Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants that have been widely used in consumer products, but that are problematic because of their environmental persistence and endocrine‐disrupting properties. To date, very little is known about PBDE degradation by aerobic microorganisms and the enzymes involved in PBDE transformation. Resting cells of the polychlorinated biphenyl‐degrading actinomycete, Rhodococcus jostii RHA1, depleted nine mono‐ through penta‐BDEs in separate assays. Extensive depletion of PBDEs occurred with cells grown on biphenyl, ethylbenzene, propane, or styrene, whereas very limited depletion occurred with cells grown on pyruvate or benzoate. In RHA1, expression of bphAa encoding biphenyl dioxygenase (BPDO) and etbAa1 and etbAc encoding ethylbenzene dioxygenase (EBDO) was induced 30‐ to 3,000‐fold during growth on the substrates that supported PBDE depletion. The BPDO and EBDO enzymes had gene expression profiles that matched the PBDE‐depletion profiles exhibited by RHA1 grown on different substrates. Using the non‐PBDE‐degrading bacterium Rhodococcus erythropolis as a host, two recombinant strains were developed by inserting the eth and bph genes of RHA1, respectively. The resultant EBDO extensively depleted mono‐ through penta‐BDEs, while the BPDO depleted only mono‐, di‐, and one tetra‐BDE. A dihydroxylated‐BDE was detected as the primary metabolite of 4‐bromodiphenyl ether in both recombinant strains. These results indicate that although both dioxygenases are capable of transforming PBDEs, EBDO more potently transforms the highly brominated congeners. The availability of substrates or inducing compounds can markedly affect total PBDE removal as well as patterns of removal of individual congeners. Biotechnol. Bioeng. 2011;108: 313–321. © 2010 Wiley Periodicals, Inc.     

The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene (etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27–33% and 32–38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCbphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.     

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.     

Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases

The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic α subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant α subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase α subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an α subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.     

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.     

Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.     

Biphenyl dioxygenase from an arctic isolate is not cold adapted

Biphenyl dioxygenase from the psychrotolerant bacterium Pseudomonas sp. strain Cam-1 (BPDO(Cam-1)) was purified and found to have an apparent k(cat) for biphenyl of 1.1 +/- 0.1 s(-1) (mean +/- standard deviation) at 4 degrees C. In contrast, BPDO(LB400) from the mesophile Burkholderia xenovorans LB400 had no detectable activity at this temperature. At 57 degrees C, the half-life of the BPDO(Cam-1) oxygenase was less than half that of the BPDO(LB400) oxygenase. Nevertheless, BPDO(Cam-1) appears to be a typical Pseudomonas pseudoalcaligenes KF707-type dioxygenase.     

Family shuffling of soil DNA to change the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 +/- 1 nmol min(-1) per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues M237 S238 and S283 outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.     

Identification of Chlorobenzene Dioxygenase Sequence Elements Involved in Dechlorination of 1,2,4,5-Tetrachlorobenzene

The TecA chlorobenzene dioxygenase and the TodCBA toluene dioxygenase exhibit substantial sequence similarity yet have different substrate specificities. Escherichia coli cells producing recombinant TecA enzyme dioxygenate and simultaneously eliminate a halogen substituent from 1,2,4,5-tetrachlorobenzene but show no activity toward benzene, whereas those producing TodCBA dioxygenate benzene but not tetrachlorobenzene. A hybrid TecA dioxygenase variant containing the large α-subunit of the TodCBA dioxygenase exhibited a TodCBA dioxygenase specificity. Acquisition of dehalogenase activity was achieved by replacement of specific todC1 α-subunit subsequences by equivalent sequences of the tecA1 α-subunit. Substrate transformation specificities and rates by E. coli resting cells expressing hybrid systems were analyzed by high-performance liquid chromatography. This allowed the identification of both a single amino acid and potentially interacting regions required for dechlorination of tetrachlorobenzene. Hybrids with extended substrate ranges were generated that exhibited activity toward both benzene and tetrachlorobenzene. The regions determining substrate specificity in (chloro)benzene dioxygenases appear to be different from those previously identified in biphenyl dioxygenases.     

Gene expression profiling of Pseudomonas putida F1 after exposure to aromatic hydrocarbon in soil by using proteome analysis

Pseudomonas putida F1 can metabolize toluene, ethylbenzene, and benzene for growth. Previously, we identified proteins involved in the utilization of these compounds by P. putida F1 through culture in liquid media. However, it was unclear whether laboratory analysis of bacterial activity and catabolism accurately reflected the soil environment. We identified proteins involved in the degradation of toluene, ethylbenzene, and benzene growth in soil using two-dimensional gel electrophoresis (2-DE) or standard SDS-PAGE combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS). According to 2-DE/LC–MS/MS analysis, 12 of 22 key enzymes involved in the degradation of toluene, ethylbenzene, and benzene were detected. In standard SDS-PAGE/LC–MS/MS analysis of soil with ethylbenzene, approximately 1,260 cellular proteins were identified in P. putida F1. All key enzymes and transporter and sensor proteins involved in ethylbenzene degradation were up-regulated similar to that noted in liquid cultures. In P. putida F1, aromatic hydrocarbon response in soil is the same as that observed in liquid media.     

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.     

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.