Biochemistry and genetics of PCB metabolism.

Biphenyl(BP)-utilizing bacteria, which include both Gram-negative and Gram-positive strains, are ubiquitously distributed in the environment. These bacteria co-metabolically degrade a variety of polychlorinated biphenyl (PCB) congeners to the corresponding chlorobenzoic acids through 2,3-dioxygenation. Certain strains degrade even highly chlorinated PCBs through 3,4-dioxygenation. The ring meta-cleavage dioxygenase purified from Pseudomonas pseudoalcaligenes KF707 is a homo-octamer containing ferrous ions as the essential cofactor. Transposon mutants revealed that the bph-encoded enzymes possess a wide range of substrate specificity for various aromatic hydrocarbons. The bphABCXD gene cluster coding for the degradation of PCBs to chlorobenzoic acids was first cloned from P. pseudocaligenes KF707 and sequenced and then was cloned from a number of BP-utilizing strains and sequenced. Some strains possess a bph operon that is very similar, if not identical, to that of KF707. Some bph genes share homologies with different degrees. Deletion and shuffling of bph genes are also found.

Polychlorinated biphenyls (PCBs) can be transformed aerobically and anaerobically by microorganisms. Aerobic PCB degraders can be isolated as biphenyl (BP)utilizing bacteria, which are widely distributed in the environment. PCB degradation by such microbes is thus the co-metabolism by BP-metabolic enzymes. The major pathway of PCB degradation by soil bacteria proceeds via 2,3-dioxygenation, in which molecular oxygen is introduced at the nonchlorinated 2,3 site (1). This group of PCB degraders shows a rather narrow range of PCB specificities for degradation. A large number of Pseudomonas strains along with some strains of Alcaligenes, Achromobacter, Acinetobacter, and Moraxella have been isolated and characterized so far (2). Gram-positive strains such as Arthrobacter and Rhodococcus are also known to degrade PCBs through the same 2,3-dioxygenation. On the other hand, Alcaligenes eutrophus H850 (3) and Pseudomonas sp. LB400 (4) degrade PCBs primarily through 3,4-dioxygenation. The same strains also seem to possess a 2,3-dioxygenase system so that these strains show a wide range of PCB-congener specificities for the degradation. Typically, PCB degraders having 2,3-dioxygenase readily degrade 4,4'-dichlorobiphenyl (4,4'-CB), but they are almost inactive to 2,5,2',5'-CB. On the other hand, the PCB degraders having 3,4-dioxygenase metabolize 2,5,2',5'-CB efficiently, but metabolize 4,4'-CB rather slowly. Yeast and fungi convert PCBs mainly to monohydroxy compounds, as in the case of the mammalian system. Reductive dechlorination occurs due to anaerobic bacteria in laboratory and field experiments (5), but these anaerobic strains have not yet been purely isolated. The 3(meta) and 4(para) positions of highly chlorinated PCBs are preferentially dechlorinated by anaerobic bacteria, resulting in the formation of less chlorinated ones with chlorines at 2(ortho) positions.
The major oxidative degradation of BP/PCBs to (chloro)benzoates through 2,3-dioxygenation is presented in Figure 1. Chlorobenzoates are not metabolized any further by BP-utilizing bacteria so that these compounds usually accumulate in the reaction mixture, but catabolic intermediates such as dihydrodiols, dihydroxy compounds, ring-meta-cleavage compounds, or unknown compounds are often produced and accumulate. The occurrence of such compounds is dependent on the chlorine substitution of PCB congeners. For example, 2,3,2',3'-CB, 2,3,2',5'-CB, and 2,4,5,2',3'-CB, in which chlorines are commonly substituted at the 2,3 position on a single ring, were metabolized rapidly but by an alternative pathway, and a large amount of unknown products always accumulated. The 2,4,6-CB was slowly converted to 2,4,6-trichlorobenzoic acid by an Alcaligenes strain, but the same compound was rapidly converted to a trihydroxy com-pound via a dihydroxy compound by an Acinetobacter strain. The metabolic intermediates such as chlorobenzoic acids, dihydrodiols, monohydroxy, dihydroxy, and trihydroxy compounds, ring-meta-cleavage compounds, and some unknown compounds were produced from commercial PCB mixtures by both Gram-negative and Gram-positive strains having 2,3-dioxygenase. The effects of these catabolic intermediates on living organisms should be evaluated from the viewpoint of environmental toxicology.
