Idengtification Of A New C-di-GMP Responding Transcription Factor And Its Regulatory Functions In Mycobacterium Smegmatis

Cyclic diguanylate monophosphate(c-di-GMP)is now recognized as one of the most well-conserved second messengers in bacteria and participates in the regulation of a wide range of physiological and pathological processes.However,the regulatory function of c-di-GMP is largely unknown in mycobacteria,and the clearly identified receptor for the signaling molecule(in particular for transcription factors receptors)is extremely rare.In addition,mycobacteria possess a distinctive cell wall that provides a barrier against adverse environment.The enzymes involved in metabolism of cell wall components are always attractive targets for developing antituberculosis drugs.However,the relationship between c-di-GMP signal and the metabolism regulation of cell wall in mycobacteria remains unclear.In this study,we identified a transcription factor in Mycobacterium smegmatis,EcaR,which can interact with both c-di-GMP and the frontline antituberculous drug ethambutol(EMB),and further explored its regulatory function.The major results are listed as follows:(1)We constructed EcaR overexpression strain and its target gene knockout strain of M.smegmatis and found that both strains appear less wrinkle and edge smoothing when growing on solid medium,indicating that EcaR could regulate the growth and colony phenotype of mycobacteria.(2)We purified the EcaR protein and confirmed the specific interaction between EcaR and its own promoter DNA by EMSA assays.(3)Using UV cross-linking and ITC assays,we verified that both EMB and c-di-GMP could interact specifically with EcaR,and both promote the binding of EcaR to DNA.(4)Light microscope and scanning electron microscope(SEM)assays found that mutation strain presented an enhanced cell wall permeability and shorter cell length if compared with wild type strain,suggesting that EcaR may participate in regulating the mycobacterial cell wall metabolism.(5)The cell wall components of M.smegmatis are successfully extracted by monosaccharide extraction and derivatization methods.Using high performance liquid chromatography(HPLC),we determined the content of arabinose(Ara)of the cell walls derived from different strains.Compared with control group,thecontent of Ara was 1.7-fold lower in EcaR overexpression strain cell wall and 1.9-fold lower in the EcaR target gene mutant strains,respectively.In summary,we have indentified EcaR as a new receptor that can bind with two different small molecule,EMB and c-di-GMP.We showed that EcaR could participate in regulating glucolipid metabolism of mycobacteria cell wall and could affect bacterial colony morphology and cell growth.Our findings provided a novel clue for further investigating c-di-GMP signal pathways and cell wall metabolic regulation in mycobacteria.