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Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a ؊24 TGGC, ؊12 TTGC sequence, characteristic of 54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this ؊24, ؊12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.Burkholderia (formerly Pseudomonas) pickettii PKO1 metabolizes benzene and toluene via a toluene-3-monooxygenase pathway (36). The initial step of this pathway involves the hydroxylation of toluene and benzene to m-cresol and phenol, respectively, by toluene-3-monooxygenase (36). The phenolic intermediates (phenol and m-cresol) are then further hydroxylated to catechol and methylcatechol, respectively, by a phenol hydroxylase, prior to ring cleavage by a meta-fission dioxygenase (25-27). We recently reported on the ability of B. pickettii PKO1 to degrade trichloroethylene (TCE) and provided evidence suggesting that this oxidation reaction was catalyzed by this same pathway (29). The toluene-3-monooxygenase is encoded by six tightly clustered chromosomal genes, tbuA1UBVA2C (6). On the basis of functional and sequence data, the toluene-3-monooxygenase is similar to the toluene-4-monooxygenase from Pseudomonas mendocina KR1 (57, 58) and the toluene/ benzene-2-monooxygenase from Pseudomonas sp. strain JS150 (24). These enzyme systems together with the toluene-2-monooxygenase fro...

The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKO1. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.Ralstonia pickettii PKO1 has been investigated by our laboratory for several years as a model microorganism representative of those bacteria capable of metabolizing alkylaromatic hydrocarbons in oxygen-limited (hypoxic) aquifer environments (23,34,42,43). The tbu pathway of R. pickettii PKO1, which encodes enzymes for utilization of benzene, toluene, and related alkylaromatic hydrocarbons as well as enabling this strain to transform trichloroethylene (TCE), has been cloned as a 26.5-kbp DNA fragment designated pRO1957 (41). The genes encoding enzymes for this catabolic pathway have been shown previously to be organized into three operons: the tbuA1UBVA2C and tbuT operon encoding the initial toluene-3-monooxygenase and the transcriptional activator TbuT (6), the tbuD operon encoding phenol/cresol hydroxylase (24, 26), and the tbuWEFGKIHJ operon encoding enzymes of the metacleavage pathway for conversion of catechol and methylcatechols to tricarboxylic acid cycle intermediates (25). We have previously shown through physiological analysis as well as through transcriptional fusion analysis of promoter regions that TbuT controls transcription of each of these operons in response to aromatic effector compounds. Moreover, it has been shown that the unique organization of tbuT, which is located immediately dow...

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The degradation of trichloroethylene (TCE) by toluene-oxidizing bacteria has been extensively studied, and yet the influence of environmental conditions and physiological characteristics of individual strains has received little attention. To consider these effects, the levels of TCE degradation by strains distinguishable on the basis of toluene and nitrate metabolism were compared under aerobic or hypoxic conditions in the presence and absence of nitrate and an exogenous electron donor, lactate. Under aerobic conditions with tolueneinduced cells, strains expressing toluene dioxygenases (Pseudomonas putida F1, Pseudomonas sp. strain JS150, Pseudomonas fluorescens CFS215, and Pseudomonas sp. strain W31) degraded TCE at low rates, with less than 12% of the TCE removed in 18 h. In contrast, strains expressing toluene monooxygenases (Burkholderia cepacia G4, Burkholderia pickettii PKO1, and Pseudomonas mendocina KR1) degraded 36 to 67% of the TCE over the same period. Under hypoxic conditions (1.7 mg of dissolved oxygen per liter) or when lactate was added as an electron donor, the extent of TCE degradation by toluene-induced cells was generally lower. In the presence of lactate, degradation of TCE by denitrifying strain PKO1 was enhanced by nitrate under conditions in which dissimilatory nitrate reduction was observed. The results of experiments performed with strains F1, G4, PKO1, and KR1 suggested that TCE or an oxidation product induces toluene degradation and that TCE induces its own degradation in the monooxygenase strains. The role of TCE as an inducer of toluene oxygenase activity in PKO1 was confirmed by performing a promoter probe analysis, in which we found that TCE activates transcription from the PKO1 3-monooxygenase operon promoter.

Our work has focused on the determination of physiological traits that may facilitate in situ degradation of xenobiotic compounds by indigenous microorganisms. For this our interests center on the following questions: What are the ambient conditions in a benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated aquifer? What is the behavior of indigenous bacteria under these conditions? What are the attributes of bacterial strains that are functional under hypoxic conditions? How do these strains compare with other BTEX-degrading strains?

