Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter

Abstract

We have developed a system for the induction of marker-free mutation of Bacillus subtilis. The system features both the advantages of the use of antibiotic-resistance markers for mutant selection, and the ability to efficiently remove the markers, leaving unmarked mutations in the genome. It utilizes both a selective marker cassette and a counter-selective marker cassette. The selective marker cassette contains a chloramphenicol-resistance gene and the araR gene, which encodes the repressor for the arabinose operon (ara) of B. subtilis. The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene (neo) fused to the ara promoter. First, the chromosomal araR locus is replaced with the counter-selective marker cassette by double-crossover homologous recombination and positive selection for Nm resistance. The selective marker cassette is connected with upstream and downstream sequences from the target locus, and is integrated into the upstream region of the target locus by a double-crossover event. This integration is also positively selected for, using chloramphenicol resistance. In the resultant strain, AraR, encoded by araR on the selective marker cassette, represses the expression of neo in the absence of L-arabinose. Finally, the eviction of the selective marker cassette together with the target locus is achieved by an intra-genomic single-crossover event between the two downstream regions of the target locus, and can be selected for based on Nm resistance, because of the excision of araR. The counter-selective marker cassette remaining in the genome, whose expression is switched on or off based on the excision or introduction of the selective marker cassette, is used again for the next round of deletion. Using this system, the 3.8 kb iolS-csbC region and the 41.8 kb hutM-csbC region have been efficiently and successfully deleted, without leaving markers in the target loci. The positive selection and simple procedure will make it a useful tool for the construction of multiple mutations.

Novel High-Molecular-Weight, R-Type Bacteriocins of Clostridium difficile

“…We suspected that production of these phage-like particles might limit diffocin production. This deletion was accomplished by the marker-free deletion method of Liu et al (18), by which we created strain BDG9. The second modification was to switch the integrant selective marker from spectinomycin to chloramphenicol, which often results in better protein expression (E. Ferrara, personal communication).…”

A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis

“…The resulting MGB874 strain displayed increased extracellular cellulase and protease productivity (Morimoto et al, 2008). The arabinose operon repressor ( araR ) has been used to generate 3.8 kb ( iolS - csbC ) and 41.8 kb ( hutM - csbC ) deletion mutant in B. subtilis precisely (Liu et al, 2008). Although these counter-selectable markers successfully removed the target gene without using antibiotics resistance markers, the system used previously constructed upp - and araR - inactivated strains ( upp :: erm and araR :: neo ) that left traces of foreign DNA on the secondary region of the chromosome.…”

Combined Effect of Improved Cell Yield and Increased Specific Productivity Enhances Recombinant Enzyme Production in Genome-Reduced Bacillus subtilis Strain MGB874

“…The DNA manipulation techniques used for the construction of mutants were described previously (36). MGB625 ⌬r1 was constructed from MGB625 as follows.…”