Brandner Christina J, Maier Richard H, Henderson Daryl S, Hintner Helmut, Bauer Johann W, Onder Kamil
Department of Cell Biology, University of Salzburg, Hellbrunner Strasse 34, A-5020 Salzburg, Austria.
BMC Genomics. 2008 Jul 7;9:321. doi: 10.1186/1471-2164-9-321.
The bacterium Staphylococcus aureus causes significant morbidity and mortality in humans, primarily due to the emergence of strains that are resistant to antibiotics - notably methicillin-resistant S. aureus (MRSA) isolates. Development of effective strategies for the control and treatment of MRSA infections may best be achieved through 'omics' approaches, which first requires cloning the entire set of S. aureus' protein-encoding open reading frames (ORFs), or ORFeome.
The complete genome sequence of S. aureus strain Mu50 has 2697 predicted protein-coding ORFs. Based on the sequence of this strain we designed PCR primers to construct from an S. aureus (non-MRSA) clinical isolate an ORFeome library that contains 2562 unique Gateway entry clones (95% coverage), each corresponding to a defined ORF. The high quality of the ORFeome library was verified by DNA sequencing and PCR amplification, and its functionality was demonstrated by expressing recombinant proteins and observing protein interactions in a yeast 2-hybrid homodimerization screen.
This first ORFeome library for S. aureus provides an essential new tool for investigating the systems biology of this important pathogen.
金黄色葡萄球菌可导致人类出现严重的发病和死亡情况，主要原因是对抗生素耐药的菌株的出现，尤其是耐甲氧西林金黄色葡萄球菌（MRSA）分离株。控制和治疗MRSA感染的有效策略的开发最好通过“组学”方法来实现，而这首先需要克隆金黄色葡萄球菌整套蛋白质编码开放阅读框（ORF），即ORFeome。
金黄色葡萄球菌Mu50菌株的完整基因组序列有2697个预测的蛋白质编码ORF。基于该菌株的序列，我们设计了PCR引物，以从一株金黄色葡萄球菌（非MRSA）临床分离株构建一个ORFeome文库，该文库包含2562个独特的Gateway入门克隆（覆盖率为95%），每个克隆对应一个确定的ORF。通过DNA测序和PCR扩增验证了ORFeome文库的高质量，并通过在酵母双杂交同二聚化筛选中表达重组蛋白和观察蛋白质相互作用证明了其功能。
这个首个金黄色葡萄球菌ORFeome文库为研究这种重要病原体的系统生物学提供了一个重要的新工具。
