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J Microbiol Methods. 2012 Sep;90(3):250-5. doi: 10.1016/j.mimet.2012.05.017. Epub 2012 May 29.

Plasmid integration method: a new tool for analysis of the essentiality and function of genes in S. aureus.

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Oncology Research Laboratories, Daiichi Sankyo Co., Ltd., Japan.


Staphylococcus aureus is a Gram-positive coccus and one of the major causes of community-acquired and hospital-acquired infections. We established the convenient and reliable experimental system for analyzing the essentiality and function of genes, the plasmid integration (PI) method. This method is based on plasmid integration into the genome by single cross-over recombination using a temperature-sensitive shuttle vector, and it was validated using known essential genes, gyrA and mvaD, and non-essential genes, sigB and hla. Then we analyzed 116 S. aureus conserved hypothetical protein genes with the PI method, and identified 28 essential genes. Moreover, applying the PI method, we confirmed the functional redundancy between the S. aureus gene (SA0865) and its ortholog human gene, the NAD kinase gene. These results show that the PI method is a powerful tool for the identification of essential genes and functional analysis by evaluation of complementarity.

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