Cloning and sequencing of a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis.
Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a halogenated organic insecticide, as a sole carbon and energy source. In a previous study, we showed that gamma-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 gamma-HCH by S. paucimobilis UT26.
Miyauchi
K
K
Department of Biotechnology, The University of Tokyo, Japan. aa67118@hongo.ecc.u-tokyo.ac.jp
Suh
S K
SK
Nagata
Y
Y
Takagi
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001482:Base Composition; D015152:Blotting, Northern; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D001556:Hexachlorocyclohexane / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics*; D006873:Hydroquinones / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D006873:Hydroquinones; D012967:Sodium Dodecyl Sulfate; D001556:Hexachlorocyclohexane; C069879:2,5-dichlorohydroquinone; D005982:Glutathione Transferase; D006867:Hydrolases; C111301:LinD protein, Pseudomonas paucimobilis; D005978:Glutathione