Cre/ lox System and PCR-Based Genome Engineering in Bacillus subtilis

Abstract

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.Bacillus subtilis is the best-characterized gram-positive bacterial organism: its biochemistry, physiology and genetics have been studied intensely for more than 50 years. B. subtilis and the closely related Bacillus species are nonpathogenic and free of endotoxins, and their fermentation technology has been well characterized, making them important cell factories for industrial enzymes, fine biochemicals, antibiotics, and insecticides (8, 31). The completion of the sequencing and annotation of the B. subtilis 168 genome supplies a complete view of the B. subtilis protein machinery, inspiring new approaches for analyzing biochemical pathways (23,25). Postgenomic studies require simple and highly efficient tools to enable genetic manipulation. Classically, these chromosomal modifications have been achieved by a method that uses a positive selection marker, usually an antibiotic resistance marker, which is generated by insertion of the marker gene into the chromosome. When introducing multiple modifications into the same background, it is better to evict the selection marker gene, usually through a single-crossover event. Selection of the strain that has lost marker gene is tedious without the aid of counterselectable markers that, under appropriate growth conditions, can promote the death of the microorganisms that harbor them. Until now, four counterselectable markers have been described (6,12,17,38) that improve the genetic manipulations of B. subtilis by allowing the subsequent excision of the selection marker, coupled with positive selection. The genetic manipulations described above, however, are mainly based on restriction enzyme and DNA ligase-dependent vector construction, which requi...

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

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Functional Characterization and Localization of a Bacillus subtilis Sortase and Its Substrate and Use of This Sortase System To Covalently Anchor a Heterologous Protein to the B. subtilis Cell Wall for Surface Display

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The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

“…The following strains were constructed in that way, and the primer pairs used for the long-flanking-region PCR are given in parentheses: ACB164 [⌬(ywcA::ery)1] (5=-CATGTTAAAA AAGATTTGGACAAAGGTTGG-3=/5=-TTTGTCATCAATCTGAAAGAA GCCTAAATC-3=), ACB165 [⌬(yodF::neo)1] (5=-TGTATCCAAAGCTTTC CGTTAACAATTGTA-3=/5=-TGTCTATTCAGAGATGAAAGACAACAAC AT-3=), and ACB274 [⌬(gabP::zeo)1] (5=-CTCTTGTTTAGCAGAAAAAAT AGATTCTCC-3=/5=-TTTTAAGCGTTTTTCCTTTGGGTCATGAAT-3=). The antibiotic resistance genes used for these gene disruption constructs were derived from plasmids pDG642 (ery), pDG783 (neo) (49), and p7Z6 (zeo) (50). The presence of the correct gene disruption constructs in the newly created mutant strains was verified by PCR using primers that flank the deleted genomic region; PCR products derived from genomic DNA of the B. subtilis wild-type strain JH642 were used for comparison.…”