title: mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis.
abstract: 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locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus.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.
authors.affiliation: 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.
authors.forename: Xiao-Zhou
authors.initials: XZ
authors.lastname: Zhang
authors.forename: Xin
authors.initials: X
authors.lastname: Yan
authors.forename: Zhong-Li
authors.initials: ZL
authors.lastname: Cui
authors.forename: Qing
authors.initials: Q
authors.lastname: Hong
authors.forename: Shun-Peng
authors.initials: SP
authors.lastname: Li
mesh_terms: 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
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C422955:MazF protein, E coli; D004722:Endoribonucleases