Harayama S, Rekik M
Department of Medical Biochemistry, University of Geneva, Switzerland.
Gene. 1989 May 15;78(1):19-27. doi: 10.1016/0378-1119(89)90310-7.
A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.
本文描述了一种将非接合型大肠杆菌质粒转移至其他革兰氏阴性菌并使其维持的简单方法。该方法包括通过反向转座介导产生非接合型大肠杆菌质粒与携带Tn10的接合型IncW广宿主范围质粒R388的共整合体。通过从含有这两种质粒的大肠杆菌供体进行接合转移至大肠杆菌受体，并选择表达大肠杆菌质粒标记的转接合子，可轻松实现此类共整合体的分离。当需要在其他革兰氏阴性菌中表达大量基于大肠杆菌载体的克隆以通过互补或重组进行功能分析时，该方法特别有用。通过对含有TOL质粒pWW0分解代谢基因的pBR322杂交质粒在恶臭假单胞菌中的功能分析，展示了该方法的实用性。