Since the BP-utilizing bacteria that degrade PCBs through 2,3-dioxygenation are widely distributed in the environment, 2,3-dihydroxybiphenyl dioxygenase, the key enzyme of PCB degradation, was purified from two Pseudomonas strains, P. pseudoalcaligenes KF707 (isolated in Kyushu, Japan) and P. paucimobilis Ql (isolated in Chicago, IL). Both enzymes showed similar molecular masses of 260 kDa for the native enzyme and 33 kDa for the subunit, and both contained ferrous ions as essential cofactors for enzymatic activity so that the structure of the holoenzyme is considered to be (xFeII)8. However, the antibody raised against the KF707 enzyme did not show any crossreactivity with the Ql enzyme, and the Ql enzyme antibody did not cross-react with the KF707 enzyme. Sixteen PCB-degrading strains, including KF707 and Ql, were grown with BP and the cell extracts were prepared. Seven cell extracts, including KF707, among the 16 tested showed fused precipitin bands; 5 other extracts also showed precipitin bands but formed a spur. Four cell extracts did not cross-react. On the other hand, when the Ql enzyme Environmental Health Perspectives FURUKAWA AND KIMURA antibody was used, no cross-reactivity was observed for any cell extracts from the other 15 PCB degraders. The ring-metacleavage compound hydrolase was purified from P. pseudoalcaligenes KF707. The molecular masses of the native enzyme and the subunit were 120 kDa and 30 kDa, respectively, indicating that this enzyme is a homo-tetramer. The antibody raised against the KF707 hydrolase cross-reacted with 10 cell extracts, including the KF707 enzyme, forming fused precipitin bands. Four other cell extracts showed precipitin bands with a spur, and two cell extracts from P. paucimobilis Ql and Gram-positive Arthrobacter sp. M5 did not crossreact.
The gene clusters coding for the conversion of PCBs to chlorobenzoic acids were first cloned from P. pseudoalcaligenes KF707 (6) and then from some other Pseudomonas strains. The organization of bph operons of P. pseudoalcaligenes KF707 and P. putida KF715 were similar, particularly in bphA region (-4 kb), which encodes biphenyl dioxygenase. The bphB [dihydrodiol dehydrogenase gene, 831 base pairs (bp) in KF707], the bphC (2,3-dihydroxybiphenyl dioxygenase gene, 894 bp in KF707 and 876 in KF715), and the bphD (hydrolase gene, 855 bp in KF707 and 858 bp in KF715) were located downstream of bphA in this order in both operons, but the bphX region (-3.5 kilobases) was present between bphC and bphD in the KF707-bph operon, but the same DNA segment was missing in the KF715-bph operon. The 11.3 kb-DNA was sequenced (Figure 1 KF707. Since biphenyl dioxygenase is a multicomponent enzyme, five open reading frames (ORF) were detected; bphAl, bphA2, bphA3, and bphA4 were assigned to be a large subunit of terminal dioxygenase, a small subunit of terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively. Another orf3 was found between bphA2 and bphA3, but its function remains unknown. The orJ3-deletion mutant still retained the ability of BP oxidation (7).
