...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of bacteriology >In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030.
【24h】

In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030.

机译：LlaI的体内限制是由共轭乳球菌质粒pTR2030上的三个基因编码的，这些基因与llaIM排列在一个操纵子中。

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The LlaI restriction and modification (R/M) system is encoded on pTR2030, a 46.2-kb conjugative plasmid from Lactococcus lactis. The llaI methylase gene, sequenced previously, encodes a functional type IIS methylase and is located approximately 5 kb upstream from the abiA gene, encoding abortive phage resistance. In this study, the sequence of the region between llaIM and abiA was determined and revealed four consecutive open reading frames (ORFs). Northern (RNA) analysis showed that the four ORFs were part of a 7-kb operon with llaIM and the downstream abiA gene on a separate transcriptional unit. The deduced protein sequence of ORF2 revealed a P-loop consensus motif for ATP/GTP-binding sites and a three-part consensus motif for GTP-binding proteins. Data bank searches with the deduced protein sequences for all four ORFs revealed no homology except for ORF2 with MerB, in three regions that coincided with the GTP-binding motifs in both proteins. To phenotypically analyze the llaI operon, a 9.0-kb fragment was cloned into a high-copy-number lactococcal shuttle vector, pTRKH2. The resulting construct, pTRK370, exhibited a significantly higher level of in vivo restriction and modification in L. lactis NCK203 than the low-copy-number parental plasmid, pTR2030. A combination of deletion constructions and frameshift mutations indicated that the first three ORFs were involved in LlaI restriction, and they were therefore designated llaI.1, llaI.2, and llaI.3. Mutating llaI.1 completely abolished restriction, while disrupting llaI.2 or llaI.3 allowed an inefficient restriction of phage DNA to occur, manifested primarily by a variable plaque phenotype. ORF4 had no discernible effect on in vivo restriction. A frameshift mutation in llaIM proved lethal to L. lactis NCK203, implying that the restriction component was active without the modification subunit. These results suggested that the LlaI R/M system is unlike any other R/M system studied to date and has diverged from the type IIS class of restriction enzymes by acquiring some characteristics reminiscent of type I enzymes.
机译：LlaI限制和修饰（R / M）系统在pTR2030（一种来自乳酸乳球菌的46.2 kb结合质粒）上编码。先前测序的IIIaI甲基化酶基因编码功能性类型的IIS甲基化酶，位于abiA基因上游约5 kb处，编码流产的噬菌体抗性。在这项研究中，确定了IlaIM和abiA之间的区域序列，并揭示了四个连续的开放阅读框（ORF）。 Northern（RNA）分析显示，四个ORF是7la操纵子的一部分，带有llaIM和下游abiA基因在单独的转录单元上。推导的ORF2蛋白质序列揭示了ATP / GTP结合位点的P环共有基序和GTP结合蛋白的三部分共有基序。用推导的所有四个ORF的蛋白质序列进行的数据库搜索显示，除了ORF2与MerB以外，在三个区域中均与两个蛋白质的GTP结合基序一致，没有同源性。为了表型分析IlaI操纵子，将9.0kb的片段克隆到高拷贝数的乳球菌穿梭载体pTRKH2中。与低拷贝数的亲本质粒pTR2030相比，所得的构建体pTRK370在乳酸乳球菌NCK203中表现出明显更高的体内限制性和修饰水平。缺失结构和移码突变的组合表明前三个ORF参与了LlaI限制，因此将其命名为llaI.1，llaI.2和llaI.3。突变IlaI.1完全取消了限制，而破坏IlaI.2或IlaI.3则使噬菌体DNA发生了无效的限制，主要表现为可变的噬菌斑表型。 ORF4对体内限制没有明显作用。 llaIM中的移码突变证明对乳酸乳球菌NCK203具有致死性，这表明限制成分在没有修饰亚基的情况下是有活性的。这些结果表明，LlaI R / M系统不同于迄今研究的任何其他R / M系统，并且通过获得一些使人联想到I型酶的特征而与IIS型限制酶有所不同。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号