Metabolism of 2,2'- and 3,3'-dihydroxybiphenyl by the biphenyl catabolic pathway of Comamonas testosteroni B-356.

The purpose of this investigation was to examine the capacity of the biphenyl catabolic enzymes of Comamonas testosteroni B-356 to metabolize dihydroxybiphenyls symmetrically substituted on both rings. Data show that 3,3'-dihydroxybiphenyl is by far the preferred substrate for strain B-356. However, the dihydrodiol metabolite is very unstable and readily tautomerizes to a dead-end metabolite or is dehydroxylated by elimination of water. The tautomerization route is the most prominent. Thus, a very small fraction of the substrate is converted to other hydroxylated and acidic metabolites. Although 2,2'-dihydroxybiphenyl is a poor substrate for strain B-356 biphenyl dioxygenase, metabolites were produced by the biphenyl catabolic enzymes, leading to production of 2-hydroxybenzoic acid. Data show that the major route of metabolism involves, as a first step, a direct dehydroxylation of one of the ortho-substituted carbons to yield 2,3,2'-trihydroxybiphenyl. However, other metabolites resulting from hydroxylation of carbons 5 and 6 of 2,2'-dihydroxybiphenyl were also produced, leading to dead-end metabolites.

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.

Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356.

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinated biphenyl degradation pathway. Based on the crystal structure of a related BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been suggested to function as a "catalytic triad" as for other members of the short-chain alcohol dehydrogenase/reductase (SDR) family. In this study, substitution of each triad residue was examined in BphB. At pH 9.0, turnover numbers relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; and K159A, 10%. Although the Michaelis constants of K159A and S142A for cis-2,3-dihydro-2,3-dihydroxybiphenyl increased about 20-fold, relatively little change was observed in the K(m) for dinucleotide. The K159A mutant, which showed little dehydrogenase activity at pH 7, was sharply activated by increasing the pH, reaching almost 25% of the activity of the wild-type enzyme at pH 9. 8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family members. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K(m)). Evidence is presented that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.

A ColE1-compatible expression vector for the production of His-tagged fusion proteins.

Plasmid pDB31 is a ColE1-compatible expression vector based on the p15A origin of replication. It is designed to express His-tagged fusion proteins in cells co-hosting a compatible expression vector. It was constructed by assembling the operator/promoter region plus the 6xHis and the multiple cloning site of pQE31 (QIAGEN) with the p15A origin of replication plus KanR of pGP1-2. The plasmid was found to be stable in Escherichia coli strains BL21 and DH11S. It was used to produce and purify the ferredoxin reductase component of Comamonas testosteroni B-356 biphenyl dioxygenase inside a clone hosting the remaining dioxygenase genes on a compatible plasmid.

Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it.

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The alpha or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp. strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(LB400)beta(P6), and ht-alpha(B-356)beta(P6). ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase alpha subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E. coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an alpha and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.

Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase.

Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2'-, 3,3'-, 4,4'-di- and 2,2',5,5'-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases.

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates.

Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively). A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase.

Degradation of polychlorinated biphenyl metabolites by naphthalene-catabolizing enzymes.

The ability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to transform 3,4-dihydroxylated biphenyl metabolites was investigated. 1,2-Dihydro-1, 2-dihydroxynaphthalene dehydrogenase was expressed as a histidine-tagged protein. The purified enzyme transformed 2, 3-dihydro-2,3-dihydroxybiphenyl, 3,4-dihydro-3,4-dihydroxybiphenyl, and 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl to 2, 3-dihydroxybiphenyl, 3,4-dihydroxybiphenyl (3,4-DHB), and 3, 4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl (3,4-DH-2,2',5,5'-TCB), respectively. Our data also suggested that purified 1, 2-dihydroxynaphthalene dioxygenase catalyzed the meta cleavage of 3, 4-DHB in both the 2,3 and 4,5 positions. This enzyme cleaved 3, 4-DH-2,2',5,5'-TCB and 3,4-DHB at similar rates. These results demonstrate the utility of the naphthalene catabolic enzymes in expanding the ability of the bph pathway to degrade polychlorinated biphenyls.

Involvement of the terminal oxygenase beta subunit in the biphenyl dioxygenase reactivity pattern toward chlorobiphenyls.

