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首页> 外文期刊>Applied Microbiology >Generation of a Stable Plasmid for In Vitro and In Vivo Studies of Staphylococcus Species
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Generation of a Stable Plasmid for In Vitro and In Vivo Studies of Staphylococcus Species

机译：对体外和体内葡萄球菌的体内研究产生稳定的质粒

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A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli - S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded ( dso and sso ) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis . Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of Staphylococcus .IMPORTANCE Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.
机译：对基于质粒的遗传工具的重大缺点是使用抗生素来确保质粒维持的必要性。虽然可选择的标记非常强大，但它们的使用并不总是实用的，例如在细菌感染的体内模型期间。在先前的研究期间，注意到，在没有已知选择的情况下，金黄色葡萄球菌中的无特征的LAC-P01质粒在没有已知选择的情况下稳定，因此可以作为葡萄球菌物种的新遗传工具的平台。 LAC-P01被遗传地操纵成大肠杆菌的金黄色葡萄球菌梭载体，其在没有抗生素选择的情况下保持至少100代的稳定性。鉴定了双链（DSO和SSO）起源，发现分别对质粒复制和维护至关重要。相反，删除分析表明，稳定性需要四个LAC-P01预测的开放阅读帧。在此之后，梭子载体被用作产生两个质粒的平台。第一个质粒pKK22含有用于质粒的所有基因，用于金黄色葡萄球菌300株，而第二个PKK30缺乏用于非USA300隔离物的四个预测的开放阅读框架。 PKK30也被确定在葡萄球菌表皮中稳定。此外，PKK22在S.UUREUS系统感染的鼠模型中占据了7天的第7天，并成功地互补了DermeCRASY模型中的HLA突变体。这些质粒，即在体外和体内实验中消除对抗生素的需要是强大的新型研究金葡萄球菌的研究工具。分量的质粒稳定性在细菌研究中存在问题，并且已经使用历史上的抗生素来确保质粒维持。这在体内研究中是一个重大限制，其中提供质粒维持的抗生素是困难的并且具有混杂的影响。在这里，我们利用来自S.UUREUS USA300菌株的天然存在的质粒LAC-P01，以构建稳定的质粒，抗生素使用。这些新改性的质粒在体外和体内不受抗生素选择的众多世代的稳定性。通过这些质粒，需要遗传互补，蛋白质表达或遗传报告系统的研究不仅将克服抗生素使用的负担，而且消除了这些抗生素的副作用。因此，我们的质粒可以用作葡萄球菌研究的强大的遗传工具。

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