mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis.
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-beta-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locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus.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.
Zhang
Xiao-Zhou
XZ
Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Jiangsu, People's Republic of China.
Yan
Xin
X
Cui
Zhong-Li
ZL
Hong
Qing
Q
Li
Shun-Peng
SP
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics*; D004722:Endoribonucleases; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D018390:Gene Targeting / Q000379:methods*; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C422955:MazF protein, E coli; D004722:Endoribonucleases