Trichloroethylene Degradation by Toluene-Oxidizing Bacteria Grown on Non-aromatic Substrates

The potential of trichloroethylene (TCE) to induce and non-aromatic growth substrates to support TCE degradation in five strains (Pseudomonas mendocina KR1, Ralstonia pickettii PKO1, Pseudomonas putida F1, Burkholderia cepacia G4, B. cepacia PR1) of toluene-oxidizing bacteria was examined. LB broth and acetate did not support TCE degradation in any of the wild-type strains. In contrast, fructose supported the highest specific levels of TCE oxidation observed in each of the strains tested, except B. cepacia G4. We discuss the potential mechanisms and implications of this observation. In particular, cells of P. mendocina KR1 degraded significant amounts of TCE during cell growth on non-aromatic substrates. Apparently, TCE degradation was not completely constrained by any given factor in this microorganism, as was observed with P. putida F1 (TCE was an extremely poor substrate) or B. cepacia G4 (lack of oxygenase induction by TCE). Our results indicate that multiple physiological traits are required to enable useful TCE degradation by toluene-oxidizing bacteria in the absence of aromatic cosubstrates. These traits include oxygenase induction, effective TCE turnover, and some level of resistance to TCE mediated toxicity.     

Toluene-degrading bacteria are chemotactic towards the environmental pollutants benzene, toluene, and trichloroethylene

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (Pseudomonas putida F1, Ralstonia pickettii PKO1, and Burkholderia cepacia G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene. Pseudomonas mendocina KR1 and P. putida PaW15 did not show a convincing response. The chemotactic responses of P. putida F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for P. putida F1, including benzene and ethylbenzene, were chemoattractants. P. putida F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by P. putida F1. Mutant strains of P. putida F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in P. putida F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.     

Chloroform mineralization by toluene-oxidizing bacteria.

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.     

Induction of toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 by chlorinated solvents and alkanes.

Toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 was induced by trichloroethylene (TCE), and induction was followed by the degradation of TCE. Higher levels of toluene oxidation activity were achieved in the presence of a supplemental growth substrate such as glutamate, with levels of activity of up to 86% of that observed with toluene-induced cells. Activity in P. mendocina KR1 was also induced by cis-1,2-dichloroethylene, perchloroethylene, chloroethane, hexane, pentane, and octane, but not by trans-1,2-dichloroethylene. Toluene oxidation was not induced by TCE in Burkholderia (Pseudomonas) cepacia G4, P. putida F1, Pseudomonas sp. strain ENV110, or Pseudomonas sp. strain ENV113.     

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure.     

Substrate inhibition kinetics for toluene and benzene degrading pure cultures and a method for collection and analysis of respirometric data for strongly inhibited cultures

We present an evaluation of the qualitative and quantitative effects that high concentrations of benzene and toluene have on the growth rate of several pure cultures that use these compounds as their sole carbon and energy source. The cultures employed were five widely studied environmental isolates: Pseudomonas putida F1, P. putida mt2, P. mendocina KR, Ralstonia pickettii PKO1, and Burkholderia cepacia G4. Three cultures degraded toluene following a pattern consistent with the kinetic model of Wayman and Tseng (1976) while the other two followed a modification of this model introduced by Alagappan and Cowan (2001). The pattern followed for benzene degradation was different than that for toluene degradation for all four capable pure cultures and consistent with that described by the model of Luong (1987). Mechanisms of substrate inhibition and solvent toxicity are discussed, used to conceptually evaluate the reasons for the differences in inhibition behavior, and used to support a call for more widespread use of the empirical, terminal substrate concentration inhibition models employed here. We also present the methodology developed to overcome a limitation commonly encountered when attempting to collect oxygen uptake data for use in quantifying substrate inhibition kinetics. The experimental method was effective for use in the collection of high quality data and the substrate inhibition models most useful in representing the growth of bacteria on these solvents are those that show a complete loss of activity at high concentration rather than the more popular asymptotic inhibition models.     

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.     

Carbon and hydrogen stable isotope fractionation during aerobic bacterial degradation of aromatic hydrocarbons

13C/(12)C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.     

Phospholipid compositional changes of five pseudomonad archetypes grown with and without toluene

Bacterial physiological responses to toluene exposure were investigated in five reference pseudomonad strains that express different toluene degradation pathways: Pseudomonas putida mt-2, Pseudomonas putida F1, Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1. The intact phospholipids of these archetypes, grown with and without toluene, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. All strains showed significant changes in phospholipid content and composition as an adaptive response to toluene exposure, as well as considerable diversity in response mechanisms. For example, the phospholipid content of toluene-grown PKO1, F1, and KR1 were 10.9–34.7% of that found in succinate-grown strains, while the phospholipid content of mt-2 and G4 increased by 56% and 94%, respectively, when grown on toluene. In addition, PKO1, F1, and mt-2 responded to the presence of toluene by synthesizing more phosphatidylglycerol, whereas G4 and KR1 synthesized phospholipids with polyunsaturated fatty acids (C18:2) on one or both of the sn-2 positions. These changes in phospholipid composition and concentration probably reflect the sensitivity and degree of tolerance of these strains to toluene, and suggest that different mechanisms are utilized by dissimilar bacteria to maintain optimal lipid ordering in the presence of such environmental pollutants. Received: 13 October 1999 / Received revision: 16 February 2000 / Accepted: 25 February 2000     

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1.

Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2+, oxidized toluene to p-cresol as the first detectable product. Experiments with p-deutero-toluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 18O2, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.     

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate

Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers-polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 degrees C and a glass transition temperature of -48.5 degrees C.     

Amplification of toluene dioxygenase genes in a hybrid Pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture

A hybrid metabolic pathway through which benzene, toluene, and p-xylene (BTX) mixture could be simultaneously mineralized was previously constructed in Pseudomonas putida TB101 (Lee, Roh, Kim, Biotechnol. Bioeng 43: 1146-1152, 1994). In this work, we improved the performance of the hybrid pathway by cloning the todC1C2BA genes in the broad-host-range multicopy vector RSF1010 and by introducing the resulting plasmid pTOL037 into P. putida mt-2 which harbors the archetypal TOL plasmid. As a result, a new hybrid strain, P. putida TB103, possessing the enhanced activity of toluene dioxygenase in the hybrid pathway was constructed. The degradation rates of benzene, toluene, and p-xylene by P. putida TB103 were increased by about 9.3-, 3.7-, and 1.4-fold, respectively, compared with those by previously constructed P. putida TB101. Apparently, this improved capability of P. putida TB103 for the degradation of BTX mixture resulted from the amplification of the todC1C2BA genes. Furthermore, a relatively long lag period for benzene degradation observed when P. putida TB101 was used for the degradation of BTX mixture at low dissolved oxygen (DO) tension disappeared when P. putida TB103 was employed. (c) 1995 John Wiley & Sons, Inc.     

Genetic engineering of a highly solvent-tolerant Pseudomonas putida strain for biotransformation of toluene to p-hydroxybenzoate

The solvent-tolerant strain Pseudomonas putida DOT-T1E has been engineered for biotransformation of toluene into 4-hydroxybenzoate (4-HBA). P. putida DOT-T1E transforms toluene into 3-methylcatechol in a reaction catalyzed by toluene dioxygenase. The todC1C2 genes encode the alpha and beta subunits of the multicomponent enzyme toluene dioxygenase, which catalyzes the first step in the Tod pathway of toluene catabolism. A DOT-T1EdeltatodC mutant strain was constructed by homologous recombination and was shown to be unable to use toluene as a sole carbon source. The P. putida pobA gene, whose product is responsible for the hydroxylation of 4-HBA into 3,4-hydroxybenzoate, was cloned by complementation of a Pseudomonas mendocina pobA1 pobA2 double mutant. This pobA gene was knocked out in vitro and used to generate a double mutant, DOT-T1EdeltatodCpobA, that was unable to use either toluene or 4-HBA as a carbon source. The tmo and pcu genes from P. mendocina KR1, which catalyze the transformation of toluene into 4-HBA through a combination of the toluene 4-monoxygenase pathway and oxidation of p-cresol into the hydroxylated carboxylic acid, were subcloned in mini-Tn5Tc and stably recruited in the chromosome of DOT-T1EdeltatodCpobA. Expression of the tmo and pcu genes took place in a DOT-T1E background due to cross-activation of the tmo promoter by the two-component signal transduction system TodST. Several independent isolates that accumulated 4-HBA in the supernatant from toluene were analyzed. Differences were observed in these clones in the time required for detection of 4-HBA and in the amount of this compound accumulated in the supernatant. The fastest and most noticeable accumulation of 4-HBA (12 mM) was found with a clone designated DOT-T1E-24.     

Effect of Trichloroethylene on the Competitive Behavior of Toluene-Degrading Bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight]⁻¹ h⁻¹). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments.

We studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. Three Pseudomonas isolates, P. pickettii PKO1, Pseudomonas sp. strain W31, and P. fluorescens CFS215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. We examined kinetic parameters (K(m) and Vmax) for catechol 2,3-dioxygenase (C230), a key shared enzyme of the toluene-degradative pathway for these strains, and compared these parameters with those for the analogous enzymes from archetypal toluene-degrading pseudomonads which did not show enhanced, nitrate-dependent toluene degradation. C230 purified from strains W31, PKO1, and CFS215 had a significantly greater affinity for oxygen as well as a significantly greater rate of substrate turnover than found for the analogous enzymes from the TOL plasmid (pWW0) of Pseudomonas putida PaW1, from Pseudomonas cepacia G4, or from P. putida F1. Analysis of the nucleotide and deduced amino acid sequences of C23O from strain PKO1 suggests that this extradiol dioxygenase belongs to a new cluster within the subfamily of C23Os that preferentially cleave monocyclic substrates. Moreover, deletion analysis of the nucleotide sequence upstream of the translational start of the meta-pathway operon that contains tbuE, the gene that encodes the C230 of strain PKO1, allowed identification of sequences critical for regulated expression of tbuE, including a sequence homologous to the ANR-binding site of Pseudomonas aeruginosa PAO. When present in cis, this site enhanced expression of tbuE under oxygen-limited conditions. Taken together, these results suggest the occurrence of a novel group of microorganisms capable of oxygen-requiring but nitrate-enhanced degradation of benzene, toluene, ethylbenzene, and xylenes in hypoxic environments. Strain PKO1, which exemplifies this novel group of microorganisms, compensates for a low-oxygen environment by the development of an oxygen-requiring enzyme with kinetic parameters favorable to function in hypoxic environments, as well as by elevating synthesis of such an enzyme in response to oxygen limitation.