Multiple Polychlorinated Biphenyl Transformation Systems in the Gram-Positive Bacterium Rhodococcus sp. Strain RHA1

The cloned bphA gene of the polychlorinated biphenyl (PCB) degrader Rhodococcus sp. strain RHA1 was expressed in Rhodococcus erythropolis IAM1399 cells, resulting in the transformation of di-, tri-, and tetrachlorobiphenyls. Disruption of the bphA1 gene in RHA1 resulted in a lack of growth on biphenyl and a loss of PCB transformation activity. However, the bphA1 insertion mutant of RHA1, designated RDA1, retained the ability to transform PCB congeners when grown on ethylbenzene as its carbon source. It also transformed 4-chlorobiphenyl to 4-chlorobenzoate, although it was suspected to be deficient in bphB and bphC gene activities as well as bphA. This suggested that an alternative PCB degradation system distinct from the one encoded by the cloned bph genes was present.     

Polychlorinated biphenyl/biphenyl degrading gene clusters in Rhodococcus sp. K37, HA99, and TA431 are different from well-known bph gene clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.     

Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature

The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15min at 80 degrees C and 50min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.     

Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.     

Characterization of two biphenyl dioxygenases for biphenyl/PCB degradation in A PCB degrader, Rhodococcus sp. strain RHA1

Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.     

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.     

Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.     

Structural alteration of linear plasmids encoding the genes for polychlorinated biphenyl degradation in Rhodococcus strain RHA1

Polychlorinated biphenyl (PCB) tolerant derivatives of a strong PCB degrader, Rhodococcus strain RHA1, were selected after growth in the presence of 100 g/ml PCBs. Some of the derivatives did not grow on biphenyl but accumulated a yellow coloured metabolite suggesting a defect in the meta-ring-cleavage compound hydrolase step encoded by the bphD gene. Other derivatives failed to grow on biphenyl and exhibited little PCB transformation activity suggesting a defect in the initial ring-hydroxylation dioxygenase step encoded by the bphA gene. These organisms had a structural alteration in the linear plasmids coding for the bph genes in RHA1, which included the bph gene deletion. When a bphD containing plasmid was introduced into a tolerant derivative, RCD1, which was shown to have a bphD deletion, the defect in the growth on biphenyl of RCD1 was overcome. The bph gene deletion seems to play a key role in these tolerant derivatives thereby suggesting that the toxic metabolic intermediate would be a main cause of the growth inhibition of RHA1 in the presence of high concentration PCBs.     

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.     

Three of the seven bphC genes of Rhodococcus erythropolis TA421, isolated from a termite ecosystem, are located on an indigenous plasmid associated with biphenyl degradation.

Rhodococcus erythropolis TA421, a polychlorinated biphenyl and biphenyl degrader isolated from a termite ecosystem, has seven bphC genes expressing 2,3-dihydroxybiphenyl dioxygenase activity. R. erythropolis TA421 harbored a large and probably linear plasmid on which three (bphC2, bphC3, and bphC4) of the seven bphC genes were located. A non-biphenyl-degrading mutant, designated strain TA422, was obtained spontaneously from R. erythropolis TA421. TA422 lacked the plasmid, suggesting that the three bphC genes were involved in the degradation of biphenyl. Southern blot analyses showed that R. erythropolis TA421 and Rhodococcus globerulus P6 have a similar set of bphC genes and that the genes for biphenyl catabolism are located on plasmids of different sizes. These results indicated that the genes encoding the biphenyl catabolic pathway in Rhodococcus strains are borne on plasmids.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.     

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.     

Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.     

Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6

Rhodococcus globerulus strain P6 contains at least three genes, bphC1, bphC2, and bphC3, coding for 2,3-dihydroxybiphenyl 1,2-dioxygenases; the latter two specify enzymes of the family of one-domain extradiol dioxygenases. In order to assess the importance of these different isoenzymes for the broad catabolic activity of this organism towards the degradation of polychlorinated biphenyls (PCBs), the capacities of recombinant enzymes expressed in Escherichia coli to transform different chlorosubstituted dihydroxybiphenyls formed by the action of R. globerulus P6 biphenyl dioxygenase and biphenyl 2,3-dihydrodiol dehydrogenase were determined. Whereas both BphC2 and BphC3 showed similar activities for 2,3-dihydroxybiphenyl and all monochlorinated 2,3-dihydroxybiphenyls, BphC1 exhibited only weak activity for 2'-chloro-2,3-dihydroxybiphenyl. More highly chlorinated 2'-chlorosubstituted 2,3-dihydroxybiphenyls were also transformed at high rates by BphC2 and BphC3 but not BphC1. In R. globerulus P6, BphC2 was constitutively expressed, BphC1 expression was induced during growth on biphenyl, and BphC3 was not expressed at significant levels under the experimental conditions. Although we cannot rule out the expression of BphC3 under certain environmental conditions, it seems that the contrasting substrate specificities of BphC1 and BphC2 contribute significantly to the versatile PCB-degrading phenotype of R. globerulus P6.     

Family shuffling of a targeted bphA region to engineer biphenyl dioxygenase

In this work we used a new strategy designed to reduce the size of the library that needs to be explored in family shuffling to evolve new biphenyl dioxygenases (BPDOs). Instead of shuffling the whole gene, we have targeted a fragment of bphA that is critical for enzyme specificity. We also describe a new protocol to screen for more potent BPDOs that is based on the detection of catechol metabolites from chlorobiphenyls. Several BphA variants with extended potency to degrade polychlorinated biphenyls (PCBs) were obtained by shuffling critical segments of bphA genes from Burkholderia sp. strain LB400, Comamonas testosteroni B-356, and Rhodococcus globerulus P6. Unlike all parents, these variants exhibited high activity toward 2,2'-, 3,3'-, and 4,4'-dichlorobiphenyls and were able to oxygenate the very persistent 2,6-dichlorobiphenyl. The data showed that the replacement of a short segment (335TFNNIRI341) of LB400 BphA by the corresponding segment (333GINTIRT339) of B-356 BphA or P6 BphA contributes to relax the enzyme toward PCB substrates.     

Multiplicity of 2,3-dihydroxybiphenyl dioxygenase genes in the Gram-positive polychlorinated biphenyl degrading bacterium Rhodococcus rhodochrous K37

Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.