Identification Of The Global Regulator,Ms6174,and Its Regulatory Mechanistic Of Mycobacterium Smegmatis Biofilms

Mycobacteria are a large class of Gram-strain-positive bacteria featuring an extraordinary thick cell envelope rich in lipids.The most wellknown mycobacteria are Mycobacterium tuberculosis,the causative agent of tuberculosis,and its model strain Mycobacterium smegmatis.Bacterial biofilms are a population of bacteria encapsulated by an extracellular matrix such as lipids,polysaccharides,DNA and proteins,and its formation generally plays an important role on stress resistance and pathogenicity of pathogenic bacteria.The formation is closely related to their cell wall lipid composition.However,the regulatory mechanisms of lipid metabolism affecting the formation of mycobacterial biofilms are currently largely unknown,and the associated transcriptional regulators remain obscure.In this paper,we characterized a widely regulated transcription factor Ms6174 which is a nucleoid-associated protein in M.smegmatis.And we also found that Ms6174 negatively regulate mycobacterial biofilm formation by regulating the expression of multiple lipid metabolism related genes.The achieved results were listed as follows:(1)The gene,ms6174,encoding a transposition factor which affected the formation of mycobacterial biofilm was characterized in M.smegmatis using transcription insertion mutant library screening as described previously.Ms6174 was expressed and purified.Subsequently,electrophoretic mobility shift assay(EMSA)and β-galactosidase activity assays confirmed that Ms6147 negatively regulated the ms6147 operon.(2)We found that Ms6174 has the characteristics of a nucleoidassociated protein by EMSA,DNase I digestion,and fluorescence colocalization assays.Namely,Ms6174 is a novel nucleoid-associated protein in M.smegmatis.(3)RNA-Seq data analysis showed that 682 genes expression were significantly changed in the ms6174 knockout strain compared with that of the wild-type strain,of which 387 genes were up-regulated and 295 genes were down-regulated.Among them,142 lipid metabolism related genes were changed.The above-mentioned results illustrate that Ms6174 is a widely regulated transcription factor that mainly regulates the expression of lipid metabolism genes in M.smegmatis.(4)By analysis of the colony morphology and the formation of airliquid interface pellicles,we found that wrinkles and air-liquid interface pellicles of the ms6174 knockout strain significantly increased compared to that of the wild-type strain.Therefore,the transcription factor,Ms6174,regulates the biofilm formation of M.smegmatis.In summary,this study identified a global regulatory function of transcription factor(Ms6174)in M.smegmatis.Firstly,the autoregulatory role of Ms6174 was identified and its nucleoid-associated protein were charactered.Secondly,Ms6174 globally regulated genes expression by transcriptomic analysis.Finally,it is found that Ms6174 negatively regulate mycobacterial biofilm formation,and it is inferred that the formation of mycobacterial biofilm is regulated by the expression of genes involved in lipid metabolism.The findings identified a new nucleoidassociated protein in mycobacteria and deepened our understanding of the regulatory mechanisms of mycobacterial biofilm formation.