Development of a new host-vector system and its application to genetic analysis in mycobacteria

分枝杆菌分子生物学技术的发展一直被认为是困难的。在分枝杆菌分子克隆系统的开发和建立过程中，取得了以下结果：1。分别以大肠杆菌质粒pACYC 177和偶发分枝杆菌质粒pMF 129为载体，构建了能在大肠杆菌和分枝杆菌（牛分枝杆菌BCG和耻垢分枝杆菌）中复制的穿梭质粒pUT 21（19.2kb）。重组质粒可以比先前开发的穿梭载体pYT 937更频繁地引入，所述穿梭载体pYT 937已经用结核分枝杆菌质粒（pMSC 262）和pACYC 177.2构建。通过将转化的细胞传代培养20次，然后在45 ℃下用溴化乙锭对质粒进行固化处理，分离出耻垢分枝杆菌的有效可转化变体。使用大肠杆菌克隆载体pHSG 298制备一些分枝杆菌物种的基因组文库，然后通过电穿孔将其引入大肠杆菌受体细胞（K12 C600）中。然而，没有获得任何表达分枝杆菌基因的转化子，表明分枝杆菌结构基因的启动子在大肠杆菌细胞中不会被识别.作为快速生长分枝杆菌的一种新表型，在偶然分枝杆菌、龟分枝杆菌和耻垢分枝杆菌的培养上清液中证实了胞外溶血活性。这一新特征似乎不足以克隆该基因，因为其活性物质不太可能是酶或其他类型的蛋白质，溶血活性的致病作用还有待研究。

Development of techniques for molecular biology of mycobacteria has long been considered to be difficult. During the experiments aimed to develop and establish molecular cloning systems in mycobacteria, the following results could be obtained :1. A recombinant shuttle vector, pUT21 (19.2kb), capable of replicating in both E.coli and mycobacteria (M.bovis BCG and M.smegmatis) was constructed using the plasmids pACYC177 and pMF129 of E.coli and M.fortuitum, respectively. The recombinant plasmid could be introduced more frequently than a previously developed shuttle vector, pYT937, which has been constructed with a M.scrofulaceum plasmid (pMSC262) and pACYC177.2. The efficiently transformable variants of M.smegmatis were isolated by subculturing the transformed cells 20 times followed by a curing treatment of the plasmid with ethidium bromide at 45ﾟC.3. The genomic libraries of some species of mycobacteria were prepared using a E.coli cloning vector, pHSG298, and then introduced into E.coli recipient cells (K12 C600) by electroporation. However, no any transformants expressing the mycobacterial genes could be obtained, suggesting that the promotors of the mycobacterial structural genes would not be recognized in E.coli cells.4. As a new phenotype of rapidly growing mycobacteria, extracellular hemolytic activity was demonstrated in culture supernatants of M.fortuitum, M.chelonae, and M.smegmatis. This new character seemed to be inadequate for cloning the gene since the active substance is not likely to be an enzyme or other type of protein.Pathogenic role of the hemolytic activity has been remained to be studied.