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Nucleic Acids Research 2006 34(9):e71; doi:10.1093/nar/gkl358
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Published online 19 May 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
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Methods Online

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis

Xiao-Zhou Zhang, Xin Yan, Zhong-Li Cui, Qing Hong and Shun-Peng Li*

Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University 6 Tongwei Road, Nanjing, Jiangsu 210095, People's Republic of China

*To whom correspondence should be addressed. Tel/Fax: +86 25 84396314; Email: lsp{at}njau.edu.cn

Received March 23, 2006. Revised April 21, 2006. Accepted April 24, 2006.

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.


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