Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.     

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.     

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.     

Biodegradation of 4-methyl-5-nitrocatechol by Pseudomonas sp. strain DNT.

Pseudomonas sp. strain DNT degrades 2,4-dinitrotoluene (DNT) by a dioxygenase attack at the 4,5 position with concomitant removal of the nitro group to yield 4-methyl-5-nitrocatechol (MNC). Here we describe the mechanism of removal of the nitro group from MNC and subsequent reactions leading to ring fission. Washed suspensions of DNT-grown cells oxidized MNC and 2,4,5-trihydroxytoluene (THT). Extracts prepared from DNT-induced cells catalyzed the disappearance of MNC in the presence of oxygen and NADPH. Partially purified MNC oxygenase oxidized MNC in a reaction requiring 1 mol of NADPH and 1 mol of oxygen per mol of substrate. The enzyme converted MNC to 2-hydroxy-5-methylquinone (HMQ), which was identified by gas chromatography-mass spectrometry. HMQ was also detected transiently in culture fluids of cells grown on DNT. A quinone reductase was partially purified and shown to convert HMQ to THT in a reaction requiring NADH. A partially purified THT oxygenase catalyzed ring fission of THT and accumulation of a compound tentatively identified as 3-hydroxy-5-(1-formylethylidene)-2-furanone. Preliminary results indicate that this compound is an artifact of the isolation procedure and suggest that 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid is the actual ring fission product.     

Cloning and characterization of plasmid-encoded genes for the degradation of 1,2-dichloro-, 1,4-dichloro-, and 1,2,4-trichlorobenzene of Pseudomonas sp. strain P51.

Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.     

Cloning and analysis of s-triazine catabolic genes from Pseudomonas sp. strain NRRLB-12227.

Pseudomonas sp. strain NRRLB-12227 degrades the s-triazine melamine by a six-step pathway which allows it to use melamine and pathway intermediates as nitrogen sources. With the plasmid pLG221, mutants defective in five of the six steps of the pathway were generated. Tn5-containing-EcoRI fragments from these mutants were cloned and identified by selection for Tn5-encoded kanamycin resistance in transformants. A restriction fragment from ammelide-negative mutant RE411 was used as a probe in colony hybridization experiments to identify cloned wild-type s-triazine catabolic genes encoding ammeline aminohydrolase, ammelide aminohydrolase, and cyanuric acid amidohydrolase. These genes were cloned from total cellular DNA on several similar, but not identical, HindIII fragments, as well as on a PstI fragment and a BglII fragment. Restriction mapping and Southern hybridization analyses of these cloned DNA fragments suggested that these s-triazine catabolic genes may be located on a transposable element, the ends of which are identical 2.2-kb insertion sequences.     

Cloning and characterization of the genes for p-nitrobenzoate degradation from Pseudomonas pickettii YH105.

Pseudomonas pickettii YH105 was isolated for its ability to utilize p-nitrobenzoate as the sole source of carbon, nitrogen, and energy. Degradation of p-nitrobenzoate by this strain proceeds through a reductive route as evidenced by the accumulation of ammonia in the culture medium during growth on p-nitrobenzoate. Enzyme assays and high-performance liquid chromatography (HPLC) analysis of culture supernatants indicate that p-nitrobenzoate is degraded through p-hydroxylaminobenzoate and protocatechuate. In order to clone the genes responsible for the initial steps in the catabolic pathway, a cosmid library was constructed with P. pickettii YH105 genomic DNA. The library was screened for clones capable of transforming p-nitrobenzoate to protocatechuate, using a plate assay specific for diphenolic compounds. HPLC analysis of culture supernatants confirmed that the cosmid clones did indeed produce protocatechuate from p-nitrobenzoate. Five positive cosmid clones that possessed this activity were identified. Restriction digests of the cosmid clones indicated that all of the clones had two EcoRI fragments in common (3.9 and 1.0 kb). One of these cosmid clones, designated pGJZ1601, was chosen for further analysis. Subcloning and activity assay experiments localized the genes responsible for the conversion of p-nitrobenzoate to protocatechuate to a 1.4-kb SalI-SphI DNA fragment. Further subcloning experiments localized the gene coding for p-nitrobenzoate reductase, responsible for the first enzymatic step in the catabolic pathway, to a 0.8-kb SalI-ApaI DNA fragment. The gene for the second step in the catabolic pathway, coding for hydroxylaminolyase, was located adjacent to the gene for the p-nitrobenzoate reductase.     

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain.

Pseudomonas sp. strain SE83 converts cephalosporin C and 7 beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA). A DNA library of this strain was constructed in Escherichia coli and screened for the ability to deacylate GL-7ACA to 7ACA. Apparently, two distinct genes, designated acyI and acyII, were cloned on 4.8- and 6.0-kilobase-pair BglII fragments, respectively. The enzymes encoded by the two genes showed different substrate specificities, and the acyII-encoded enzyme was found to yield 7ACA from cephalosporin C by direct deacylation. Expression of the two genes in E. coli was strongly dependent on a promoter of the vector. The coding regions for acyI and acyII were localized on the 2.5- and 2.8-kilobase-pair fragments, respectively, by subcloning experiments, and high expression of both genes was obtained by placing them under the control of the lacUV5 promoter. The acyII-encoded enzyme was purified and shown to be composed of two nonidentical subunits with molecular weights of 26,000 and 57,000. Maxicell analysis revealed three acyII-specific polypeptides, two of which corresponded to the above subunits. The third polypeptide with a molecular weight of 83,000 was suggested to be the precursor of both subunits.     

Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation.

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs). A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introduced into Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the 2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability to transform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for further study. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a 12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with that of the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410 derivative) approached that of LB400 and was significantly greater than degradation by the original recombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organism on biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbon source, PCB degradation by FM4560 was markedly superior to that by LB400.     

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10.

Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.     

Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation

Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.     

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.     

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90.

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.     

Monooxygenase-mediated 1,2-dichloroethane degradation by Pseudomonas sp. strain DCA1.

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a Km value below the detection limit of 0.5 microM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.