A Novel CYCLIC DI-GMP Responding Regulator And Its Regulatory Function For Bacterial Resistance To Oxidative Stress In Mycobacterium Smegmatis

Bis-（3’-5’）-cyclic dimeric guanosine monophosphate（c-di-GMP）is an important second messenger in bacteria.It regulates various physiological processes including biofilm formation,virluence gene expression and bacterial motility and so on.However,the role and regulatory function of the signaling molecule remain quite unclear in mycobacterial species.Using Mycobacterium smegmatis as a model,we previously performed a screen of its transcription factor expression library and identified some potential c-di-GMP receptor regulators.In this study,a Tet R family receptor,Ms5860,was selected from them,and both its regulatory mechanism and function were further carefully characterized.The major results are as follows:1)Using ³²P radioactive-labelled method and UV cross-linking in combination with SDS-PAGE assays,c-di-GMP molecule was found to specifically bind with Ms5860protein.2)Using EMSA and bacterial one-hybrid experiments and Ch IP assays,we confirmed that the transcription factor can specifically binding to its target promoter DNA.Subsequently,footprint experiments found that the DNA substrate recognized by Ms5860contains a reverse palindrome sequence nmotif GGACACATGTCC.3)By determining theβ-galactosidase activities we confirmed that Ms5860negatively regulated expression of a redox gene cluster.Further bacterial growth assays found that the Ms5860-overexpressed strain is more sensitive to H₂O₂than the wildtype strain in the 7H9 medium containing H₂O₂.By contrast,the Ms5860-deleted strain is more resistant to H₂O₂under the similar growth conditions.Consistently,the complemented strain re-obtained a similar growth phenotype to the wildtype strain.（4）By constructing a overexpression strain containing recombinant DGC gene and subsequent determination of bacterial growth curve,we found that high level of c-d-GMP in bacterial cell could significantly increase mycobacterial resistance to H₂O₂.In summary,this study reveals a novel c-di-GMP receptor transcriptional factor which is involved in regulation of mycobacterial antioxidant activity.The finding provides important insight into the c-di-GMP-mediated antioxidant mechanism in mycobacteria.