耻垢分枝杆菌MSMEG_6281 过表达对菌株生长和形态的影响

Effect of overexpression of MSMEG_6281 on morphology of Mycobacterium smegmatis

摘要:

【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 pVV16-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/pVV16-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。

Abstract:

[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.