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Applied and Environmental Microbiology, April 2004, p. 2508-2513, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2508-2513.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Targeted Isolation of a Designated Region of the Bacillus subtilis Genome by Recombinational Transfer

Satoshi Tomita,1 Kenji Tsuge,2 Yo Kikuchi,1 and Mitsuhiro Itaya2*

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580,1 Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo 194-8511, Japan2

Received 20 October 2003/ Accepted 17 December 2003

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.

* Corresponding author. Mailing address: Mitsubishi Kagaku Institute of Life Sciences, Minamiooya 11, Machida-shi, Tokyo 194-8511, Japan. Phone: 81-42-724-6254. Fax: 81-42-724-6316. E-mail: ita{at}libra.ls.m-kagaku.co.jp.

Applied and Environmental Microbiology, April 2004, p. 2508-2513, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2508-2513.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kuroki, A., Toda, T., Matsui, K., Uotsu-Tomita, R., Tomita, M., Itaya, M. (2008). Reshuffling of the Bacillus subtilis 168 Genome by Multifold Inversion. J Biochem 143: 97-105 [Abstract] [Full Text]  
  • Sakaya, N., Kaneko, S., Matsunaga, S., Itaya, M. (2006). Experimental Basis for a Stable Plasmid, pLS30, to Shuttle between Bacillus subtilis Species by Conjugational Transfer.. J Biochem 139: 557-561 [Abstract] [Full Text]  


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