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Applied and Environmental Microbiology, December 2004, p. 7241-7250, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7241-7250.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers

Alain Brans,{dagger} Patrice Filée,{dagger} Andy Chevigné, Aurore Claessens, and Bernard Joris*

Centre for Protein Engineering, Institut de Chimie, Université de Liège, Sart-Tilman, B4000 Liège, Belgium

Received 6 April 2004/ Accepted 20 July 2004

The novel method described in this paper combines the use of blaI, which encodes a repressor involved in Bacillus licheniformis BlaP ß-lactamase regulation, an antibiotic resistance gene, and a B. subtilis strain (BS1541) that is conditionally auxotrophic for lysine. We constructed a BlaI cassette containing blaI and the spectinomycin resistance genes and two short direct repeat DNA sequences, one at each extremity of the cassette. The BS1541 strain was obtained by replacing the B. subtilis PlysA promoter with that of the PblaP ß-lactamase promoter. In the resulting strain, the cloning of the blaI repressor gene confers lysine auxotrophy to BS1541. After integration of the BlaI cassette into the chromosome of a conditionally lys-auxotrophic (BS1541) strain by homologous recombination and positive selection for spectinomycin resistance, the eviction of the BlaI cassette was achieved by single crossover between the two short direct repeat sequences. This strategy was successfully used to inactivate a single gene and to introduce a gene of interest in the Bacillus chromosome. In both cases the resulting strains are free of selection marker. This allows the use of the BlaI cassette to repeatedly further modify the Bacillus chromosome.

* Corresponding author. Mailing address: Centre for Protein Engineering, Institut de Chimie B6a, Université de Liège, Sart-Tilman, B4000 Liège, Belgium. Phone: (32) 366 2954. Fax: (32) 366 3364. E-mail: bjoris{at}ulg.ac.be.

{dagger} A.B. and P.F. contributed equally to this work and are listed in alphabetical order.

Applied and Environmental Microbiology, December 2004, p. 7241-7250, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7241-7250.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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