMP and C-terminal GGDEF domain,respectively.The presence of the N-terminal cNMP domain suggests this protein(Lys385)most probably senses a yet-to-be-identified nucleic acid to regulate the enzymatic activity expressed by the GGDEF domain located in the N terminus.To explore the potential regulation of this protein in the biosynthesis of HSAF and WAP-8294A2,we generated an in-frame deletion mutant,and the corresponding complemented strain as well as the overexpression strain.The quantitative determination showed that mutation of lys385did not affect the production of HSAF and WAP-8294A2,implying this gene was not involved in the regulation of the biosynthesis of these two antibiotics under the test condition.However,overexpression of lys385 in OH 11 can increase HSAF production.Furthermore,the lys385 deletion mutant or its overexpression strain also exhibited wild-type antimicrobial activity against Rhizoctonia solani.To reveal other putative roles of this gene,we tested and compared the phenotypes of lytic-enzyme production and colony morphology among wild-type OH11,lys385 deletion mutant and its overexpression strain,and we found mutation of lys385 or overexpression of this gene also did not affect the production and/or secretion of chitinase,protease and cellulose,three major lytic enzymes produced by L.enzyogenes,suggesting this gene was not involved in the regulation of the biosynthesis of the tested three lytic enzymes.However,we found overexpression of lys385 resulted in a moist colony,revealing this gene was probably involved in the biosynthesis of extracellular polysaccharide biosynthesis in L.enzymogenes.