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Identification and sequence analysis of two regulatory genes involved in anaerobic toluene metabolism by strain T1.

ABSTRACT: T1 is a denitrifying bacterium isolated for its ability to grow with toluene serving as the sole carbon source. Mutants of this strain that have defects in the toluene utilization pathway have been isolated and have been separated into classes based on growth phenotypes. A cosmid clone has been isolated by complementing the tutB16 (for toluene utilization) mutation. The complementing gene has been localized to a 3.3-kb DNA fragment. An additional open reading frame upstream of the tutB gene has also been identified and is designated tutC. The nucleotide sequence and the predicted amino acid translation of the 6.4-kb DNA fragment that contains these genes are presented. The tutB and tutC gene products of strain T1 have homology to members of the two-component sensor-regulator family and are proposed to play a role in the regulation of toluene metabolic genes of strain T1. To our knowledge, this is the first published report of the isolation of mutants defective in anaerobic aromatic hydrocarbon degradation. Additionally, we report for the first time the cloning of genes involved in an anaerobic aromatic hydrocarbon degradation pathway.

SUBMITTER: Coschigano PW

PROVIDER: S-EPMC168355 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Identification and sequence analysis of two regulatory genes involved in anaerobic toluene metabolism by strain T1.

Coschigano P W PW Young L Y LY

Applied and environmental microbiology 19970201 2

T1 is a denitrifying bacterium isolated for its ability to grow with toluene serving as the sole carbon source. Mutants of this strain that have defects in the toluene utilization pathway have been isolated and have been separated into classes based on growth phenotypes. A cosmid clone has been isolated by complementing the tutB16 (for toluene utilization) mutation. The complementing gene has been localized to a 3.3-kb DNA fragment. An additional open reading frame upstream of the tutB gene has ...[more]

PMID: 9023943

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Project description:Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium Thauera aromatica have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor ("phenylphosphate synthase") and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate ("phenylphosphate carboxylase"). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the ubiD gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the ubiX gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different Thauera and Azoarcus strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the mutT gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of Pseudomonas putida. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.

Project description:It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.

Dataset Information

Identification and sequence analysis of two regulatory genes involved in anaerobic toluene metabolism by strain T1.

Publications

Identification and sequence analysis of two regulatory genes involved in anaerobic toluene metabolism by strain T1.

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