The KF707 bphAIA2A3A4BC genes are very similar to the todCI C2BADE genes coding for toluene/benzene metabolism in P. putida Fl (8) in terms of gene organization as well as the size and homology of the corresponding enzymes and despite their discrete substrate specificities for metabolism. Studies of the gene components responsible for substrate specificity between the bph and tod operons revealed that the large subunit of the terminal dioxygenase (encoded by bphAl and todCl) and the hydrolase (bphD and todF) were critical for their discrete metabolic specificities (9). Introduction of todCl C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism), or even of only todCl, into the biphenyl-utilizing strain KF707 allowed it to grow on toluene/benzene. On the other hand, introduction of bphD into toluene-utilizing P. putida Fl permitted growth on biphenyl. Thus, the other components of ferredoxins (encoded by bphA31todB) and ferredoxin reductases (encoded by bphA41todD) were complementary with one another (9). Furthermore, Ecsherichia coli cells carrying a hybrid gene cluster of todCl::bphA2A3A4BC (constructed by replacing bphAl with todCl) converted toluene to a ring-meta-cleavage compound) indicating clearly that TodCG formed a functionally active multicomponent dioxygenase associated with BphA2, BphA3, and BphA4 (10). The bphXregion (3.5 kb) between bphC and bphD in the KF707 bph operon has been sequenced, and at least three ORFs (BphXl, BphX2, and BphX3) were found. The amino acid sequences of BphXl showed approximately 70% identity with DmpE or XylJ, which is a 2-hydroxypenta-2,4-dienoate hydratase in phenol/3,4dimethylphenol catabolism of Pseudomonas sp. strain CF600 (11) and the meta-cleavage pathway in the xyl operon of pWWO (12). Similarly, BphX2 showed approximately 56% identity with DmpF and XylQ of acetaldehyde dehydrogenase (acylating), and BphX3 showed approximately 57% identity with DmpG and XylK (4hydroxy-2-oxovalerate aldolase). Since 2hydroxypenta-2,4-dienoate is formed as a counterpart product of benzoic acid in the hydrolysis of BP-meta-cleavage compound, it is likely that the bphX region could be involved in the metabolism of 2-hydroxypenta-2,4-dienoate to acetyl coenzyme A via 4-hydroxy-2-oxovalerate and acetaldehyde ( Figure 1).
The molecular relationships of bph genes in various PCB degraders were investigated using bphABC genes of P. pseudoalcaligenes KF707 as the DNA probe (2). Among 15 strains tested, five Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on a XhoI 6.8-kb fragment corresponding to that of P. pseudoalcaligenes KF707. The restriction profiles of these Xhol 6.8-kb fragments containing bphABC genes were almost identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous to those of KF707, but the restriction profiles among them were different. Five other strains showed weak or no significant homology with the KF707 bphABC genes. On the other hand, the bphC gene of P. paucimobilis Ql lacked genetic homology with any of the other 15 PCB degraders. These data corresponded well with the immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenase (2). The existence of the nearly identical chromosomal genes among various stains suggests that the bphDNA segment of this group has a mechanism for transferring the genes from one strain to another. It is surprising to note that the organization as well as the nucleotide sequences of the bph operon of P. putida LB400 (12), which converts PCBs through 3,4-dioxygenase, is almost identical to those of P. pseudoalcaligenes KF707, having 2,3-dioxygenase. The bph operon of Pseudomonas sp. strain KKS 102 has been recently sequenced (13). The bph genes are greatly shuffled, compared with those of P. pseudoalcaligenes KF707. In the KKS bph operon, bphAIA2A3BCD is organized in this order but bphA4 is located downstream of bphD, and the bphXregion is located upstream of bphAl ( Figure 1). A transposon, Tn5-B21 (Tcr), was genespecifically inserted into the bph operon of P. pseudoalcaligenes KF707 (14). First, the cloned bphA, bphB, and bphC genes were mutagenized by random insertion of Tn5-B2 1. The mutagenized bphABC DNA, carried by a suicide plasmid, was then introduced back into the parent strain KF707, resulting in the appearance of mutants in which Tn5-B21 was gene-specifically inserted in the chromosomal bph operon. The bphA::Tn5-B21 mutant thus obtained did not attack 4-chlorobiphenyl at all; the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant accumulated dihydroxy compound. The transposon mutants revealed that the bphencoded enzymes possessed very relaxed substrate specificities for a variety of biphenyl-related compounds. P. pseudoalcaligenes KF707 Bph enzymes convert biphenyl with various substituents, such as halogen, hydroxyl, methyl, and nitro, and also biphenyl-related compounds, including biphenylmethane, dibenzyl, diphenylether, and benzalacetophenone.
However, the KF707 enzyme system is almost inactive to benzene and its derivatives. Another Pseudomonas sp., KF712, produced Bph enzymes that showed much wider substrate ranges, compared with the KF707 enzymes. The bphC::Tn5-B21 mutant of KF712 converted many benzene derivatives, as well as various BP derivatives and BP-related compounds, to the corresponding dihydroxy compounds. The relaxed substrate ranges of Bph enzymes, along with the fact that BP/PCB degraders are widely distributed in the environment, indicate that BP/PCB-degrading bacteria might be primarily involved in the degradation of plant lignin at the final stage, since plant lignin is massively distributed in the environment and is the source of various aromatic compounds, which include many benzene and biphenyl derivatives.