We have previously reported on the organization of a unique toluene-3-monooxygenase pathway for the degradation of alkyl-substituted petroleum hydrocarbons including characteristics of the second step in the pathway transforming phenols to catechols. In the present work we have focused on the regulation and unusual genetic organization of this metabolic step. In particular, we have sequenced the 3-kb DNA interval between the region encoding the tbuD gene product (phenol/cresol hydroxylase) and part of the toluene-3-monooxygenase operon of strain PKO1. Then, various regions of this DNA were fused to a LacZ expression system to ascertain the location of the tbuD gene promoter and the binding site for its regulator, TbuT. The 5' end for transcripts for the putative promoter of the tbuD gene was also analyzed using primer extension analysis. Collectively, these results revealed that the promoter was located 2.5-kb upstream of the region encoding the tbuD gene product whose N-terminal region had been previously determined by peptide sequencing. Remarkably, the intervening 2.5-kb region showed sequence identity to results we reported previously for a multi-subunit toluene-2-monooxygenase cloned from a different bacterium, strain JS150, for which phenols are also substrates and effectors. When the DNA sequence for the tbuD gene and its contiguous 2.5-kb upstream region were compared to the entire toluene-2-monooxygenase sequence cloned from strain JS150, a promoter proximal region encoding three reading frames showed 99% identity to subunits for the toluene-2-monooxygenase operon. Within the contiguous tbuD gene region, however, DNA sequence homology was reduced to 64% overall identity and deduced amino acid sequence homology was only 21% similar. Although regions internal to the tbuD gene showed homology to corresponding toluene-2-monooxygenase subunits, domains associated with the putative functions proposed for such subunits were deleted. We believe that these results suggest that through evolution either tbuD was derived from the 2-monooxygenase pathway by deletions and molecular rearrangements, or alternatively the tbuD gene recruited part of the 2-monooxygenase pathway and its regulatory system which is activated by benzene, alkyl-substituted benzenes and phenols.

Our work has focused on the determination of physiological traits that may facilitate in situ degradation of xenobiotic compounds by indigenous microorganisms. For this our interests center on the following questions: What are the ambient conditions in a benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated aquifer? What is the behavior of indigenous bacteria under these conditions? What are the attributes of bacterial strains that are functional under hypoxic conditions? How do these strains compare with other BTEX-degrading strains? -Environ Health Perspect 103 (Suppl 59:4-51 (1995) Key words: biodegradation, hypoxia, Pseudomonas picketti, benzene, toluene, ethylbenzene, xylenes, groundwater, nitrate, oxygen Aquifer CharacteristicsFor several years we sampled three sites in which groundwater was contaminated by petroleum products, including benzene, toluene, ethylbenzene, and xylene (BTEX) components (1,2). All three of these contaminated aquifers were characterized by their low dissolved oxygen levels in the contaminated regions (-1-2 mg/l ), in contrast to uncontaminated adjacent regions in the aquifer (-3-4 mg/1). The occurrence of dissolved nitrate also showed a corresponding relationship when the contaminated and uncontaminated regions were compared. The nitrate concentrations were low or undetectable in the contaminated regions, but were higher in the uncontaminated regions. These relationships were observed for all three aquifers, leading to the conclusion that oxygen growth on BTEX were observed when cores of aquifer material that had been sampled vertically were tested for microbial activity as a function of depth and location within the plume regions. Bacterial strains growing on BTEX aerobically were observed for all samples independent of depth or BTEX concentration. However, when such samples were incubated under hypoxic conditions (i.e., < 2 mg/l dissolved oxygen), bacterial growth and denitrification matched BTEX degradation observed for microcosm cultures inoculated with bacteria eluted from the soil core samples. Therefore, differences were observed between bacteria that would degrade BTEX under hypoxic-denitrifying conditions and those that grow on BTEX under aerobic conditions. Diversity of Bacteria from Hypoxic EnvironmentsSamples from the BTEX-contaminated aquifers were incubated in mineral salts buffer overnight to elute bacteria from particles. These eluates were then plated on solid mineral salts medium followed by incubation aerobically or under hypoxic conditions in the presence of BTEX vapors. Aerobic cultures were incubated 48 hr and hypoxic cultures were incubated 1 week. Colonies were replated onto the same medium and incubated as before. These cultures then became the source for subsequent tests for the mineralization of individual BTEX components under both aerobic and hypoxic conditions. For this work, p-xylene was used as a representative xylene. The colonies selected for such tests were selected on the basis of their distinctive morphologies and colony size to fa...