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Antimicrobial Agents and Chemotherapy, November 2005, p. 4641-4648, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4641-4648.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Horizontal Transfer of Iturin A Operon, itu, to Bacillus subtilis 168 and Conversion into an Iturin A Producer

Kenji Tsuge,1,2 Satoka Inoue,1 Takashi Ano,1 Mitsuhiro Itaya,2 and Makoto Shoda1*

Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan,1 Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan2

Received 17 December 2004/ Returned for modification 30 January 2005/ Accepted 23 August 2005

Iturin A and its derivatives are lipopeptide antibiotics produced by Bacillus subtilis and several closely related bacteria. Three iturin group operons (i.e., iturin A, mycosubtilin, and bacillomycin D) of those antibiotic-producing strains have been cloned and sequenced thus far, strongly implying the horizontal transfer of these operons. To examine the nature of such horizontal transfer in terms of antibiotic production, a 42-kb region of the B. subtilis RB14 genome, which contains a complete 38-kb iturin A operon, was transferred via competent cell transformation to the genome of a non-iturin A producer, B. subtilis 168, using a method based on double-crossover homologous recombination with two short landing pad sequences (LPSs) in the genome. The recombinant was positively selected by confirming the elimination of the cI repressor gene, which was localized between the two LPSs and substituted by the transferred segment. The iturin A operon-transferred strain 168 was then converted into an iturin A producer by the introduction of an sfp gene, which encodes 4'-phosphopantetheinyl transferase and is mutated in strain 168. By inserting the pleiotropic regulator degQ, the productivity of iturin A increased sevenfold and was restored to about half that of the donor strain RB14, without the transfer of additional genes, such as regulatory or self-resistance genes.

* Corresponding author. Mailing address: 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan. Phone: 81-45-924-5274. Fax: 81-45-924-5276. E-mail: mshoda{at}res.titech.ac.jp.

Antimicrobial Agents and Chemotherapy, November 2005, p. 4641-4648, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4641-4648.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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