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Journal of Bacteriology, November 1999, p. 6882-6888, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Random Transposition by Tn916 in Desulfitobacterium dehalogenans Allows for Isolation and Characterization of Halorespiration-Deficient Mutants

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

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

Received 21 June 1999/Accepted 20 August 1999

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

* Corresponding author. Mailing address: Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Phone: 31-(0)-317483110. Fax: 31-(0)-317483829. E-mail: hauke.smidt{at}algemeen.micr.wau.nl.

dagger Present address: Wuhan Institute of Virology, Chinese Academy of Science, Wuhan 430071, Hubei, The Peoples Republic of China.

Journal of Bacteriology, November 1999, p. 6882-6888, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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