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (alpha and beta) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3'-dichlorobiphenyl over the double-para-substituted congener 4,4'-dichlorobiphenyl or the double-ortho-substituted congener 2,2'-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2'-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2',5,5'-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH alpha or beta subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the alpha subunit of ISPBPH of strain LB400 and the beta subunit of ISPBPH of strain B-356 (the alphaLB400 betaB-356 chimera) and the alphaB-356betaLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH beta subunit influences BPH dox's reactivity pattern toward chlorobiphenyls. Thus, if the alpha subunit were the sole determinant of the enzyme reactivity pattern, the alphaB-356betaLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alphaLB400 betaB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2'- and 3, 3'-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2',5,5'-tetrachlorobiphenyl.

Biphenyl-associated meta-cleavage dioxygenases from Comamonas testosteroni B-356.

In addition to 2,3-dihydroxybiphenyl 1,2-dioxygenase (B1,2O), biphenyl-grown cells of Comamonas testosteroni B-356 were shown to produce a catechol 2,3-dioxygenase (C2,3O). B1,2O showed strong sequence homology with B1,2Os found in other biphenyl catabolic pathways, while partial sequence analysis of the C2,3O of B-356 suggested a relationship with xylEII-encoded C2,3O. The coexistence of two meta-cleavage dioxygenases in this strain prompted a comparison between the catalytic properties of the two enzymes. C2,3O has a much broader substrate specificity than native or His-tagged B1,2O: both enzymes were inhibited by chlorocatechols, but B1,2O was more sensitive than C2,3O. The results are discussed in terms of the physiological implications of interaction between metabolites from the lower biphenyl-chlorobiphenyl pathway and enzymes of the upper pathway.

Sequencing of Comamonas testosteroni strain B-356-biphenyl/chlorobiphenyl dioxygenase genes: evolutionary relationships among Gram-negative bacterial biphenyl dioxygenases.

In a previous work, all three components of Comamonas testosteroni B-356 biphenyl (BPH)/chlorobiphenyls (PCBs) dioxygenase (dox) have been purified and characterized. They include an iron-sulphur protein (ISPBPH) which is the terminal oxygenase composed of two subunits (encoded by bphA and bphE), a ferredoxin (FERBPH) encoded by bphF and a reductase (REDBPH) encoded by bphG. bphG Is not located in the neighbourhood of bphAEF in B-356. We are reporting the cloning of B-356-bphG and the sequencing of B-356-BPH dox genes. Comparative analysis of the genes provided genetic evidence showing that two BPH dox lineages have emerged in Gram-negative bacteria. The main features of the lineage that includes B-356 are the location of bphG outside the bph gene cluster and the structure of REDBPH which is very distinct from all other aryl dioxygenase-reductases.

Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB).

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Characterization of active recombinant his-tagged oxygenase component of Comamonas testosteroni B-356 biphenyl dioxygenase.

Biphenyl (BPH) dioxygenase oxidizes BPH to 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356. The enzyme comprises a two-subunit iron-sulfur protein (ISPBPH), a ferredoxin FERBPH, and a ferredoxin reductase REDBPH. REDBPH and FERBPH transfer electrons from NADH to an Fe-S active center of ISPBPH which activates molecular oxygen for insertion into the substrate. In this work B-356 ISPBPH complex and its alpha and beta subunits were purified from recombinant Escherichia coli strains using the His-bind QIAGEN system. His-tagged B-356 ISPBPH construction carrying a single His tail on the N-terminal portion of the alpha subunit was active. Its major features were compared to the untagged enzyme. In both cases, the native form is an alpha3beta3 heteromer, with each alphabeta unit containing a [2Fe-2S] Rieske center (epsilon455 = 8,300 M-1 cm-1) and a mononuclear Fe2+. Although purified His-tagged alpha subunit showed the characteristic absorption spectra of Rieske-type protein, reassociation of this enzyme component and His-tagged beta subunit to reconstitute active ISPBPH was weak. However, when His-tagged alpha and beta subunits were reassembled in vitro in crude cell extracts from E. coli recombinants, active ISPBPH could be purified on Ni-nitrilotriacetic acid resin.

Purification and characterization of the Comamonas testosteroni B-356 biphenyl dioxygenase components.

In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISPBPH) made up of an alpha subunit (51 kDa) and a beta subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FERBPH; 12 kDa) encoded by bphF; and a ferredoxin reductase (REDBPH; 43 kDa) encoded by bphG. ISPBPH subunits were purified from B-356 cells grown on BPH. Since highly purified FERBPH and REDBPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISPBPH in the presence of NADH. FERBPH and REDBPH are thought to pass electrons from NADH to ISPBPH, which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (epsilon 460, 7,455 M-1 cm-1) which was readily lost from the protein during purification and storage. In the presence of REDBPH and FERBPH, ISPBPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.

Antimicrobial activity of fusidic acid and disk diffusion susceptibility testing criteria for gram-positive cocci.

