Buckley N D, Lee L N, LeBlanc D J
University of Texas Health Science Center at San Antonio 78284-7758, USA.
J Bacteriol. 1995 Sep;177(17):5028-34. doi: 10.1128/jb.177.17.5028-5034.1995.
The virulence factors of the cariogenic bacterium Streptococcus sobrinus have been difficult to assess because of a lack of tools for the genetic manipulation of this organism. The construction of an Escherichia coli-Streptococcus shuttle vector, pDL289, that can be mobilized into S. sobrinus by the conjugative plasmid pAM beta 1 was described in a previous report. The vector contains pVA380-1 for replication and mobilization in streptococci, the pSC101 replicon for maintenance in E. coli, a kanamycin resistance marker that functions in both hosts, and the multiple cloning site and lacZ from pGEM7Zf(-). pDL289 is stable with or without selection in several species of Streptococcus. In this study, a derivative with a deletion in the minus origin of the pVA380-1 component of pDL289 was constructed. This derivative, pDL289 delta 202, was less stable than pDL289 in Streptococcus gordonii Challis, Streptococcus mutans, and S. sobrinus. Both pDL289 and pDL289 delta 202 were mobilizable by pAM beta 1 into S. sobrinus, with frequencies of 3 x 10(-6) and 1 x 10(-7) transconjugants per recipient CFU, respectively. The cloned scrA gene of S. sobrinus 6715-10 coding for the EIISuc of the sucrose-specific phosphoenolpyruvate phosphotransferase system was interrupted by the insertion of a streptococcal spectinomycin resistance gene active in E. coli and streptococci. The interrupted scrA gene was subcloned into both pDL289 and pDL289 delta 202. Each recombinant plasmid was introduced into the DL1 strain of S. gordonii Challis, which was then used as a recipient for the conjugative transfer of pAM beta 1. The latter plasmid was used to mobilize each recombinant plasmid from S. gordonii Challis DL1 to S. sobrinus 6715-10RF. Subsequently, recombinants derived from a double-crossover event were isolated on the basis of resistance to spectinomycin and susceptibility to kanamycin. Recombinational events were confirmed by Southern hybridization, and the inactivation of the EII Suc in double crossovers was confirmed by phosphotransferase system assays. This is the first report of allelic replacement in S. sobrinus.
由于缺乏对致龋菌远缘链球菌进行基因操作的工具，其毒力因子一直难以评估。先前的一份报告描述了一种大肠杆菌-链球菌穿梭载体pDL289的构建，该载体可通过接合质粒pAMβ1转入远缘链球菌。该载体包含用于在链球菌中复制和转移的pVA380-1、用于在大肠杆菌中维持的pSC101复制子、在两种宿主中均起作用的卡那霉素抗性标记，以及来自pGEM7Zf(-)的多克隆位点和lacZ。pDL289在几种链球菌中，无论有无选择压力都很稳定。在本研究中，构建了pDL289的pVA380-1成分负链起始位点缺失的衍生物。该衍生物pDL289δ202在戈登链球菌Challis株、变形链球菌和远缘链球菌中的稳定性低于pDL289。pDL289和pDL289δ202均可被pAMβ1转入远缘链球菌，转导频率分别为每受体CFU 3×10(-6)和1×10(-7)个转接合子。远缘链球菌6715-10编码蔗糖特异性磷酸烯醇丙酮酸磷酸转移酶系统的EIISuc的scrA基因，被插入在大肠杆菌和链球菌中均有活性的链球菌壮观霉素抗性基因中断。中断的scrA基因被亚克隆到pDL289和pDL289δ202中。每种重组质粒都被导入戈登链球菌Challis的DL1菌株，然后用作pAMβ1接合转移的受体。后一种质粒用于将每种重组质粒从戈登链球菌Challis DL1转移到远缘链球菌6715-10RF。随后，根据对壮观霉素的抗性和对卡那霉素的敏感性，分离出双交换事件产生的重组体。通过Southern杂交确认重组事件，通过磷酸转移酶系统测定确认双交换中EII Suc的失活。这是远缘链球菌等位基因替换的首次报道。
