Antioxidant compounds improved PCB-degradation by Burkholderia xenovorans strain LB400

Polychlorobiphenyls (PCBs) are toxic and persistent organic pollutants that are widely distributed in the environment. Burkholderia xenovorans LB400 is capable of degrading aerobically an unusually wide range of PCBs. However, during PCB-degradation B. xenovorans LB400 generates reactive oxygen species (ROS) that affect its viability. The aim of this study was to increase the efficiency of PCB-degradation of B. xenovorans LB400 by adding antioxidant compounds that could increase tolerance to oxidative stress. The effect of antioxidant compounds on the growth, morphology and PCB-degradation by B. xenovorans LB400 was evaluated. α-Tocopherol or vitamin E (vitE) and berry extract (BE) increased slightly the growth of strain LB400 on biphenyl, whereas in presence of ascorbic acid or vitamin C (vitC) an inhibition of growth was observed. The growth of B. xenovorans LB400 in glucose was inhibited by the addition of 4-chlorobiphenyl (4-CB). Interestingly, in presence of α-tocopherol the growth of strain LB400 was less affected by 4-CB. By transmission electronic microscopy it was observed that α-tocopherol preserved the cell membranes and improved cell integrity of glucose-grown LB400 cells exposed to 4-CB, suggesting a protective effect of α-tocopherol. Notably, α-tocopherol increased biphenyl and 4-CB degradation by B. xenovorans LB400 in an aqueous solution. The effect of antioxidants compounds on PCB-bioremediation was evaluated in agricultural soil spiked with 2-chlorobiphenyl (2-CB), 4-CB and 2,4'-chlorobiphenyl (2,4'-CB). For bioaugmentation, LB400 cells grown on biphenyl and subsequently incubated with pyruvate were added to the soil. Native soil microbiota was able to remove PCBs. Bioaugmentation with strain LB400 increased strongly the PCB-degradation rate. Bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract increased further the PCB degradation. Half-life of 2,4'-CB decreased by bioaugmentation from 24 days to 4 days and by bioaugmentation in presence of α-tocopherol and berry extract to 2 days. By bioaugmentation with strain LB400, 85% of 2,4'-CB was degraded in 20 days, whereas bioaugmentation with strain LB400 and biostimulation with α-tocopherol or berry extract reduced the time to less than 13 days. This indicates that antioxidant compounds stimulated PCB-degradation in soil. Therefore, the addition of antioxidant compounds constitutes an attractive strategy for the scale-up of aerobic PCB-bioremediation processes.     

Enhancement of PCB degradation by Burkholderia xenovorans LB400 in biphasic systems by manipulating culture conditions

Two-phase partitioning bioreactors (TPPBs) can be used to biodegrade environmental contaminants after their extraction from soil. TPPBs are typically stirred tank bioreactors containing an aqueous phase hosting the degrading microorganism and an immiscible, non-toxic and non-bioavailable organic phase functioning as a reservoir for hydrophobic compounds. Biodegradation of these compounds in the aqueous phase results in thermodynamic disequilibrium and partitioning of additional compounds from the organic phase into the aqueous phase. This self-regulated process can allow the delivery of large amounts of hydrophobic substances to degrading microorganisms. This paper explores the reactor conditions under which the polychlorinated biphenyl (PCB) degrader Burkholderia xenovorans LB400 can degrade significant amounts of the PCB mixture Aroclor(R) 1242. Aroclor(R) degradation was found to stall after approximately 40 h if no carbon source other than PCBs was available in the reactor. Sodium pyruvate was found to be a suitable carbon source to maintain microbial activity against PCBs and to function as a substrate for additional cell growth. Both biphenyl (while required during the inoculum preparation) and glucose had a negative effect during the Aroclor(R) degradation phase. Initial Aroclor(R) 1242 degradation rates in the presence of pyruvate were high (6.2 mg L(-1) h(-1)) and 85% of an equivalent concentration of 100 mg Aroclor(R) 1242 per L aqueous phase could be degraded in 48 h, which suggest that solvent extraction of PCBs from soil followed by their biodegradation in TPPBs might be a feasible remediation option.     

