Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis.
Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis.
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作者：
Monk IR;Shah IM;Xu M;Tan MW;Foster TJ
金黄色葡萄球菌和表皮葡萄球菌中存在的强烈限制障碍限制了对一小部分能够进行基因操作的菌株的功能基因组分析。最近，一种被称为SauUSI(特异性识别胞嘧啶甲基化DNA)的保守的IV型限制性内切酶系统被发现是转化外源DNA的主要障碍。在这里，我们在一种广泛使用的金黄色葡萄球菌实验室菌株中独立证实了这些发现。此外，我们还在高效的大肠杆菌克隆菌株DH10B(简称DC10B)中构建了DNA胞嘧啶甲基转移酶突变体。从DC10B中分离出的质粒可直接转化金黄色葡萄球菌和表皮葡萄球菌临床分离株。我们还表明，金黄色葡萄球菌USA300菌株的限制性内切酶丧失(I型和IV型)不会对毒力产生影响。绕过SauUSI限制障碍，结合改进的缺失和转化方案，允许对这些重要的机会性病原体以前不可转化的菌株进行基因操作。葡萄球菌感染的重要性给卫生保健部门带来了巨大的负担，这既是因为其严重性，也是因为长期住院治疗感染所造成的经济影响。为了提高对金黄色葡萄球菌和表皮葡萄球菌感染的了解，我们开发了一系列改进的技术，允许对以前难以转化的菌株进行基因操作。这些进展将加快突变构建的进程，并增加我们对这些物种作为一个整体的了解，而不仅仅是以前可以操纵的一小部分菌株。葡萄球菌感染给卫生保健部门带来了巨大的负担，这既是因为其严重性，也是因为长期住院治疗感染所造成的经济影响。为了提高对金黄色葡萄球菌和表皮葡萄球菌感染的了解，我们开发了一系列改进的技术，允许对以前难以转化的菌株进行基因操作。这些进展将加快突变构建的进程，并增加我们对这些物种作为一个整体的了解，而不仅仅是以前可以操纵的一小部分菌株。
The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens. IMPORTANCE Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated. Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.