| Mycobacteria represent a large family of gram-positive bacteria which are rich in lipids and possess envelope structures.Mycobacterial species include the causative agent of tuberculosis,Mycobacterium tuberculosis,and the nonpathogenic Mycobacterium smegmatis.The lipid homeostasis of mycobacterial envelope has a critically important effect on bacterial growth,cellular morphology,biofilm formation and pathogenicity.However,the research on regulation network of lipid metabolism in mycobacteria is just beginning,and the potentially global regulatory factors remain to be uncovered.In this study,we identified a novel transcriptional factor,designated as Mmb R,and systematically proved its global regulatory effects on colony morphology,biofilm formation,lipid homeostasis and drug resistance in the model strain of M.smegmatis.In our previous screening experiments,the expression level of a Tet R-like transcriptional factor gene was found to significantly affect bacterial colony phenotype.Based on this finding,the mmb R deletion strain was constructed,and the growth differences among the deletion,the complementary and the wild type strains were compared and analyzed.We confirmed that the deletion of mmb R led to the colony phenotype of M.smegmatis changed from the typically wrinkled rough to smooth,the defect in biofilm formation and bacterial length.The phenotypes of the complementary strain were restored similarly to that of the wild-type when mmb R was reintroduced into the deletion strain.These results suggest that Mmb R affects bacterial growth and biofilm formation.In order to further analyze the regulatory effects of Mmb R,the different gene expression profile between the mmb R deletion and wild type strain was firstly compared by transcriptomic analysis.The results showed that the deletion of mmb R led to the expression of 2006 genes,almost 1/3 of the mycobacterial genome,were significantly upregulated,indicating that Mmb R globally and negatively regulated gene expression.Gene clustering analysis showed that most of differentially expressed genes were involved in fatty acid metabolism and ? oxidation.Furthermore,we confirmed that Mmb R possessed the characteristics of nucleoid-associated proteins.For example,it can protect DNA from being digested by Top A and DNase I in vitro,and it can colocalize with chromatin DNA in vivo,which are consistent with its global regulatory function.Interestingly,the distribution of differently expressed genes obviously formed two highly up-regulated regions within the genome.The in vitro EMSA and in vivo Ch IP assays confirmed that Mmb R could directly bind to promoters of these gene clusters.The footprint and EMSA assays shown that Mmb R specifically bound to these promoters by recognizing conserve binding motif,and that long chain acyl-Co A or fatty acid molecules could inhibit its DNA binding activity.Strikingly,the binding affinity of Mmb R to promoters of adjacent target genes were significantly higher than that of the distant promoters,indicating an obvious polarity effect.These results suggested that Mmb R had both the characteristic of a nucleoid-associated protein and the polarity effect of DNA-binding activity,and it could globally regulate the expressions of genes participated in fatty acid metabolism and ? oxidation processes in M.smegmatis.Next,the effect of Mmb R on lipid metabolism was determined through comparative lipidomic assays.The data showed that mmb R deletion led to a significant decrease in the contents of 21 saturated fatty acids and 13 unsaturated fatty acids,indicating that mmb R knockout inhibited fatty acid accumulation in M.smegmatis.Comparative lipidomic assay between the biofilms formed by wild type and mmb R deletion M.smegmatis strains revealed that the mycobacterial biofilm lipid components,including 6 mycolic acids,8 fatty acids and 4 mycobactins were significantly decreased,while 6 methyl-branched fatty acids and 8 glucose monomycolates were significantly increased in the mmb R knockout strain.A further analysis of transcriptomic in combination with lipidomic assay showed that mmb R deletion induced an enhanced fatty acid metabolism,which led to an increased biosynthesis of methyl-branched fatty acid and its derivatives.By contrast,the mmb R deletion decreased the de nevo synthesis of mycolic acids,which also resulted in a decrease in mycobatin synthesis.The alteration of mycobacterial envelope lipid composition usually affects the bacterial sensitivity to antibiotics.We determined the survival rates and MICs of mmb R recombinant strains to two anti-TB drugs,Rif and INH,and the results showed that the mmb R-deleted strain became more sensitive to both drugs than the wild type strain.These results suggested that the expression of mmb R significantly affected the fatty acid accumulation,biofilm lipid composition and bacterial sensitive to anti-tuberculosis drugs in M.smegmatis.Therefore,in this study,we successfully characterized a transcriptional factor Mmb R,who globally regulates the fatty acid ?-oxidation in M.smegmatis,and dissected the molecular mechanism on lipid homeostasis maintenance mediated by Mmb R.A model in which Mmb R regulates bacterial lipid metabolism to maintain lipid homeostasis and triggers biofilm formation has been proposed.These works have enhanced greatly our understandings on the regulation mechanism of mycobacterial biofilm formation and lipid metabolism. |
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