and several of its close relatives have long been exploited for industrial and biotechnological applications (1
). The completion of the sequencing and annotation of the B.subtilis
168 strain genome supplied a complete view of the B.subtilis
protein machinery, and this knowledge stimulated new approaches to the analysis of its biochemical pathways (2
). Post-genomic studies require simple and highly efficient tools to enable genetic manipulations. The most prominent and widely used systems are delivery plasmids, which allow the insertion of any type of genetic information into the bacterial chromosome. Classically, these chromosomal modifications have been achieved by a method using a positive selection marker, usually an antibiotic-resistance marker generated by the insertion of a selection marker gene into the chromosome. According to this strategy, the introduction of a second chromosomal modification requires a second resistance gene; alternatively, if the same resistance gene is used, the eviction of the gene by a single-crossover event prior to further genetic manipulation is required. In the first case, the number of chromosomal modifications is limited by the number of available resistance genes; moreover, the multi-antibiotic pressure could modify the physiology of the manipulated strain. In the second case, selection of the strain that has lost resistance is time-consuming due to the relatively low frequencies and the absence of positive selection. Counter-selectable markers are often instrumental in the construction of clean and unmarked mutations in bacteria (3
). Under appropriate growth conditions, a counter-selectable gene can promote the death of the microorganisms that harbor it. Hence, transformants that have integrated a suicide vector containing a counter-selectable marker through a single-crossover or double-crossover event retain a copy of the counter-selectable marker in the chromosome, and are therefore eliminated in the presence of the counter-selective compound.
Until now, only two similar methods have been described that allow the subsequent excision of the selection marker coupled with positive selection in B.subtilis
; these approaches use the upp
gene and the blaI
gene, respectively, as a counter-selectable marker (4
). However, for the two abovementioned methods to be effective, it is essential to have a strain with a mutation for a specific gene in the chromosome. When these methods are applied to different strains, new mutants must also be prepared. The two existing methods can therefore only be used in strains that have a clear genetic background for preparing mutations of a specific gene, which limits their application. The novel method described here can satisfy the strong demand for a universal unmarked delivery system that can be applied in any Bacillus
species without requiring any prior modification to the host.
Toxin–antitoxin (TA) systems comprise pairs of adjacent genes in which a stable toxic peptide is neutralized by an unstable antitoxin (6
). The mazEF
cassette in the Escherichia coli
chromosome is a well-characterized TA locus; ectopic expression of the MazF toxin inhibits cell growth and the MazE antitoxin neutralizes the cognate toxin (7
). The latest research has revealed that MazF is an endoribonuclease that specifically cleaves free mRNAs at ACA sequences (8
). Here, we describe a procedure based on the use of mazF
as a novel counter-selectable maker in B.subtilis
, which has enabled us to inactivate a single gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene into the B.subtilis
chromosome. In these cases, the resulting strains were free of selection markers, thus allowing the repeated use of the method for further manipulations of the Bacillus
chromosome. No prerequisite strain is needed for this newly developed method, so it will have wide applications.