A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome.
A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome.
复制标题
单个副本集成向量，该矢量在金黄色葡萄球菌染色体上的工程地点集成。
DOI：
10.1186/1756-0500-5-5
复制
发表时间：
2012-01-05
影响因子：
1.8
通讯作者：
Lee CY
中科院分区：
文献类型：
--
作者：
Lei MG;Cue D;Alba J;Junecko J;Graham JW;Lee CY
基于噬菌体位点特异性重组系统的单拷贝整合载体是研究细菌发病机制的宝贵工具。然而，由于整合常常导致细菌基因失活或对基因转录产生不良影响，这种载体的效用往往受到限制。本研究的目的是开发一种整合载体，在整合时对基因转录没有可检测的影响。我们已经开发了一种单拷贝整合系统，使克隆载体能够在金黄色葡萄球菌染色体中未转录的基因间区域内的特定工程位点进行整合。该系统基于溶原噬菌体L54a位点特异性重组系统，其中L54a噬菌体（attP）和染色体（attB）的附着位点具有相同的18 bp核心序列，并被相同的突变修饰。构建了整合载体pLL102，内含核心序列中5个核苷酸位置发生改变的修饰L54a attP位点（attP2）。在受体菌株中，类似修饰的attB位点（attB2）被插入到没有可检测到的转录通读的基因间区域。无法在金黄色葡萄球菌染色体外复制的载体整合是通过在受体的质粒中提供L54a整合酶基因来实现的。我们发现，pLL102特异性地在工程位点而不是在原生L54a attB位点进行整合，并且这种整合对整合位点上游或下游的基因转录没有显著影响。在这项工作中，我们描述了大肠杆菌。金黄色葡萄球菌穿梭载体，可用于在不影响基因表达的情况下将任何克隆基因导入金黄色葡萄球菌染色体的选择位点。该载体可用于金黄色葡萄球菌的遗传操作和在体内研究中标记菌株。
Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site. In this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.