Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134.
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 (FADH2)-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 FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-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 FADH2 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.
Louie
Tai Man
TM
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234, USA.
Webster
Christopher M
CM
Xun
Luying
L
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001673:Biodegradation, Environmental; D002733:Chlorophenols / Q000378:metabolism*; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D008298:Maleates / Q000378:metabolism; D005810:Multigene Family; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D002733:Chlorophenols; D008298:Maleates; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; C079154:2-chloromaleylacetate; D010105:Oxygenases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; C024564:2,4,6-trichlorophenol