Biphenyl uptake by psychrotolerant Pseudomonas sp. strain Cam-1 and mesophilic Burkholderia sp. strain LB400

We investigated the uptake of biphenyl by the psychrotolerant, polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain Cam-1 and the mesophilic PCB-degrader, Burkholderia sp. strain LB400. The effects of growth substrates, metabolic inhibitors, and temperature on [14C]biphenyl uptake were studied. Biphenyl uptake by both strains was induced by growth on biphenyl, and was inhibited by dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which are metabolic uncouplers. The Vmax and Km for biphenyl uptake by Cam-1 at 22 degrees C were 5.4 +/- 1.7 nmol x min(-1) x (mg of cell protein)(-1) and 83.1 +/- 15.9 micromol x L(-1), respectively. The Vmax and Km for biphenyl uptake by LB400 at 22 degrees C were 3.2 +/- 0.3 nmol x min(-1) x (mg of cell protein(-1)) and 51.5 +/- 9.6 micromol x L(-1), respectively. At 15 degrees C, the maximum rate for biphenyl uptake by Cam-1 and LB400 was 3.1 +/- 0.3 nmol x min(-1) x (mg of cell protein)(-1) and 0.89 +/- 0.1 nmol x min(-1) x (mg of cell protein)(-1), respectively. Thus, the maximum rate for biphenyl uptake by Cam-1 at 15 degrees C was more than 3 times higher than that for LB400.     

Regiospecificity of Dioxygenation of Di- to Pentachlorobiphenyls and Their Degradation to Chlorobenzoates by the bph-Encoded Catabolic Pathway of Burkholderia sp. Strain LB400

Burkholderia sp. strain LB400 is one of the most potent aerobic polychlorobiphenyl (PCB)-degrading microorganisms that have been characterized. Its PCB-dioxygenating activity originates predominantly or exclusively from the biphenyl dioxygenase encoded by its bph gene cluster. Analysis of the dioxygenation products of several di- to pentachlorinated biphenyls formed by this enzyme revealed a complex dependence of the regiospecificity and the yield of dioxygenation on the substitution patterns of both the oxidized and the nonoxidized rings. No dioxygenolytic attack involving chlorinated meta or para carbons was observed. Therefore, the ability of the enzyme to hydroxylate chlorinated carbons appears to be limited to the ortho position. However, it is not limited to monochlorinated rings, as evidenced by dioxygenation of the 2,4-disubstituted ring at carbons 2 and 3. This site of attack is strikingly different from that of the 2,5-dichlorinated ring, which has been shown to be dihydroxylated at positions 3 and 4 (J. D. Haddock, J. R. Horton, and D. T. Gibson, J. Bacteriol. 177:20–26, 1995). These results demonstrate that a second substituent of ortho-chlorinated rings crucially influences the site of dioxygenation at this ring and thereby determines whether or not the initial chlorobiphenyl oxidation product is further metabolized through the bph-encoded pathway. The 2,4-dichlorinated ring can alternatively be attacked at carbons 5 and 6. The preferred site crucially depends on the substitution pattern of the other ring. The formation of more than a single dioxygenation product was found predominantly with congeners that contain two chlorinated rings, both of which are similarly prone to dioxygenation or one is substituted only at carbon 3.     

Mineralization of low-chlorinated biphenyls by Burkholderia sp. strain LB400 and by a two-membered consortium upon directed interspecies transfer of chlorocatechol pathway genes

The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4^′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction. Received: 15 April 1998 / Received revision: 12 June 1998 / Accepted: 19 June 1998     

Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400

Abstract In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho -dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.     

Influence of chlorine substituents on rates of oxidation of chlorinated biphenyls by the biphenyl dioxygenase of Burkholderia sp. strain LB400

Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3',4'-CB, 2,3,2',5'-CB, 2,3,4,5-CB, 2,3,2',3'-CB, 2,4, 5,2',5'-CB, 2,5,3'-CB, 2,5,4'-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2',5'-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4'-CB, 2,4,2',4'-CB, 3,4,5, 2'-CB, 3,4,5,3'-CB, 3,5,3',5'-CB, and 3,4,5,2',5'-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.     

