Allelic replacement in Staphylococcus aureus with inducible counter-selection
Allelic replacement in Staphylococcus aureus with inducible counter-selection
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DOI：
10.1016/j.plasmid.2005.05.005
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发表时间：
2006-01-01
期刊：
影响因子：
2.6
通讯作者：
Schneewind, O
中科院分区：
文献类型：
--
作者：
Bae, T;Schneewind, O
描述了一种在金黄色葡萄球菌染色体中快速选择等位基因替换突变的方法。质粒pKOR1是一种大肠杆菌 - 金黄色葡萄球菌穿梭载体，可通过λ重组和ccdB选择进行快速克隆。葡萄球菌的质粒转化以及在43℃（pKOR1复制的非许可条件）下生长，可选择出同源重组以及pKOR1整合到细菌染色体中。通过Pxyl/tetO启动子由脱水四环素介导诱导pKOR1编码的secY反义转录物，这种条件与葡萄球菌生长不相容，可选择出染色体切除和质粒丢失。利用这种策略，在金黄色葡萄球菌rocA中产生等位基因替换的频率使得无需进行抗生素标记选择。（c）2005爱思唯尔公司。保留所有权利。
A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Plasmid pKOR1, an Escherichia colilS. aureus shuttle vector, permits rapid cloning via lambda recombination and ccdB selection. Plasmid transformation of staphylococci and growth at 43 degrees C, a non-permissive condition for pKOR1 replication, selects for homologous recombination and pKOR1 integration into the bacterial chromosome. Anhydrotetracycline-mediated induction of pKOR1-encoded secY antisense transcripts via the Pxyl/tetO promoter, a condition that is not compatible with staphylococcal growth, selects for chromosomal excision and loss of plasmid. Using this strategy, allelic replacements in S. aureus rocA were generated at frequencies that obviated the need for antibiotic marker selection. (c) 2005 Elsevier Inc. All rights reserved.