期刊
JOURNAL OF MICROBIOLOGICAL METHODS
卷 87, 期 1, 页码 76-81出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2011.07.010
关键词
Staphylococcus aureus; Gene deletion; Markerless; Plasmid
资金
- Japan Society for the Promotion of Science
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Ministry of the Health, Labor and Welfare
Staphylococcus aureus is a Gram-positive pathogen that causes opportunistic infections and a wide variety of diseases. Methicillin-resistant S. aureus (MESA) is frequently isolated as multidrug-resistant in nosocomial and community infections. Molecular genetic manipulation is an important tool for understanding the molecular mechanism of S. aureus infection. However the number of available antibiotic markers is limited due to multidrug resistance. In this study, we constructed two Escherichia coli-S. aureus shuttle vectors, pKFT and pKFC, that carry a temperature-sensitive origin of replication in S. aureus, lacZ(a) enabling a simple blue-white screening in E. coli, an ampicillin resistant gene, and either a tetracycline resistance gene or a chloramphenicol resistance gene. We report a simple technique using pKFT to construct a markerless gene deletion mutant in S. aureus by allelic replacement without the use of a counter-selection marker. Subculture twice at 25 degrees C was critical to promote an allelic exchange rate in S. aureus. This technique is very simple and useful to facilitate genetic research on S. aureus. (C) 2011 Elsevier B.V. All rights reserved.
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