Enzymatic Activity And Functional Characterization Of The Second Messenger C-di-GMP Metabolizing Proteins In Lysobacter Enzymogenes

Lysobacter,belonging to the Xanthomonadaceae family,is a group of environmental friendly and Gram-negative bacteria with difficulties in isolation.L.enzymogenes,a typical species of Lyosbacter,is a well-studied biological control agent.This species produces HSAF(Heat Stable Antifungal Factor),which is a secondary antifungal metabolite with new structure and mode of action.The properties of HSAF in low toxicity,broad-spectrum antimicrobial activity and heat stability make it to have a great potential in developing as a new type of antifungal drug or biopesticide.Cyclic diguanylate(C-di-GMP)is a ubiquitous intracellular second messenger in bacteria.This molecule is synthesized by the GGDEF-domain diguanylate cyclase(DGC)using 2-molecule GTP as substrate,while it is degraded into pGpG or GMP by the phosphodiesterases(PDEs)with EAL or HD-GYP domain proteins,respectively.Specific signals activate or inhibit the activity of corresponding PDEs or DGCs and hence modulate the intracellular c-di-GMP level.The changes in c-di-GMP level lead to exert a series of important physiological and biochemical functions via the downstream c-di-GMP-specific receptors in bacteria.In the past decade,despite the c-di-GMP signaling pathway has been well studied in a variety of plant and animal pathogenic bacteria,less is known,however,about this molecule signaling in bacterial biological control agents,in particular in small-molecule antimicrobial compound biosynthesis.The objective of this study is to explore the enzymatic activity of c-di-GMP metabolizing proteins(PDEs and DGCs)and their potential regulation on the HSAF biosynthesis using L.enzymogenes strain OH11,an isolate from our laboratory as the working organism.The results gained from the present study were provided as follows:A computational analysis led to identification of 26 c-di-GMP metabolizing proteins in the complete genome of strain OH11,including 14 GGDEF-domain proteins(putative DGCs);6 dual GGDEF-EAL proteins(putative DGCs or PDEs);1 EAL-domain protein(putative PDE);5 HD-GYP proteins(putative PDEs).These 26 proteins were further selected for testing their potential activity in synthesizing or degrading c-di-GMP.In the present study,a well-developed,swimming-motility based system involving E.coli MG 1655 and its derivative ?yhjH was adopted for testing the DGC or PDE activity of each target protein.In this system,if the test protein has DGC activity,its expression in the wild-type MG 1655 will lead to a significant decrease of swimming-motility halo,while expression of a target protein having PDE activity will cause a full or at least an increase of swimming motility of ?yhjH,which is completely abolished in this ability.The sensitivity and reliability of this system was validated by expression of a characterized DGC(Slr)and PDE(YjhH)in MG 1655 and ?yhjH,respectively.On the basis of these findings,each of the 26 proteins were tested using such an approach.Our results showed that 9 of 14 GGDEF-domain proteins possessed a strong DGC activity.Their expression in MG 1655 caused a significant decrease in swimming mobility halo,compared to the case from MG 1655 carrying an empty vector.Only 1 of 6 dual GGDEF-EAL proteins were detected to have DGC activity,the 5 remaining proteins had no detectable DGC or PDE activity.Similarly,only 1 of the 6 PDEs was shown to have low PDE activity.This overexpressed PDE having a HD-GYP domain partially rescued the deficiency of AyhjH in swimming motility.The above results promote us to investigate the role of each c-di-GMP protein in producing the antibiotic HSAF.To achieve this point,each gene was overexpressed under the respective native promoter in the wild-type OH 11 in order to alter the intracellular c-di-GMP level in each transformed strain.The results of the HSFA quantifications showed that overexpression of 10 DGC coding genes resulted in a significant decrease in HSAF production,suggesting high c-di-GMP level decreased the HSAF yield.This further reveals that the intracellular c-di-GMP level suppressed the antibiotic HSAF biosynthesis in L.enzymogenes.In agreement with this finding,we observed that expression of the known DGC,Slr coding gene under a constitutive promoter led to a remarkable decrease in HSAF yield.In addition,we also found that each of 5 degenerated GGDEF proteins(no DGC activity was note detected in this study)at overexpressed state caused a significant decrease in the HSAF production,suggesting these 5 proteins probably act as c-di-GMP receptor to perform their respective regulation in the HSAF biosynthesis.Taken together,this study paves the way for future in-depth study on how c-di-GMP signaling regulates the HSAF biosynthesis in L.enzymogenes.