Preliminary Study On The Function Of Mycobacterium Smegmatis MSMEG＿3150 Gene

Objective:Using Mycobacterium smegmatis as model bacteria,investigating the function of the protein encoded by MSMEG＿3150 gene,including the effects of MSMEG＿3150 on ultrastructure,growth characteristics,surface hydrophobicity,fatty acids content and biofilm formation of Mycobacterium smegmatis.Methods:1.Bioinformatics analysis1.1 The amino acid sequence of Mycobacterium tuberculosis(MTB)Fab G1 was searched onGen Bank website.The protein was input into Ex PASy and the physicochemical properties of Fab G1 were analyzed online.1.2 Gene sequences and amino acid sequences of MTB Fab G1 and Mycobacterium smegmatis mc~2155(MS-MC~2155)MSMEG＿3150 were obtained from Gen Bank,and homology comparison of genes and proteins were performed by Blast and DNAMAN.2.Construction of mutant strains2.1 Construction of MSMEG＿3150 overexpressed strain:The target gene MSMEG＿3150 was recombined with free vector PMV261,and transformed into E.coli DH5α.The overexpressed plasmid PMV261-MSMEG＿3150 was constructed.The positive plasmid was electrically transformed into MS-MC~2155 to obtain MSMEG＿3150 overexpressed strain.2.2 Construction of MSMEG＿3150 gene silent strain:The strain was constructed by CRSIPRi technique.Plasmid p Tet-d Cas9 was transferred to Mycobacterium smegmatis to obtain MS-d Cas9 strain,which was verified by PCR.Two single-stranded sg RNA Oligo sequences targeting MSMEG＿3150 were designed,and the double stranded DNA was formed by annealing and phosphorylated.The double stranded DNA was connected to the p Grna vector to construct the plasmid p Grna-MSMEG＿3150.The positive plasmid was transferred to the receptive state of MS-d Cas9 to obtain MSMEG＿3150 silent strain.3.Functional study of MSMEG＿3150 protein(1)MS-MC~2155 was used as the control group,and MSMEG＿3150 overexpressed strain and silent strain were used as the experimental group.Acid-fast staining was performed for each strain.(2)The bacterial morphology and cell wall structure of each strain was observed by optical microscope,scanning and transmission electron microscopy.(3)The surface hydrophobicity of bacteria was detected by bacterial aggregation experiment and Congo red experiment.(4)The composition and content of fatty acids were determined by gas chromatography-mass spectrometry(GC-MS).(5)The growth curve of bacteria under different conditions was measured and the viability of each strain was tested.(6)Each strain was cultured to form biofilm,and the formation of biofilm was quantified by crystal violet staining.Results:1.Fab G1 protein was predicted to be hydrophobic and stable.The gene sequence homology of MTB Fab G1 and MS-MC~2155 MSMEG＿3150 reached 77.56%,while the protein homology reached 81.18%.2.MSMEG＿3150 overexpressed strain and MSMEG＿3150 silent strain were successfully constructed.3.Functional studies of MSMEG＿3150 protein(1)Acid-fast staining results of each strain showed that the overexpressed strains were longer and the staining was significantly deepened.(2)The results of scanning electron microscopy showed that overexpression of MSMEG＿3150resulted in longer thalli and increased surface folds of silent strains.Transmission electron microscopy showed that the cell wall of overexpressed strain was significantly thickened,while that of silent strain was thinner than that of MS-MC~2155.(3)The results of bacterial aggregation experiment showed that in the absence of Tween80,the precipitation bacteria of overexpressed strains were significantly more than those of MS-MC~2155 and silent strains,indicating that the overexpressed strains were easier to aggregate.Congo red test showed that the overexpressed strain had the deepest staining,that is,the surface hydrophobicity was enhanced.(4)The growth curves of each strain were plotted,and it was found that the survival ability of the overexpressed strain was stronger than that of the wild strain and the silent strain after 60h culture under normal conditions.However,there was no significant difference in the growth curve of the strain cultured under hypoxia.(5)The results of GS-MS showed that the content of C16:0,C18:0,C18:1n9,C24:0 was higher than that of the other strains.(6)After 72h and 120h of culture,the overexpressed strain formed more folds and dense biofilm than the other two strains.On the contrary,the silent strain could only form a thin biofilm and the membrane was incomplete.(7)Crystal violet quantification of biofilm showed that the biofilm formation of overexpressed plants increased significantly,and that of silent plants formed the least biofilm.Conclusion:1.The gene sequence homology of MTB Fab G1 and MS-MC~2155 MSMEG＿3150 is up to 77.56%,while the protein homology is up to 81.18%.2.The protein encoded by MSMEG＿3150 gene of MS-MS~2155 is related to the ultrastructure (including cell length and cell wall thickness),growth characteristics and surface hydrophobicity of the strain.3.The protein encoded by MSMEG＿3150 gene is related to fatty acid synthesis and biofilm formation of MS-MS~2155.