Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723

Abstract

The first three enzymes of the pentachlorophenol (PCP) degradation pathway in Sphingobium chlorophenolicum (formerly Sphingomonas chlorophenolica) ATCC 39723 have been characterized, and the corresponding genes, pcpA, pcpB, and pcpC, have been individually cloned and sequenced. To search for new genes involved in PCP degradation and map the physical locations of the pcp genes, a 24-kb fragment containing pcpA and pcpC was completely sequenced. A putative LysR-type transcriptional regulator gene, pcpM, and a maleylacetate reductase gene, pcpE, were identified upstream of pcpA. pcpE was found to play a role in PCP degradation. pcpB was not found on the 24-kb fragment. The four gene products PcpB, PcpC, PcpA, and PcpE were responsible for the metabolism of PCP to 3-oxoadipate in ATCC 39723, and inactivational mutation of each gene disrupted the degradation pathway. The organization of the pcp genes is unusual because the four PCP-degrading genes, pcpA, pcpB, pcpC, and pcpE, were found to be located at four discrete locations. Two hypothetical LysR-type regulator genes, pcpM and pcpR, have been identified; pcpM was not required, but pcpR was essential for the induction of pcpB, pcpA, and pcpE. The coinducers of PcpR were PCP and other polychlorinated phenols. The expression of pcpC was constitutive. Thus, the organization and regulation of the genes involved in PCP degradation to 3-oxoadipate were documented.Pentachlorophenol (PCP) has been released into the environment as a wood preservative (8, 13). This compound is a major environmental pollutant due to its toxicity and recalcitrance, and it is regulated as one of the priority pollutants by the U.S. Environmental Protection Agency (16, 30). Microorganisms have been used to remove PCP from the environment (16, 17), and several aerobic PCP-degrading bacteria have been isolated from contaminated soils (7). Sphingobium chlorophenolicum (31) (formerly Sphingomonas chlorophenolica) strain ATCC 39723 is one of the bacteria capable of completely mineralizing PCP (24). The biochemistry of PCP degradation by ATCC 39723 has been extensively studied (Fig. 1). PCP 4-monooxygenase (PcpB) oxidizes PCP to 2,3,5,6-tetrachlorop-hydroquinone (TeCH) (22,35,37,38). TeCH reductive dehalogenase (PcpC) converts TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone (DiCH) by reductive dechlorination (20,39,40). DiCH is subject to ring cleavage by DiCH 1,2-dioxygenase (PcpA), producing 2-chloromaleylacetate (2-CMA) (19, 33). The corresponding genes, pcpB, pcpC, and pcpA, have been individually cloned and sequenced (21,22,36). pcpB was found to be physically linked with two other putative pcp genes, pcpD and pcpR (21). pcpR is a hypothetical LysR-type regulator. Northern hybridization and enzymatic activity analysis suggest that PcpB and PcpA are PCP inducible in strain ATCC 39723 (22, 33), while PcpC is constitutively produced (20,40). However, the overall organization and regulation of PCP-degrading genes have not been reported, and the metabolic steps beyond rin...

Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A

“…It is evident from the present study that lin genes are scattered within the S. indicum B90A genome, a situation similar for a number of catabolic genes found in sphingomonads 27,28 . In S. japonicum UT26, lin genes were found to be dispersed on three circular replicons (linA-linC on chromosome I, linF on chromosome II, and linDER on the 2) and HindIII (lanes 3 and 4), prepared for Southern hybridization, and hybridized separately against the linA (B), linC (C), linX (D), linD (E), linE (F) and linR (G) probes.…”

Characterization of a gene cluster encoding the maleylacetate reductase from Ralstonia eutropha 335T, an enzyme recruited for growth with 4-fluorobenzoate

“…It has been reported for the 2,4,5-trichlorophenoxyacetate conversion via 5-chloro-2-hydroxyquinol and hydroxyquinol by B. cepacia AC1100 (Daubaras et al, , 1996Zaborina et al, 1998) as well as for pentachlorophenol degradation via 2,6-dichloroquinol by Sphingomonas chlorophenolica ATCC 39723 (Cai & Xun, 2002). It has also been shown for pathways that use chlorocatechols as their central intermediates.…”

“…The outgroups were removed afterwards and resulting column gaps were eliminated. References for the published sequences (order as in the alignment): AF130250 (this study); U19883 ; AF169302 (Johnson et al, 2002); NZ_AAAI01000075, Ralstonia metallidurans Reut_75 genome shotgun sequence; AF512952 (Cai & Xun, 2002); NC_003304, positions 2 501 013-2 502 068, Agrobacterium tumefaciens C58 chromosome (Wood et al, 2001); NC_004463, positions 2 938 247-2 939 326, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al, 2002); AJ536297 (Fritsche, 1998); X72850 (Armengaud et al, 1999); U32188 (Vedler et al, 2000); AF030176 (Seibert et al, 1998);M57629 (van der Meer et al, 1991); AF019038 (Kasberg et al, 1997); M35097 (Perkins et al, 1990); AB050198 (Liu et al, 2001); P94135 (Laemmli et al, 2000); NC_004369, positions 3 077 929-3 079 059, Corynebacterium efficiens YS-314 T genome; NC_003450, positions 1 214 871-1 215 941 and 3 257 401-3 258 492, Corynebacterium glutamicum ATCC 13032 T genome; NC_004463, positions 1 096 165-1 097 388, Bradyrhizobium japonicum USDA 110 genome (Kaneko et al, 2002). Fig.…”

The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses

“…These activities include not only the catabolism of complex compounds to provide energy and carbon sources but also mechanisms of detoxification relevant to the bioremediation of cold environments and other biotechnological applications. For instance, C. psychrerythraea possesses a homolog to the 2,4,6-trichlorophenol monooxygenase of Ralstonia eutropha (CPS2047) along with two homologs to reductive dehalogenases involved in the degradation of pentachlorophenol (CPS1905 and CPS1668) (37). Genome analyses also suggest the presence of putative dioxygenases (CPS1846 and CPS4358) and monooxygenases (CPS3582, CPS3527, and CPS1273) critical to the cleavage of ring bearing and aliphatic compound degradation.…”