Abe M, Tsuda M, Kimoto M, Inouye S, Nakazawa A, Nakazawa T
Department of Microbiology, Yamaguchi University School of Medicine, Japan.
Gene. 1996 Oct 3;174(2):191-4. doi: 10.1016/0378-1119(96)00038-8.
A genetic analysis system of Burkholderia cepacia (Bc) was developed which included transposon mutagenesis and complementation of mutation with the cloned genes of interest. To deliver the transposon in this multidrug-resistant microorganism, two plasmids, pKN30 and pKN31, were constructed which contained Tn5 derivatives, Tn5-30Tp and Tn5-31Tp, respectively, carrying KmR and TpR genes. The plasmids have the origin of ColE1 replication and the mobilization gene of RP4. Tn5-31Tp was mobilized to Bc KF1, a strain isolated from a pneumonia patient, by the transfer system of RP4 integrated in the chromosome of Escherichia coli (Ec). Selection with trimethoprim resulted in generation of a number of transposants of Bc KF1. Fourteen protease-deficient mutants were isolated, all of which contained a single transposon marker in the chromosome. Thirteen protease-deficient mutants were also lipase deficient. An Ec-Bc shuttle plasmid, pTS1209, was constructed that consists of oriColE1, oripSa, ApR and CmR genes, and several unique restriction sites for cloning. Plasmid pTS1209 was successfully employed for cloning genes of Bc involved in protease production.
构建了洋葱伯克霍尔德菌（Bc）的遗传分析系统，该系统包括转座子诱变以及用感兴趣的克隆基因对突变进行互补。为了在这种多重耐药微生物中导入转座子，构建了两个质粒pKN30和pKN31，它们分别含有携带卡那霉素抗性（KmR）基因和甲氧苄啶抗性（TpR）基因的Tn5衍生物Tn5 - 30Tp和Tn5 - 31Tp。这些质粒具有ColE1复制起点和RP4的转移基因。通过整合在大肠杆菌（Ec）染色体中的RP4转移系统，将Tn5 - 31Tp导入从一名肺炎患者分离出的菌株Bc KF1中。用甲氧苄啶进行筛选导致产生了许多Bc KF1转座子。分离出14个蛋白酶缺陷型突变体，所有这些突变体在染色体中都含有单个转座子标记。13个蛋白酶缺陷型突变体也缺乏脂肪酶。构建了一个Ec - Bc穿梭质粒pTS1209，它由oriColE1、oripSa、氨苄青霉素抗性（ApR）和氯霉素抗性（CmR）基因以及几个用于克隆的独特限制性酶切位点组成。质粒pTS1209成功用于克隆Bc中参与蛋白酶产生的基因。
