Sanchez R
Center for Oral Biology, University of Rochester, Rochester, New York, 14642, USA.
Plasmid. 1998 Nov;40(3):247-51. doi: 10.1006/plas.1998.1371.
We have constructed a plasmid useful for insertional mutagenesis in Streptococcus mutans. The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates in Escherichia coli, does not replicate in S. mutans, contains an erythromycin-resistance marker which can be selected in E. coli or the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library of S. mutans DNA in E. coli and show that the clones can be reintegrated into the S. mutans chromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of the E. coli drpA gene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked to drpA in S. mutans with high homology to IS861.
我们构建了一种可用于变形链球菌插入诱变的质粒。该分子pSU20Erm基于pACYC184的一种衍生物pSU20。此处描述的质粒大小约为3.7 kb，具有以下特性：它在大肠杆菌中复制，在变形链球菌中不复制，含有一个可在大肠杆菌或链球菌中选择的红霉素抗性标记，在分子其余部分含有一个多克隆位点且限制酶切位点较少，并且可在含X - Gal的培养基上筛选多克隆位点是否有插入片段。我们已使用该质粒在大肠杆菌中构建了变形链球菌DNA文库，并表明克隆可通过同源重组重新整合到变形链球菌染色体中，从而中断天然基因。该质粒已用于克隆大肠杆菌drpA基因同源物的一部分，drpA基因编码一种RNA合成的全局调节元件。此外，我们在变形链球菌中鉴定出一个与drpA紧密连锁的元件，它与IS861具有高度同源性。
