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。在开发和建立分枝杆菌分子克隆系统的实验过程中，获得了以下结果：1。以大肠杆菌的pACYC177质粒和福氏分枝杆菌的pMF129质粒为载体，构建了能在大肠杆菌和分枝杆菌（牛卡介苗和耻垢杆菌）中复制的重组穿梭载体pUT21 （19.2kb）。该重组质粒可以比先前开发的穿梭载体pYT937更频繁地引入，pYT937是用m.s rofulaceum质粒（pMSC262）和pACYC177.2构建的。将转化后的细胞传代培养20次，然后用溴化乙啶（ethidium）在45 C.3下固化质粒，分离出高效转化的耻垢芽孢杆菌。利用大肠杆菌克隆载体pHSG298制备了部分分支杆菌的基因组文库，并通过电穿孔法导入大肠杆菌受体细胞K12 C600。然而，没有获得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会被识别。作为一种快速生长的分枝杆菌的新表型，在福氏分枝杆菌、龙分枝杆菌和耻垢分枝杆菌的培养上清液中发现了细胞外溶血活性。这个新性状似乎不足以克隆基因，因为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.