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Applied and Environmental Microbiology, June 2007, p. 3490-3496, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02751-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Novel Methods for Genetic Transformation of Natural Bacillus subtilis Isolates Used To Study the Regulation of the Mycosubtilin and Surfactin Synthetases ^,

Erwin H. Duitman,^1,2 Dobek Wyczawski,¹ Ludolf G. Boven,¹ Gerard Venema,¹ Oscar P. Kuipers,^1* and Leendert W. Hamoen^1,3*

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands,¹ Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany,² Institute for Cell and Molecular Biosciences, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom³

Received 24 November 2006/ Accepted 26 March 2007

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin (myc) and surfactin (srfA) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated.

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* Corresponding author. Mailing address for Leendert Hamoen: Institute for Cell and Molecular Biosciences, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44-191-2228983. Fax: 44-191-2227424. E-mail: l.hamoen{at}ncl.ac.uk. Mailing address for Oscar Kuipers: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31-50-3632093. Fax: 31-50-3632348. E-mail: o.p.kuipers{at}rug.nl

Published ahead of print on 6 April 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, June 2007, p. 3490-3496, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.02751-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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