A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes.
A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes.
复制标题
DOI：
10.1128/mbio.00537-12
复制
发表时间：
2013-02-12
期刊：
影响因子：
6.4
通讯作者：
Bayles KW
中科院分区：
文献类型：
--
作者：
Fey PD;Endres JL;Yajjala VK;Widhelm TJ;Boissy RJ;Bose JL;Bayles KW
为提高对金黄色葡萄球菌感兴趣的研究人员的研究能力，内布拉斯加葡萄球菌研究中心（CSR）构建了一个序列明确的转座子突变体文库，该文库由1952个菌株组成，每个菌株在流行性社区相关耐甲氧西林金黄色葡萄球菌（CA - MRSA）分离株USA300的一个非必需基因中都含有一个单一突变。为证明该文库在大规模筛选表型改变方面的实用性，我们将文库点种在指示平板上以评估溶血潜力、蛋白酶产生、色素沉着和甘露醇利用情况。正如预期的那样，我们鉴定出了许多已知在这些过程中起作用的基因，从而验证了这种方法的实用性。重要的是，我们还鉴定出了以前与这些表型无关的基因。总共有71个突变体显示出不同的溶血活性，其中大多数以前并不知道会影响溶血素的产生。此外，62个突变体在蛋白酶活性方面存在缺陷，其中只有14个以前被证明与细胞外蛋白酶的产生有关。另外，38个突变影响色素形成，而只有7个影响甘露醇发酵，这突出了这种方法在鉴定罕见表型方面的敏感性。最后，579个开放阅读框未被转座子中断，从而为后续的抗菌发现提供了潜在的新的必需基因靶点。总体而言，内布拉斯加转座子突变体文库是研究界的一种有价值的新资源，它将极大地促进对这种重要的人类病原体的研究。
金黄色葡萄球菌引起的感染在社区和医院环境中都导致显著的发病率和死亡率。研究这种生物体的生物学需要特定等位基因替换突变体；然而，这个过程既昂贵又耗时。我们描述了一个序列明确的转座子突变体文库的构建和验证，该文库可通过金黄色葡萄球菌抗菌药物耐药性网络（NARSA）菌株库供科学界使用。此外，还生成了一些辅助资源，包括一个网站（）以及一些基因工具，这些工具可加速用有用的选择标记和荧光报告基因融合对突变体中转座子进行等位基因替换。总体而言，这个文库及相关工具将对研究金黄色葡萄球菌发病机制和生物学的研究产生重大影响，并可作为研究其他细菌系统的有用范例。
To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen. Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website () and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.