Development of a gene cloning and inactivation system for the halorespiring Desulfitobacterium dehalogenans

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 ?g 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 x 104 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