Idengtification Of A Novel C-di-GMP Receptor In Mycobacterium Smegmatis

Mycobacterium tuberculosis,an important human pathogen,has its own c-di-GMP signaling system.However,the c-di-GMP-involved signaling pathways remain largely unclear.In this study,using M.smegmatis as a model species,we screened and identified its methionyl-tRNA synthetase（MetG）as a novel c-di-GMP receptor protein in mycobacteria.The detailed results are as follows:（1）Using ³²P radioactive-labelled method as well as UV cross-linking assays,we confirmed MetG can directly bind with c-di-GMP molecule.Further,we found that mycobacterial MetG can specifically interact with c-di-GMP based on our competition experiment together with comparative assays for the various MetGs derived from different bacterial species.（2）Utilizing bioinformatics tools,we predicted four potential amino acids residues essential for the interaction between c-di-GMP and MetG.We,therefore,further designed sevseal mutant MetGs and examined the interactions between c-di-GMP and these mutant proteins.Finally,the 294 tryptophan,which is located on the catalytic center,was shown to play an important role in the binding of MetG with c-di-GMP.（3）By measuring the catalytic activity of the protein both in the presence and absence of c-di-GMP,we found that the c-di-GMP could inhibit the enzyme activity of MetG.Further more,we found that both c-di-GMP and MetG could obviously influence mycobacterial various phenotypes such as mobility and biofilm formation by constructing recombinant strains with different expression levels of met G or c-di-GMP.In summary,this study identified the mycobacterial methionyl-tRNA synthetase as a novel c-di-GMP receptor for the first time,and also investigated its regulation on the activity of MetG.Our results provided a novel clue for further exploring the regulatory mechanisms of c-di-GMP in mycobacteria.