Singer M E, Finnerty W R
Department of Microbiology, University of Georgia, Athens 30602.
J Bacteriol. 1988 Feb;170(2):638-45. doi: 10.1128/jb.170.2.638-645.1988.
A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.
利用本研究构建的大肠杆菌-红球菌穿梭质粒，开发了一种用于红球菌属菌株H13-A的质粒转化系统。红球菌属菌株H13-A含有三个隐蔽的内源质粒，分别命名为pMVS100、pMVS200和pMVS300，大小分别为75、19.5和13.4千碱基(kb)。将pMVS300的一个3.8-kb限制性片段克隆到pIJ30中，pIJ30是一个6.3-kb的pBR322衍生物，含有大肠杆菌复制起点(ori)和氨苄青霉素抗性决定子(bla)，以及一个抗硫链丝菌素的链霉菌基因tsr。从大肠杆菌DH1(pMVS301)中分离得到10.1-kb的重组质粒，命名为pMVS301，并通过聚乙二醇辅助的红球菌原生质体转化和抗硫链丝菌素转化子的筛选，将其转化到红球菌属菌株AS-50（H13-A菌株的衍生物）中。抗硫链丝菌素转化子也对氨苄青霉素有抗性，并显示含有pMVS301，随后将其分离并重新转化回大肠杆菌。pMVS301中克隆的3.8-kb红球菌DNA片段含有一个红球菌复制起点，因为该杂种质粒能够在两个属中复制。通过限制性分析确定，该质粒在大肠杆菌和红球菌转化子中是相同的，并且在两种生物体中都作为一个稳定的独立复制子得以维持。对转化程序的优化导致在红球菌属菌株H13-A及其衍生菌株中，每微克pMVS301 DNA的转化频率在10(5)个转化子范围内。该质粒的宿主范围扩展到红平红球菌、球形红球菌和马红球菌菌株，而以红色红球菌或几种经测试作为受体的棒状杆菌未获得稳定的转化子。限制性图谱显示pMVS301中有14个独特的限制性位点，其中一些位点可能对红球菌属和其他放线菌的分子克隆有用。这是关于红球菌属中质粒转化和异源基因表达的首次报道。
