The Function Of MSMEG＿0372 In Mycobacterium Smegmatis

Purpose: The gene Rv0242 c from Mycobacterium tuberculosis(M.tuberculosis)encodes 3-oxoacyl-acp reductase 4(Fab G4)that is conserved in mycobacteria.It is speculated that Fab G4 is involved in the fatty acid synthetic pathway type Ⅱ(FAS-Ⅱ),and then participates in the synthesis of mycolic acid and cord factor,which affects the biological characteristics and pathogenicity of bacteria.MSMEG＿0372 from Mycobacterium smegmatis(M.smegmatis)mc2 155 is an orthologue of Rv0242 c.To explore the function of Fab G4,we investigated the role of MSMEG＿0372 in M.smegmatis.Methods: 1.Analysis of the genetic organization of MSMEG＿0372 in M.smegmatis mc2 155 and its gene and protein homology to M.tuberculosis H37 Rv Rv0242c using bioinformatics methods.2.MSMEG 0372 gene was amplified by polymerase chain reaction(PCR).The plasmid p MV361-MSMEG＿0372 was transformed into the wild-type(WT)M.smegmatis to construct the overexpression(OE)strain.3.The proliferation rate of the WT parent M.smegmatis mc2 155 and its derivatives MSMEG＿0372 knockout(KO),complementation(COM),and OE strains were compared by plotting the growth curve under shaking culture or static culture.4.The growth characteristics of each strain were compared by observing the colony morphology formed on the Luria-Bertani(LB)medium and the MB7H10 plates,the colony binding ability of Congo red,and colony sliding motility.5.Biofilms were visualized with crystal violet(CV)staining and quantified using the CV method.6.The ultrastructure of the cell envelope was observed by scanning electron microscopy(SEM).Fatty acid and trehalose levels were detected by gas chromatography-mass spectrometry(GC-MS)and high-performance liquid chromatography(HPLC),respectively.Results: 1.The MSMEG＿0372 gene is an orthologous gene of Rv0242 c of the H37 Rv strain and the gene homology between MSMEG＿0372 and Rv0242 c is 78.9% and their protein homology is 82.6%.MSMEG＿0372 is located in region 414922-416274 of the M.smegmatis genome.2.The recombinant plasmid p MV361-MSMEG＿0372 and the overexpression strain were successfully constructed.3.Under shaking or static culture conditions,the turbidity of the OE strain was significantly higher(P<0.05),while the turbidity of the KO strain was lower(P<0.05)than that of the WT strain.The turbidity of the COM strain is between that of WT and OE strains(P<0.05).4.Compared to the WT strain,the colonies of the OE strain became larger and the surface was flat and smooth,the colonies of the KO strain became smaller and more wrinkled,while the colony size and surface roughness of the COM strain were intermediate between those of the WT and the OE strains.Compared to the WT strain,the OE strain had a wider "waxy skirt",while the KO strain had almost no "waxy skirt",and the COM strain had a "waxy skirt" width between those of the WT and OE strains.Compared to the WT strain,the Congo red staining of the KO strain was darker,the Congo red staining of the OE strain was lighter,and the Congo red binding ability of the COM strain was intermediate between those of the WT and OE strains.Compared with the WT strain,the colony sliding mobility of the OE strain was increased,that of the KO strain decreased,and the colony sliding mobility of the COM strain was intermediate between those of the WT and OE strains.5.Biofilms of the WT strain were map-shaped,and biofilms of the KO strain were relatively aggregated.Biofilms of the OE strain were cord-shaped,and biofilms of the COM strain were relatively sparse.Compared to the WT strain,the biofilm content of the KO strain increased(P<0.05)and the biofilm content of the OE and COM strains decreased(P<0.05),but their area of coverage increased.6.Compared to the WT strain,the OE strain has a more uneven cell envelope surface,the KO strain has a smoother one,and the COM strain has a smoothness between those of the WT and OE strains.Compared to the WT strain,the fatty acid and trehalose levels of the KO strain were increased(P<0.05),those of the OE strain were decreased(P<0.05),and the fatty acid and trehalose levels of the COM strain were intermediate between those of the WT and OE strains(P<0.05).Conclusion: 1.MSMEG＿0372 is a conserved homolog of Rv0242 c.2.MSMEG＿0372 promotes mycobacterial growth,changes the colony morphology from rough to smooth,and improves sliding mobility.It reduces biofilm formation and trehalose level and alters lipid profiles.