1887

Abstract

We have developed a system for the induction of marker-free mutation of . 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 gene, which encodes the repressor for the arabinose operon () of . The counter-selective marker cassette consists of a promoterless neomycin (Nm)-resistance gene () fused to the promoter. First, the chromosomal 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 on the selective marker cassette, represses the expression of in the absence of -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 . 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 region and the 41.8 kb 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.

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2008-09-01
2019-10-17
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References

  1. Anagnostopoulos, C. & Spizizen, J. ( 1961; ). Requirements for transformation in Bacillus subtilis. J Bacteriol 81, 741–746.
    [Google Scholar]
  2. Bloor, A. E. & Cranenburgh, R. M. ( 2006; ). An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72, 2520–2525.[CrossRef]
    [Google Scholar]
  3. Brans, A., Filee, P., Chevigne, A., Claessens, A. & Joris, B. ( 2004; ). New integrative method to generate Bacillus subtilis recombinant strains free of selection markers. Appl Environ Microbiol 70, 7241–7250.[CrossRef]
    [Google Scholar]
  4. Fabret, C., Ehrlich, S. D. & Noirot, P. ( 2002; ). A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46, 25–36.[CrossRef]
    [Google Scholar]
  5. Guerout-Fleury, A. M., Shazand, K., Frandsen, N. & Stragier, P. ( 1995; ). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335–336.[CrossRef]
    [Google Scholar]
  6. Harwood, C. R. & Wipat, A. ( 1996; ). Sequencing and functional analysis of the genome of Bacillus subtilis strain 168. FEBS Lett 389, 84–87.[CrossRef]
    [Google Scholar]
  7. Harwood, C. R., Coxon, R. D. & Hancock, I. C. ( 1990; ). The Bacillus cell envelope and secretion. In Molecular Biological Methods for Bacillus, pp. 327–390. Edited by C. R. Harwood & S. M. Cutting. New York: John Wiley & Sons.
  8. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  9. Horinouchi, S. & Weisblum, B. ( 1982; ). Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150, 815–825.
    [Google Scholar]
  10. Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S. & other authors ( 2003; ). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100, 4678–4683.[CrossRef]
    [Google Scholar]
  11. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A. & other authors ( 1997; ). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef]
    [Google Scholar]
  12. Leenhouts, K., Buist, G., Bolhuis, A., ten Berge, A., Kiel, J., Mierau, I., Dabrowska, M., Venema, G. & Kok, J. ( 1996; ). A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253, 217–224.[CrossRef]
    [Google Scholar]
  13. Liu, S., Endo, K., Ara, K., Ozaki, K. & Ogasawara, N. ( 2007; ). The accurate replacement of long genome region more than several hundreds kilobases in Bacillus subtilis. Genes Genet Syst 82, 9–19.[CrossRef]
    [Google Scholar]
  14. Malaga, W., Perez, E. & Guilhot, C. ( 2003; ). Production of unmarked mutations in mycobacteria using site-specific recombination. FEMS Microbiol Lett 219, 261–268.[CrossRef]
    [Google Scholar]
  15. McKenzie, T., Hoshino, T., Tanaka, T. & Sueoka, N. ( 1986; ). The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93–103.[CrossRef]
    [Google Scholar]
  16. Mota, L. J., Tavares, P. & Sa-Nogueira, I. ( 1999; ). Mode of action of AraR, the key regulator of l-arabinose metabolism in Bacillus subtilis. Mol Microbiol 33, 476–489.[CrossRef]
    [Google Scholar]
  17. Mota, L. J., Sarmento, L. M. & de Sa-Nogueira, I. ( 2001; ). Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping. J Bacteriol 183, 4190–4201.[CrossRef]
    [Google Scholar]
  18. Sa-Nogueira, I. & Mota, L. J. ( 1997; ). Negative regulation of l-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene. J Bacteriol 179, 1598–1608.
    [Google Scholar]
  19. Sa-Nogueira, I. & Ramos, S. S. ( 1997; ). Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in l-arabinose utilization. J Bacteriol 179, 7705–7711.
    [Google Scholar]
  20. Sa-Nogueira, I., Nogueira, T. V., Soares, S. & de Lencastre, H. ( 1997; ). The Bacillus subtilis l-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology 143, 957–969.[CrossRef]
    [Google Scholar]
  21. White, A. P., Allen-Vercoe, E., Jones, B. W., DeVinney, R., Kay, W. W. & Surette, M. G. ( 2007; ). An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species. Can J Microbiol 53, 56–62.[CrossRef]
    [Google Scholar]
  22. Zhang, X. Z., Yan, X., Cui, Z. L., Hong, Q. & Li, S. P. ( 2006; ). mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res 34, e71 [CrossRef]
    [Google Scholar]
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