Phenotypic and Phylogenetic Characterization of Burkholderia (Pseudomonas) sp. Strain LB400

The relevant phenotypic traits and phylogenetic relationships between Burkholderia (Pseudomonas) sp. strain LB400 and B. cepacia ATCC 25416^T were compared to determine the degree to which these two strains might be related. Strain LB400 degrades chlorinated biphenyls and has been a model system for potential use in the bioremediation of polychlorinated biphenyls, while some strains of B. cepacia are plant and human pathogens. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that strain LB400 was a member of the genus Burkholderia. The 16S rRNA gene sequence showed that this strain was not as closely related to B. cepacia as previously suspected or to other known pathogens of this genus, but is closely related to B. phenazinium, B. caribensis, B. graminis, and three unnamed Burkholderia spp. not known to be pathogenic. Received: 16 August 2000 / Accepted: 27 September 2000     

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.     

Characterization of hybrid biphenyl dioxygenases obtained by recombining Burkholderia sp. strain LB400 bphA with the homologous gene of Comamonas testosteroni B-356.

The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 alpha subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO alpha subunit with that of LB400 alpha subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.     

A catabolic pathway for the degradation of chrysene by Pseudoxanthomonas sp. PNK-04

The chrysene-degrading bacterium Pseudoxanthomonas sp. PNK-04 was isolated from a coal sample. Three novel metabolites, hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid and salicylic acid, were identified by TLC, HPLC and MS. Key enzyme activities, namely 1-hydroxy-2-naphthoate hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase, were noted in the cell-free extract. These results suggest that chrysene is catabolized via hydroxyphenanthroic acid, 1-hydroxy-2-naphthoic acid, salicylic acid and catechol. The terminal aromatic metabolite, catechol, is then catabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed catabolic pathway for chrysene degradation by strain PNK-04 is chrysene → hydroxyphenanthroic acid → 1-hydroxy-2-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol →cis,cis-muconic acid.     

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.     

Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707.

Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Degradation of aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. strain RHA1(fcb)

Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10(4) or 10(6) cells g(-1) sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.     

Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.     

Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 was examined. The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs. cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate. Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates. Catechols were produced when the dioxygenase-catalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate. Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol. Compounds resulting from both types of reaction were produced during oxidation of 2,5,2'-trichlorobiphenyl. The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp. strain LB400.     

Multiple pathways for toluene degradation in Burkholderia sp. strain JS150.

Burkholderia (Pseudomonas) sp. strain JS150 uses multiple pathways for the metabolism of catechols that result from degradation of aromatic compounds. This suggests that the strain also uses multiple upstream pathways for the initial hydroxylation of aromatic substrates. Two distinct DNA fragments that allowed Pseudomonas aeruginosa PAO1c to grow with benzene as a sole carbon source were cloned from strain JS150. One of the recombinant plasmids containing the initial steps for the degradative pathway contained a 14-kb DNA insert and was designated pRO2016. We have previously shown that the DNA insert originated from a plasmid carried by strain JS150 and contained genes encoding a multicomponent toluene-2-monooxygenase (tbmABCDEF) as well as the cognate regulatory protein (tbmR) that controls expression of the 2-monooxygenase (G. R. Johnson and R. H. Olsen, Appl. Environ. Microbiol. 61:3336-3346, 1995). Subsequently, we have identified an additional region on this DNA fragment that encodes toluene-4-monooxygenase activity. The toluene-4-monooxygenase activity was also regulated by the tbmR gene product. A second DNA fragment that allowed P. aeruginosa to grow with benzene was obtained as a 20-kb insert on a recombinant plasmid designated pRO2015. The DNA insert contained genes encoding toluene-4-monooxygenase activity but no toluene-2-monooxygenase activity. The pRO2015 insert originated from the chromosome of strain JS150, unlike the region cloned in pRO2016. Southern blots and restriction map comparisons showed that the genes for the individual 4-monooxygenases were distinct from one another. Thus, strain JS150 has been shown to have at least three toluene/benzene monooxygenases to initiate toluene metabolism in addition to the toluene dioxygenase reported previously by others.     

Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400

Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDH(C) (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDH(C) based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDH(C) and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDH(C) promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400.     

Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.