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J Bacteriol, March 1998, p. 1354-1359, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning and Sequencing of a 2,5-Dichlorohydroquinone Reductive
Dehalogenase Gene Whose Product Is Involved in Degradation of
-Hexachlorocyclohexane by Sphingomonas paucimobilis
Keisuke
Miyauchi,1,*
Seug-Kyo
Suh,2
Yuji
Nagata,1 and
Masamichi
Takagi1
Department of Biotechnology, The University
of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan,1
and
Department of Environmental Management, Shinil Christian
College, Susung-ku, Taegu 706-020, Korea2
Received 7 August 1997/Accepted 7 January 1998
Sphingomonas (formerly Pseudomonas)
paucimobilis UT26 utilizes
-hexachlorocyclohexane
(
-HCH), a halogenated organic insecticide, as a sole carbon and
energy source. In a previous study, we showed that
-HCH is degraded
to 2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol.
176:3117-3125, 1994). In the present study, we cloned and
characterized a gene, designated linD, directly involved in
the degradation of 2,5-DCHQ. The linD gene encodes a
peptide of 343 amino acids and has a low level of similarity to
proteins which belong to the glutathione S-transferase
family. When LinD was overproduced in Escherichia coli, a
40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. Northern blot analysis revealed that expression of
the linD gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography and gas
chromatography-mass spectrometry analyses with the LinD-overexpressing E. coli cells revealed that LinD converts 2,5-DCHQ rapidly
to chlorohydroquinone (CHQ) and also converts CHQ slowly to
hydroquinone. LinD activity in crude cell extracts was increased
3.7-fold by the addition of glutathione. All three of the
Tn5-induced mutants of UT26, which lack 2,5-DCHQ
dehalogenase activity, had rearrangements or a deletion in the
linD region. These results indicate that LinD is a
glutathione-dependent reductive dehalogenase involved in the
degradation of
-HCH by S. paucimobilis UT26.
*
Corresponding author. Mailing address: Department of
Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan. Phone: 81-3-3812-2111, ext. 5178. Fax: 81-3-3812-9246. E-mail: aa67118{at}hongo.ecc.u-tokyo.ac.jp.
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