Interplay Between The Bacterial Protein Deacetylase CobB And The Second Messenger C-di-GMP

As a ubiquitous bacterial secondary messenger,Cyclic diguanylate(c-di-GMP)playskey regulatory roles in processes such as bacterial motility and transcription regulation,and also can activate the innate immune pathway of human body,which plays an important role in the host-pathogen interaction.Bacterial energy metabolism is the basic metabolic pathway to maintain the growth and survival of bacteria,which regulates a series of important physiological functions such as bacterial cell cycle and bacterial virulence.The energy metabolism of bacteria is closely regulated by protein acetylation,and most of the enzymes involved in energy metabolism in bacteria are regulated by acetylation.However,as two important regulatory factors in bacteria,whether there is the regulatory relationship between c-di-GMP and acetylation is not known.In this study,we applied an E.coli proteome microarray to globally identify the effector of c-di-GMP,and then explored the regulatory relationship between c-di-GMP and energy metabolism and acetylation pathway.Firstly,we identified 52 c-di-GMP interacting proteins by using the E.coli proteome microarray.One of them is the protein deacetylase CobB,which has many known protein substrates and plays central role in protein acetylation,thus regulates a variety of biological functions,such as the tricarboxylic acid cycle.Secondly,the interaction between c-di-GMP and CobB was confirmed by UV cross-linking assay in vitro,the binding affinity of c-di-GMP and CobB was determined as4.7?M by ITC,which is close to the physiological level of c-di-GMP in bacterial cells.The CobB activity assay showed that c-di-GMP inhibits CobB deacetylase activity,and the inhibition constant(K_i)was determined as 10.6?M.To verify that c-di-GMP can regulate the energy metabolism of bacteria through inhibiting CobB,we measured the level of Acetyl coenzyme A(Ac-CoA)in vivo,and found that high level of c-di-GMP can reduce the level of Ac-CoA in cells by 50%,and consequently inhibit the growth of bacteria.In addition,we demonstrated that c-di-GMP can globally affect the acetylation of CobB's substrates by SILAC MS assay.Thirdly,to identify the binding sites of c-di-GMP on CobB,we employed protein truncation and point mutation,the results showed that Cobb R8,R17 and E21 are the key sites of c-di-GMP binding.Through sequence alignment,we found that the three binding sites are highly conserved among bacteria.We showed that c-di-GMP could also bind to and inhibit the activity of Salmonella Typhimurium CobB.Hence,our results suggested that the inhibition of CobB by c-di-GMP is conserved in bacteria.Finally,to study whether CobB has the negative feedback regulation on the synthesis of c-di-GMP.We tested the acetylation and deacetylation of the enzymes related to c-di-GMP metabolism,we found that c-di-GMP synthetase DgcZK4 site can be acetylated in vivo and deacetylated by CobB.Furthermore,the activity assay showed that the deacetylation of K4 could increase the activity of DgcZ by 1.7-fold.To reveal the mechanism that how K4 acetylation affecting the activity of DgcZ's DGC activity,we used Absolute Quantification(AUQA)peptide to measure the stoichiometric extent of the acetylation modification on DgcZK4.The results showed that 1.4%of K4 was acetylated in vivo.Furthermore,we found that after the acetylation of DgcZK4,DgcZ became unstable and easy to aggregation/precipitation,while after the deacetylation of K4 by CobB,the stability of DgcZ protein was significantly improved.In conclusion,this research revealed that c-di-GMP can inhibit the activity of the protein deacetylase CobB and regulate the energy metabolism of bacteria,meanwhile,CobB regulates the biosynthesis of c-di-GMP by deacetylation of c-di-GMP synthetase DgcZ on K4.Therefore,this study revealed the interplay between bacterial deacetylase CobB and second messenger c-di-GMP,these two key molecules form a negative feedback regulation pathway in vivo.This finding also connects the acetylation-based energy metabolism pathway and the second messenger c-di-GMP pathway.