多氯联苯降解菌Burkholderia xenovorans LB400研究进展

Burkholderia xenovorans LB400是一株多氯联苯(polychlorinated biphenyls,PCBs)降解菌,可以氧化含有1?6个氯取代基的多氯联苯。近年来,由于其广泛的底物谱和优异的降解性能,菌株LB400已成为研究原核生物降解多氯联苯的生物化学和分子生物学方面的模式生物。目前关于PCBs的微生物降解研究已不再局限于对微生物资源的挖掘,而是更多地聚焦在LB400等降解菌的PCBs降解基因、降解酶的酶学特性以及酶的人工分子进化等方面。同时,LB400作为早期发现的降解菌,其对多氯联苯的降解途径、底物范围及相关机制也被广泛探讨;但是对于PCBs降解相关基因的调控研究较少。因此,本文以Burkholderia xenovorans LB400对多氯联苯降解为核心,通过综述其代谢途径、代谢相关基因和酶系以及降解应用等方面的研究进展,以期为深入探讨Burkholderia xenovorans LB400的应用以及进一步在遗传、分子和生化水平研究其他多氯联苯降解菌株提供借鉴。     

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400

Burkholderia species are free-living bacteria with a versatile metabolic lifestyle. The genome of B. fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system. The other Burkholderia species for which genome sequences are available, B. mallei, B. pseudomallei, and B. cepacia, are predicted to contain only the first two of these pathways. The roles of the three putative formaldehyde oxidation pathways in B. fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts. The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate. Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions.     

Metabolism of dibenzofuran and dibenzo-p-dioxin by the biphenyl dioxygenase of Burkholderia xenovorans LB400 and Comamonas testosteroni B-356

We examined the metabolism of dibenzofuran (DF) and dibenzo-p-dioxin (DD) by the biphenyl dioxygenase (BPDO) of Comamonas testosteroni B-356 and compared it with that of Burkholderia xenovorans LB400. Data showed that both enzymes oxygenated DF at a low rate, but Escherichia coli cells expressing LB400 BPDO degraded DF at higher rate (30 nmol in 18 h) compared with cells expressing B-356 BPDO (2 nmol in 18 h). Furthermore, both BPDOs produced dihydro-dihydroxy-dibenzofuran as a major metabolite, which resulted from the lateral oxygenation of DF. 2,2,3-Trihydroxybiphenyl (resulting from angular oxygenation of DF) was a minor metabolite produced by both enzymes. Deuterated DF was used to demonstrate the production of 2,2,3-dihydroxybiphenyl through angular oxygenation of DF. When tested for their ability to oxygenate DD, both enzymes produced as sole metabolite, 2,2,3-trihydroxybiphenyl ether at about the same rate, indicating similar catalytic properties toward this substrate. Altogether, although LB400 and B-356 BPDOs oxygenate a different range of chlorobiphenyls, their metabolite profiles toward DF and DD are similar. This suggests that co-planarity influences the regiospecificity of BPDO toward DF and DD to a higher extent than the presence of an ortho substituent on the molecule.     

Distinct roles for two CYP226 family cytochromes P450 in abietane diterpenoid catabolism by Burkholderia xenovorans LB400

The 80-kb dit cluster of Burkholderia xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinate-grown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 ± 0.01 μM) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.     

Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

Coping with Polychlorinated Biphenyl (PCB) Toxicity: Physiological and Genome-Wide Responses of Burkholderia xenovorans LB400 to PCB-Mediated Stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.     

Coping with polychlorinated biphenyl (PCB) toxicity: Physiological and genome-wide responses of Burkholderia xenovorans LB400 to PCB-mediated stress

The biodegradation of polychlorinated biphenyls (PCBs) relies on the ability of aerobic microorganisms such as Burkholderia xenovorans sp. LB400 to tolerate two potential modes of toxicity presented by PCB degradation: passive toxicity, as hydrophobic PCBs potentially disrupt membrane and protein function, and degradation-dependent toxicity from intermediates of incomplete degradation. We monitored the physiological characteristics and genome-wide expression patterns of LB400 in response to the presence of Aroclor 1242 (500 ppm) under low expression of the structural biphenyl pathway (succinate and benzoate growth) and under induction by biphenyl. We found no inhibition of growth or change in fatty acid profile due to PCBs under nondegrading conditions. Moreover, we observed no differential gene expression due to PCBs themselves. However, PCBs did have a slight effect on the biosurface area of LB400 cells and caused slight membrane separation. Upon activation of the biphenyl pathway, we found growth inhibition from PCBs beginning after exponential-phase growth suggestive of the accumulation of toxic compounds. Genome-wide expression profiling revealed 47 differentially expressed genes (0.56% of all genes) under these conditions. The biphenyl and catechol pathways were induced as expected, but the quinoprotein methanol metabolic pathway and a putative chloroacetaldehyde dehydrogenase were also highly expressed. As the latter protein is essential to conversion of toxic metabolites in dichloroethane degradation, it may play a similar role in the degradation of chlorinated aliphatic compounds resulting from PCB degradation.