Journal article
Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134
Journal of bacteriology, Vol.184(13), pp.3492-3500
07/2002
Handle:
https://hdl.handle.net/2376/101252
PMCID: PMC135155
PMID: 12057943
Abstract
Ralstonia eutropha
JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH
2
)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (
tcpABC
) from JMP134 by using primers designed from conserved regions of FADH
2
-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that
tcpA
,
tcpB
, and
tcpC
encoded an FADH
2
-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The
tcpA
mutant failed to degrade 2,4,6-TCP, while both
tcpB
and
tcpC
mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of
tcpB
may have led to a polar effect on downstream
tcpC
, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH
2
was supplied by an
Escherichia coli
flavin reductase. TcpC produced in
E. coli
oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that
tcpB
may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.
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Details
- Title
- Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134
- Creators
- Tai Man Louie - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234Christopher M Webster - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234Luying Xun - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234
- Publication Details
- Journal of bacteriology, Vol.184(13), pp.3492-3500
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- American Society for Microbiology
- Identifiers
- 99900546515801842
- Language
- English
- Resource Type
- Journal article