Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Bioremediation of Aroclor 1242 by a consortium culture in marine sediment microcosm

Plant terpenes have proven to be effective in stimulation of polychlorinated biphenyls (PCBs) biodegradation in soil systems. However, data on the application of plant terpenes in marine sediments contaminated with PCBs remains limited. The aim of this study was to ascertain the roles of a PCB degrading consortium and plant terpenes in stimulation of PCB biodegradation in marine sediments. The consortium culture 1-2Mix (strains 1-2M and 1-2T in commensalism), a utilizer of biphenyl and a natural substrate was enriched and isolated from marine sediments from the Busan coast, South Korea. PCB degradation by this culture was shown to be more effectively induced by tangerine peel extract than other known substrates (limonene, pinene, and cymene). Coastal sediment microcosms inoculated with 1-2Mix were set up to elucidate the effect of the consortium and plant terpenes on degradation of Aroclor 1242. After four weeks, the highest removal rates of PCBs, compared with the control (autoclaved sediment and no inoculation of 1-2Mix), were observed in order of the inducers tested; biphenyl (71.1%), tangerine peel extract (69.5%), surfactant (66.0%), and limonene (63.0%). Bioaugmentation effect was doubled in the presence of natural substrates such as tangerine peel extract and limonene, indicating effectiveness of these substrates in biostimulation. It was concluded that the tangerine peel extract could replace biphenyl as a feasible induction substrate for effective remediation of PCBs in the marine sediment.     

Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans

Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Structure of the terminal oxygenase component of angular dioxygenase, carbazole 1,9a-dioxygenase

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.     

Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4

Sphingomonas sp. strain A4 is capable of utilizing acenaphthene and acenaphthylene as sole carbon and energy sources, but it is unable to grow on other polycyclic aromatic hydrocarbons (PAHs). The genes encoding terminal oxygenase components of ring-hydroxylating dioxygenase (arhA1 and arhA2) were isolated from this strain by means of the ability to oxidize indole to indigo of the Escherichia coli clone containing electron transport proteins from phenanthrene-degrading Sphingobium sp. strain P2. The translated products of arhA1 and arhA2 exhibited moderate sequence identity (less than 56%) to large and small subunits of dioxygenase of other ring-hydroxylating dioxygenases. Biotransformation with recombinant E. coli clone revealed the broad substrate specificity of this oxygenase toward several PAHs including acenaphthene, acenaphthylene, naphthalene, phenanthrene, anthracene and fluoranthene. Southern hybridization analysis revealed the presence of a putative arhA1 homologue on a locus different from that of the arhA1 gene. Insertion inactivation of the arhA1 gene in strain A4 suggested that the gene but not the putative homologue one was involved in the degradation of acenaphthene and acenaphthylene in this strain.     

Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution

The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE_LB400) are largely unknown. BphAE_p4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE_LB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE_LB400 and BphAE_p4 and examine the biochemical properties of two BphAE_LB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.     

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.     

Expression of bacterial biphenyl-chlorobiphenyl dioxygenase genes in tobacco plants

Optimized plant-microbe bioremediation processes in which the plant initiates the metabolism of xenobiotics and releases the metabolites in the rhizosphere to be further degraded by the rhizobacteria is a promising alternative to restore contaminated sites in situ. However, such processes require that plants produce the metabolites that bacteria can readily oxidize. The biphenyl dioxygenase is the first enzyme of the bacterial catabolic pathway involved in the degradation of polychlorinated biphenyls. This enzyme consists of three components: the two sub-unit oxygenase (BphAE) containing a Rieske-type iron-sulfur cluster and a mononuclear iron center, the Rieske-type ferredoxin (BphF), and the FAD-containing ferredoxin reductase (BphG). In this work, based on analyses with Nicotiana benthamiana plants transiently expressing the biphenyl dioxygenase genes from Burkholderia xenovorans LB400 and transgenic Nicotiana tabacum plants transformed with each of these four genes, we have shown that each of the three biphenyl dioxygenase components can be produced individually as active protein in tobacco plants. Therefore, when BphAE, BphF, and BphG purified from plant were used to catalyze the oxygenation of 4-chlorobiphenyl, detectable amounts of 2,3-dihydro-2, 3-dihydroxy-4'-chlorobiphenyl were produced. This suggests that creating transgenic plants expressing simultaneously all four genes required to produce active biphenyl dioxygenase is feasible.     

The bphC gene-encoded 2,3-dihydroxybiphenyl-1,2-dioxygenase is involved in complete degradation of dibenzofuran by the biphenyl-degrading bacterium Ralstonia sp. SBUG 290

AIMS: Biphenyl-degrading bacteria are able to metabolize dibenzofuran via lateral dioxygenation and meta-cleavage of the dihydroxylated dibenzofuran produced. This degradation was considered to be incomplete because accumulation of a yellow-orange ring-cleavage product was observed. In this study, we want to characterize the 1,2-dihydroxydibenzofuran cleaving enzyme which is involved in dibenzofuran degradation in the bacterium Ralstonia sp. SBUG 290. METHODS AND RESULTS: In this strain, complete degradation of dibenzofuran was observed after cultivation on biphenyl. The enzyme shows a wide substrate utilization spectrum, including 1,2-dihydroxydibenzofuran, 2,3-dihydroxybiphenyl, 1,2-dihydroxynaphthalene, 3- and 4-methylcatechol and catechol. MALDI-TOF analysis of the protein revealed a strong homology to the bphC gene products. We therefore cloned a 3.2 kb DNA fragment containing the bphC gene of Ralstonia sp. SBUG 290. The deduced amino acid sequence of bphC is identical to that of the corresponding gene in Pseudomonas sp. KKS102. The bphC gene was expressed in Escherichia coli and the meta-fission activity was detected using either 2,3-dihydroxybiphenyl or 1,2-dihydroxydibenzofuran as substrate. CONCLUSIONS: These results demonstrate that complete degradation of dibenzofuran by biphenyl degraders can occur after initial oxidation steps catalysed by gene products encoded by the bph-operon. The ring fission of 1,2-dihydroxydibenzofuran is catalysed by BphC. Differences found in the metabolism of the ring fission product of dibenzofuran among biphenyl degrading bacteria are assumed to be caused by different substrate specificities of BphD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows for the first time that the gene products of the bph-operon are involved in the mineralization of dibenzofuran in biphenyl degrading bacteria.     

Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites have a strong effect on the bacterial community structure, activity, and associated degradative ability.     

Characterization of the 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation system in Janibacter sp. TYM3221

Bacterial degradation of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) has been previously reported, however, its degradation enzyme system has not been characterized. In this study, a DDE-degrading bacterium, Janibacter sp. TYM3221, was isolated and characterized. Transformation of DDE was demonstrated by TYM3211 resting cells grown in LB in the presence and absence of biphenyl. Gas chromatography-mass spectrometry analysis revealed five metabolites of DDE containing a meta-ring cleavage product and 4-chlorobenzoic acid, suggesting that TYM3221 degrades DDE to 4-chlorobenzoic acid via a meta-ring cleavage product. A gene cluster, bphAaAbAcAd, which codes for biphenyl dioxygenase subunits, was cloned from TYM3221. A mutant strain with a bphAa-gene inactivation did not grow on biphenyl, and showed no DDE degradation activity. These results indicate that in strain TYM3221, the bphAa-coded biphenyl dioxygenase is involved not only in the metabolism of biphenyl but also in the degradation of DDE.     

Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.     

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.     

Characterization of the 5-Carboxyvanillate Decarboxylase Gene and Its Role in Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.