The in vitro activity of fusidic acid was assessed and was compared with those of cloxacillin, cefamandole, vancomycin, teicoplanin, ofloxacin, ciprofloxacin, pefloxacin, and fleroxacin against 500 gram-positive cocci: 151 Staphylococcus aureus, 197 coagulase-negative staphylococci, and 152 Enterococcus faecalis strains. All clinical isolates were concomitantly tested by disk diffusion and agar dilution procedures as outlined by the National Committee for Clinical Laboratory Standards. The results with fusidic acid were further analyzed by regression line and error rate-bounded methods. With control American Type Culture Collection organisms, the values were within the limits of the National Committee for Clinical Laboratory Standards or published limits. The incidence of resistance to fusidic acid was 0.7% for S. aureus, 2.5% for coagulase-negative staphylococci, and 99.3% for E. faecalis. The correlation coefficient between the results of disk diffusion and agar dilution tests with fusidic acid was 0.90. Current interpretive criteria for susceptibility to fusidic acid (i.e., MIC of < 2 micrograms/ml and inhibitory zone of 20 mm) gave 1% false susceptibility (all strains being E. faecalis). This error rate is practically eliminated if a zone diameter of 21 mm is considered the breakpoint for susceptibility.

Identification and mapping of the gene translation products involved in the first steps of the Comamonas testosteroni B-356 biphenyl/chlorobiphenyl biodegradation pathway.

In this study, we have mapped Comamonas testosteroni B-356 genes encoding enzymes for the conversion of biphenyl and 4-chlorobiphenyl into the corresponding meta-cleavage compounds onto a 6.3-kb DNA fragment, and we have determined the subunit composition of the enzymes involved in this pathway. The various proteins encoded by this 6.3-kb DNA fragment and by subclones derived from it were overexpressed and selectively labelled using the T7 polymerase promoter system in Escherichia coli. They were then analyzed using SDS-PAGE, which allowed the encoding locus of each polypeptide to be mapped. Despite apparent dissimilarity in the congener selectivity patterns of the initial oxygenase of strain B-356 with those of Pseudomonas sp. strain LB400, the number and sizes of the polypeptides involved in the enzymatic conversion of biphenyl or 4-chlorobiphenyl into the meta-cleavage product appear to be similar in the two strains. In both strains, the bph operon encodes the following: the large (51-kDa polypeptide encoded by bphA) and the small (22-kDa polypeptide encoded by bphE) subunits of the iron sulphur protein, which is thought to interact directly with the substrate to introduce the oxygen molecule; the ferredoxin (12-kDa polypeptide encoded by bphF) involved in electron transfer from the reduced ferredoxin reductase to the oxidized iron sulphur protein; the 29-kDa polypeptide of the 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase encoded by bphB; and the 32-kDa polypeptide of the 2,3-dihydroxybiphenyl-1,2-dioxygenase encoded by bphC, which catalyzes meta-1,2 fission of the aromatic ring. A major difference between strain B-356 and strain LB400 is that the bphG gene encoding biphenyl dioxygenase ferredoxin reductase is located outside the bph gene cluster in strain B-356. Several lines of evidence indicate that bphG is absent in clones carrying the bph operon from strain B-356.

Factors affecting PCB degradation by an implanted bacterial strain in soil microcosms.

Pseudomonas testosteroni B-356 was able to degrade approximately 50% of the Aroclor 1242 mixture in shaken culture. The aims of the present study were to evaluate the capabilities of this bacterial strain to degrade PCBs in soil microcosms and to identify some of the factors likely to favor the degradative performance of the implanted bacteria. The presence of biphenyl as cosubstrate was the most important factor affecting PCB degradation in soil. However, because biphenyl was rapidly depleted in soil microcosms, repeated addition of small amounts of biphenyl to maintain a constant level of the cosubstrate allowed the achievement of a higher degree of degradation of the tetrachlorinated components of Aroclor 1242 than was achieved with a single addition at the time of inoculation. Degradation of di- and tri-chlorinated PCB congeners was less affected by repeated addition of biphenyl because these congeners were degraded very fast and complete degradation was achieved before biphenyl was depleted in the soil. Biodegradation was also related to bioavailability of the substrate. We observed that the proportion of each congener degraded was higher in the microcosms receiving both the producer of the surface-active agent, Alcaligenes faecalis B-556, and strain B-356. Under the best conditions (presence of a constant level of biphenyl and of strain B-556) P. testosteroni B-356 was able to degrade approximately 30% of the Aroclor 1242 added to soil microcosms; some other factors reducing the PCB degradation capabilities of the implanted bacteria are also discussed.