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金黄色葡萄球菌中具有诱导性反选择的等位基因替换

Allelic replacement in Staphylococcus aureus with inducible counter-selection.

作者信息

Bae Taeok, Schneewind Olaf

机构信息

Department of Microbiology, University of Chicago, Chicago, IL 60637, USA.

出版信息

Plasmid. 2006 Jan;55(1):58-63. doi: 10.1016/j.plasmid.2005.05.005. Epub 2005 Jul 26.

Abstract

A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Plasmid pKOR1, an Escherichia coli/S. 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.

摘要

本文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,它允许通过λ重组和ccdB选择进行快速克隆。将葡萄球菌进行质粒转化并在43℃生长,这是pKOR1复制的非允许条件,可选择同源重组并将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反义转录本,这种条件与葡萄球菌生长不兼容,可选择染色体切除和质粒丢失。使用该策略,在金黄色葡萄球菌rocA中产生等位基因替换的频率无需抗生素标记选择。

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