title: Development of a gene cloning and inactivation system for halorespiring Desulfitobacterium dehalogenans.
abstract: 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 10(3) to 10(4) transformants per microg 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 degrees C, whereas the replicon was not functional at 40 degrees 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 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.
authors.affiliation: Laboratory of Microbiology, Wageningen University, NL-6703 CT Wageningen, The Netherlands. hauke.smidt@algemeen.micr.wag-ur.nl
authors.forename: H
authors.initials: H
authors.lastname: Smidt
authors.forename: J
authors.initials: J
authors.lastname: van der Oost
authors.forename: W M
authors.initials: WM
authors.lastname: de Vos
mesh_terms: D000693:Anaerobiosis; D003001:Cloning, Molecular; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D018274:Electroporation; D017353:Gene Deletion; D005822:Genetic Vectors; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D013385:Succinate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D013385:Succinate Dehydrogenase