Shiroza T, Shinozaki N, Hayakawa M, Fujii T, Oguma T, Kobayashi M, Fukushima K, Abiko Y
Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Gene. 1998 Jan 30;207(2):119-26. doi: 10.1016/s0378-1119(97)00611-2.
A novel transformation technique, resident plasmid integration, for the cloning of foreign DNA in oral streptococci was described recently (T. Shiroza and H.K. Kuramitsu, Plasmid 34 (1995) 85-95. This technique is based on the integration of linearized foreign genes by recombination-proficient bacteria onto a resident plasmid, if an appropriate selection marker is flanked by the same anchor sites present in the resident plasmid. Since the transforming vehicles for this system included a pUC-derived replication origin, the high level expression in Escherichia coli cells hindered the cloning of certain genes. In the present study, new plasmids were constructed, two resident plasmids, four integration plasmids, and four cloning plasmids, all of which possess the medium-copy number replication origin, p15A ori, isolated from pACYC177. The resident plasmids consisted of the following three components: the p15A ori (0.65-kb Bg/II fragment), the pVA380-1 basic replicon functional in mutans streptococci (2.5-kb BamHI fragment), and either an erythromycin resistance or a spectinomycin resistance gene (0.9- or 1.1-kb BamHI fragment, respectively). Most of the basic replicon of pVA380-1, except for the 3'-portion of the 0.2-kb region, in the resident plasmid was replaced with a kanamycin resistance gene to construct the four integration plasmids. Therefore, the upstream and downstream anchor sites for the double cross-over event in this new system were 0.65-kb p15A ori and the 0.2-kb portion of the 3'-end of pVA380-1 replicon, respectively. This system was used to clone the gene coding for cycloisomaltooligosaccharide glucanotransferase which produces cycloisomaltooligosaccharide, a potent inhibitor of oral streptococcal glucosyltransferase, isolated from Bacillus circulans chromosome, into Streptococcus gordonii, and its gene product was successfully secreted into the culture media. Plasmids described here should be useful tools for introducing heterologous DNA into resident plasmids following integration in oral streptococci.
最近描述了一种用于在口腔链球菌中克隆外源DNA的新型转化技术——常驻质粒整合技术（T. Shiroza和H.K. Kuramitsu，《质粒》34卷（1995年）85 - 95页）。该技术基于重组能力强的细菌将线性化的外源基因整合到常驻质粒上，前提是合适的选择标记两侧有与常驻质粒中相同的锚定位点。由于该系统的转化载体包含源自pUC的复制起点，在大肠杆菌细胞中的高水平表达阻碍了某些基因的克隆。在本研究中，构建了新的质粒，包括两个常驻质粒、四个整合质粒和四个克隆质粒，它们都具有从中pACYC177分离的中拷贝数复制起点p15A ori。常驻质粒由以下三个部分组成：p15A ori（0.65 kb的Bg/II片段）、在变形链球菌中起作用的pVA380 - 1基本复制子（2.5 kb的BamHI片段）以及红霉素抗性或壮观霉素抗性基因（分别为0.9 kb或1.1 kb的BamHI片段）。常驻质粒中pVA380 - 1的大部分基本复制子，除了0.2 kb区域的3'端部分，被卡那霉素抗性基因取代，构建了四个整合质粒。因此，这个新系统中双交换事件的上游和下游锚定位点分别是0.65 kb的p15A ori和pVA380 - 1复制子3'端的0.2 kb部分。该系统用于将编码环异麦芽寡糖葡糖基转移酶的基因克隆到戈登链球菌中，该酶可产生环异麦芽寡糖，一种从环状芽孢杆菌染色体中分离的口腔链球菌葡糖基转移酶的有效抑制剂，并且其基因产物成功分泌到培养基中。这里描述的质粒应该是在口腔链球菌整合后将异源DNA引入常驻质粒的有用工具。
