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Applied and Environmental Microbiology, February 2001, p. 591-597, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.591-597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development of a Gene Cloning and Inactivation System for Halorespiring Desulfitobacterium dehalogenans

Hauke Smidt,* John van der Oost, and Willem M. de Vos

Laboratory of Microbiology, Wageningen University, NL-6703 CT Wageningen, The Netherlands

Received 6 September 2000/Accepted 2 November 2000

Efficient host-vector systems have been developed for the versatile, strictly anaerobic, halo- and fumarate-respiring gram-positive bacterium Desulfitobacterium dehalogenans. An electroporation-based transformation procedure resulting in approximately 103 to 104 transformants per µg of the cloning vector pIL253 was developed and validated. The broad-host-range vector pG+host9 was shown to replicate at a permissive temperature of 30°C, whereas the replicon was not functional at 40°C. The D. dehalogenans frdCAB operon, predicted to encode a fumarate reductase, was cloned, characterized, and targeted for insertional inactivation by pG+host9 carrying a 0.6-kb internal frdA fragment. Single-crossover integration at the frdA locus occurred at a frequency of 3.3 × 10-4 per cell and resulted in partially impaired fumarate reductase activity. The gene cloning and inactivation systems described here provide a solid basis for the further elucidation of the halorespiratory network in D. dehalogenans and allow for its further exploitation as a dedicated degrader.

* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Phone: 31-317-483118. Fax: 31-317-483829. E-mail: hauke.smidt{at}algemeen.micr.wag-ur.nl.

Applied and Environmental Microbiology, February 2001, p. 591-597, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.591-597.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.