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耐辐射异常球菌SARK隐蔽质粒最小复制子的表征及通用型大肠杆菌-耐辐射异常球菌穿梭载体的构建

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors.

作者信息

Meima R, Lidstrom M E

机构信息

Departments of Chemical Engineering, University of Washington, Seattle, Washington 98195-1750, USA.

出版信息

Appl Environ Microbiol. 2000 Sep;66(9):3856-67. doi: 10.1128/AEM.66.9.3856-3867.2000.

Abstract

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

摘要

测定了耐辐射异常球菌SARK隐蔽质粒pUE10一个12 kb片段的核苷酸序列,以指导开发用于耐辐射异常球菌的小型通用克隆系统。序列注释揭示了12个可能的开放阅读框。其中包括repU和resU基因,其预测产物分别与复制蛋白和位点特异性解离酶具有相似性。使用大肠杆菌中的过表达系统证明了这两个基因的产物。发现RepU是复制所必需的,而ResU是pUE10衍生物稳定维持所必需的。使用纯化的His标签RepU进行凝胶迁移分析确定了推定的结合位点,并表明RepU可能参与复制起始和repU表达的自动调节。此外,发现了一个编码可能的抗限制蛋白的基因,该基因被证明是高转化频率所必需的。来自耐辐射异常球菌菌株SARK的该质粒的复制区域和推定的复制基因的排列与嗜热栖热菌中发现的质粒相似,但与耐辐射异常球菌菌株R1中发现的45.7 kb质粒不同。通过从12 kb片段中顺序缺失片段来确定耐辐射异常球菌中自主复制所需的最小区域。由此产生的由约2.6 kb组成的最小复制子用于构建大肠杆菌和耐辐射异常球菌的穿梭载体。该载体pRAD1是一种方便的通用克隆载体。此外,pRAD1用于生成启动子探针载体,并且含有lacZ和耐辐射异常球菌启动子的质粒被证明能有效表达LacZ。

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