To have neighbour's fare:: extending the molecular toolbox for Streptococcus pneumoniae
To have neighbour's fare:: extending the molecular toolbox for Streptococcus pneumoniae
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DOI：
10.1099/mic.0.28521-0
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发表时间：
2006-02-01
期刊：
影响因子：
2.8
通讯作者：
Kuipers, OP
中科院分区：
文献类型：
--
作者：
Kloosterman, TG;Bijlsma, JJE;Kuipers, OP
在过去的几年里，一些有用的遗传工具已经开发用于研究肺炎链球菌的分子生物学。为了扩大现有工具的范围，利用了最初为密切相关的乳酸乳球菌开发的工具箱，该工具箱适用于肺炎链球菌的操作。修改后的工具如下。(i)改良的nisin诱导（过）表达系统（NICE）。nisRK基因编码的双组分系统对nisin应答的转录激活至关重要，该基因被整合到肺炎链球菌D39的bgaA位点。在这个菌株D39nisRK中，加入nisin导致了在nisin诱导启动子控制下的几个基因的过表达，而在没有nisin的情况下没有检测到表达。lacZ报告系统。利用缺乏内源性β -半乳糖苷酶活性的菌株D39nisRK，证明了lacZ报告载体pORI13对染色体转录融合的有用性。此外，将pORI13复制所需的repA基因引入bgaA位点，从而为基于质粒的启动子表达研究提供了背景。（iii）一种简化的化学定义培养基，支持所有测序的肺炎链球菌菌株生长到与复杂培养基相当的水平。（四）将未标记的缺失和突变引入染色体的系统，该系统与目标菌株的基因型无关。这些系统在菌株R6和TIGR4中也获得了成功的应用。此外，与现有工具相比，这些工具还提供了一些改进和优势。因此，肺炎链球菌的分子工具箱已成功扩展。
In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous beta-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.