耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

【目的】耻垢分枝杆菌(Mycobacterium smegmatis mc2155,mc2155)MSMEG_6281为结核分枝杆菌自溶素Rv3717的同源蛋白,通过建立过表达MSMEG_6281的耻垢分枝杆菌菌株,推测该蛋白对耻垢分枝杆菌肽聚糖代谢的影响。【方法】利用RT-PCR方法检测乙胺丁醇(Ethambutol,EMB)作用后MSMEG_6281基因的表达变化;以耻垢分枝杆菌基因组DNA为模板,采用PCR技术克隆MSMEG_6281基因,构建分枝杆菌表达质粒p VV16-MSMEG_6281,进一步建立MSMEG_6281过表达的耻垢分枝杆菌菌株;利用生长曲线检测MSMEG_6281过表达对耻垢分枝杆菌生长的影响;利用扫描电子显微镜分析MSMEG_6281过表达引起的耻垢分枝杆菌形态变化。【结果】EMB处理引起MSMEG_6281基因表达上调;构建了过表达MSMGE_6281的耻垢分枝杆菌菌株(mc2155/p VV16-MSMEG_6281);过表达MSMGE_6281的耻垢分枝杆菌生长缓慢,菌体形态由短杆状转变为长杆状。【结论】MSMGE_6281的过表达可改变耻垢分枝杆菌形态。MSMGE_6281的功能与细胞壁肽聚糖水解相关,在mc2155细胞壁形态维持方面发挥重要作用。

Effect of overexpression of MSMEG_6281 on morphology of Mycobacterium smegmatis

Objective] In order to predict the function of MSMEG_6281 in Mycobacterium smegmatis mc2155, we analyzed the effect of overexpression of MSMEG_6281 on cellular growth and morphological characteristics. Methods] Ethambutol, one of the first anti-tuberculosis drugs, can destroy structure of cell wall of mc2155 and cause the changes of cellular morphology. The expression change of MSMEG_6281 is determined after ethambutol treatment by RT-PCR. The intact MSMEG_6281 gene was amplified from M. smegmatis mc2155 genomic DNA . After analyzing by enzymatic digestion and DNA sequencing, the MSMEG_6281 gene was cloned into vector pVV16 to constitute the recombinant plasmid of pVV16-MSMEG_6281. The expression of MSMEG_6281 for the recombinant mc2155 strain was confirmed by SDS-PAGE and western blotting. Results] RT-PCR results show that gene expression of MSMEG_6281 was up-regulated under the condition of ethambutol treatment for 6 h and 12 h. The recombinant mc2155 strain over-expressing MSMEG_6281 demonstrated a lag growth, and the cellular morphological turns longer than wild type mc2155. Conclusion] MSMEG_6281 must be related with peptidoglycan metabolism of the cell wall.