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

长期以来，分支杆菌分子生物学技术的发展一直被认为是困难的。在建立和发展分枝杆菌分子克隆体系的实验中，获得了以下结果：1.分别以大肠杆菌和偶发分枝杆菌的pACYC177和pMF129为载体，构建了能在大肠杆菌和牛分枝杆菌(M.bovis BCG)和耻垢分枝杆菌中复制的重组穿梭载体pUT21(19.2kb)。与以前开发的穿梭载体pYT937相比，该重组载体的导入频率更高。pYT937是用丛枝杆菌pMSC262和pACYC177.2构建的穿梭载体。通过对转化细胞进行20次继代培养，然后用溴化乙锭在45ﾟ条件下进行固化处理，获得了可有效转化的耻垢分枝杆菌变异株。利用大肠杆菌克隆载体pHSG298制备了几种分枝杆菌的基因组文库，并通过电穿孔的方法将其导入到大肠杆菌受体细胞K12 C600中。然而，没有获得任何表达分枝杆菌基因的转化子，这表明分枝杆菌结构基因的启动子在大肠杆菌细胞中不被识别。作为一种新的快速生长的分枝杆菌表型，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均显示出胞外溶血活性。这一新特性似乎不足以克隆该基因，因为活性物质不太可能是一种酶或其他类型的蛋白质。溶血活性的致病作用仍有待研究。

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.