Genetic and Functional Analysis of the
            <i>tbc</i>
            Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in
            <i>Burkholderia</i>
            sp. Strain JS150

257

195

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 ؋ 10 2 copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.

Section: Resultsmentioning

confidence: 99%

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Baldwin

Nakatsu

Nies

2003

257

195

“…Thus, PH is more restricted in its regioselectivity than ToMO, but it is more relaxed than other group 1 BMMs, like T2MO, which produce only o-cresol (16,17,24).…”

Section: Discussionmentioning

confidence: 99%

Regiospecificity of Two Multicomponent Monooxygenases from Pseudomonas stutzeri OX1: Molecular Basis for Catabolic Adaptation of This Microorganism to Methylated Aromatic Compounds

Cafaro

Notomista

Capasso

et al. 2005

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.

“…Analysis of the sequences from nucleotide and protein databases indicates that most bacterial strains possess only one BMM, but a few cases (3 out of 31) of bacterial genomes coding for more than one monooxygenase have been found (21). Two group 1 (phenol hydroxylases) and one group 2 (toluene-benzene monooxygenases) BMMs have been found in the genome of Ralstonia metallidurans CH34 (21), and three BMMs belonging to groups 1 and 2 were detected in Burkholderia cepacia (formerly Pseudomonas cepacia) JS150 (16,21). B. cepacia JS150 instead is endowed with the ability to use different pathways for the metabolism of substituted aromatic compounds (16).…”

mentioning

confidence: 99%

“…Two group 1 (phenol hydroxylases) and one group 2 (toluene-benzene monooxygenases) BMMs have been found in the genome of Ralstonia metallidurans CH34 (21), and three BMMs belonging to groups 1 and 2 were detected in Burkholderia cepacia (formerly Pseudomonas cepacia) JS150 (16,21). B. cepacia JS150 instead is endowed with the ability to use different pathways for the metabolism of substituted aromatic compounds (16). This led to the suggestion that in B. cepacia JS150, toluene metabolism is initiated by one dioxygenase and two distinct BMMs with different specificities (a toluene 4-monooxygenase [T4MO] belonging to group 2 and a toluene 2-monooxygenase [T2MO] belonging to group 1), whereas a third BMM acts downstream of the other two monooxygenases (16).…”

mentioning

confidence: 99%

“…B. cepacia JS150 instead is endowed with the ability to use different pathways for the metabolism of substituted aromatic compounds (16). This led to the suggestion that in B. cepacia JS150, toluene metabolism is initiated by one dioxygenase and two distinct BMMs with different specificities (a toluene 4-monooxygenase [T4MO] belonging to group 2 and a toluene 2-monooxygenase [T2MO] belonging to group 1), whereas a third BMM acts downstream of the other two monooxygenases (16).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Phenol Hydroxylase and Toluene/ o -Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

Cafaro

Izzo

Scognamiglio

et al. 2004

118

122

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.