• Korean Journal of Microbiology
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• v.30 no.1
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• pp.8-14
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• 1992

Nucleotide Sequence of phnE Gene Encoding Extradiol Dioxygenase fromPseudomonas sp. Strain DJ77Kim, Young-Chang'.", Myeong-Su Shin1, Kil-Sang Younl, Young-Soon Park1, andUg-Hyeon Kim'.' (Department of Microbiology, C'hungbuk National University.Cheongju 360-763, KOREA. and 'Research Center for Molecular Microbiology,Seoul National University)nal University)

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.259-271
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• 2021

Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain Pseudomonas putida ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the ortho- or meta-cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes (catBICIAI and catBIICIIAII) on the chromosome play an important role. The catI and catII operons are co-transcribed, whereas catAI and catAII are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of cis-cis-muconate, a well-studied inducer of the cat gene cluster, CatRI and CatRII occupy an additional downstream site, designated as the activation binding site. Notably, CatRI binds to both the catI and catII promoters with high affinity, while CatRII binds weakly. This is likely caused by a T to G mutation in the G/T-N11-A motif. Specifically, we found that CatRI and CatRII regulate catBICIAI and catBIICIIAII in a cooperative manner, which provides new insights into naphthalene degradation.

• KSBB Journal
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• v.18 no.3
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• pp.174-179
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• 2003

The bacterium NFQ-1 capable of utilizing quinoline (2,3-benzopyridine) as the sole source of carbon, nitrogen and energy was enriched and isolated from soil samples of dead coal pit areas. Strain NFQ-1 was identified as Pseudomonas nitroreducens NFQ-1 by BIOLOG system, and assigned to Pseudomonas sp. NFO-1. Pseudomonas sp. NFQ-1 was used with the concentration range of 1 to 10 mM quinoline. Strain NFQ-1 could degrade 2.5 mM quinoline within 9 hours of incubation. Initial pH 8.0 in the culture was reduced to 6.8, and eventually 7.0 as the incubation was proceeding. 2-Hydroxyquinoline, the first intermediate of the degradative pathway, accumulated transiently in the growth medium. The highest concentration of quinoline (15 mM) in this work inhibited cell growth and quinoline degradation. Pseudomonas sp. NFQ-1 was able to utilize various quinoline derivatives and aromatic compounds including 2-hydroxyquinoline, p-comaric acid, benzoic acid, p-cresol, p-hydroxybenzoate, protocatechuic acid, and catechol. The specific activity of catechol oxygenases was determined to approximately 184.7 unit/㎎ for catechol 1.2-dioxygenase and 33.19 unit/㎎ for catechol 2,3-dioxygenase, respectively. As the result, it showed that strain NFQ-1 degraded quinoline via mainly orthp-cleavage pathway, and in partial meta-cleavage pathway.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.104-111
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• 2007

In this study, aniline dioxygenase genes responsible for initial catabolism of aniline in Burkholderia sp. HY1 and Delftia sp. HY99 were cloned and the amino acid sequences were comparatively analyzed, which already have been reported as bacteria utilizing aniline as a sole source of carbon and nitrogen, B. sp. HY1 was found to have at least a plasmid, and the plasmld-cured strain, B. sp. HY1-PC obtained using mitomycin C was tested with wild type strain to investigate whether the former maintained the degradability for aniline. This proved that the aniline oxygenase gene from B. sp. HY1 was located in chromosomal DNA, not in plasmid DNA. Aniline dioxygenase small subunits from B. sp. HY1 and D. sp. HY99 were found, based on 146 amino acids, to share 79% similarity. Notably, ado2 genes from B. sp. HY1 and D. sp. HY99 which were found to be terminal dioxygenase of aniline dioxygenase small subunit showed 99% similarity in the deduced amino acid sequences with tdnA2 of Frateuria sp. ANA-18 and danA2 of D. sp. AN3, respectively. Besides, enzyme assay and amino acid sequence analysis of catechol dioxygenase supported the previous report that B. sp. HY1 might occupy ortho-cleavage pathway using catechol 1,2-dioxygenase, while D. sp. HY99 might occupy catechol 2,3-dioxygenase for meta-cleavage pathway.

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.68-73
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• 1995

Pseudomonas sp. P20 was shown to be capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce the corresponding benzoic acids wnich were not further degraded. But the potential of the strain for biodegradation of 4CB was shown to be excellent. The pcbA, B, C and D genes responsible for the aromatic ring-cleavage of biphenyl and 4CB degradation were cloned from the chromosomal DNA of the strain. In this study, the pebC and D genes specifying degradation of 2, 3-dihydroxybiphenyl (2, 3-DHBP) produced from biphenyl by the pebAB-encoded enzymes were cloned by using pBluescript SK(＋) as a vector. From the pCK102 (9.3 kb) containing pebC and D genes, pCK1022 inserted with a EcoRI-HindIII DNA fragment (4.1 kb) carrying pebC and D and a pCK1092 inserted with EcoRI-XbaI fragment (1.95 kb) carrying pebC were constructed. The expression of pcbC and D' in E. coli CK102 and pebC in E. coli CK1092 was examined by gas chromatography and UV-vis spectrophotometry. 2.3-dihydroxybiphenyl was readily degraded to produce meta-cleavage product (MCP) by E. coli CK102 after incubation for 10 min, and then only benzoic acid(BA) was detected in the 24-h old culture. The MCP was detected in E. coli CK1022 containing pebC and 0 genes (by the resting cells assay) for up to 3 h after incubation and then diminished completely in 8 h, whereas the MCP accumulated in the E. coli CK1092 culture even after 6 h of incubation. The 2, 3-DHBP dioxygenases (product of pebC gene) produced by E. coli CK1, CK102, CK1023, and CK1092 strains were measured by native PAGE analysis to be about 250 kDa in molecular weight, which were about same as those of Pseudomonas sp. DJ-12, P. pseudoa1caligenes KF707, and P. putida OU83.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.393-399
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• 2004

Useful genes can be Screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs r were extracted by the SDS-based freezing-thawing method, and then further purified using an $UltraClean^{TM}$ kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoR I, BamH I, and Sac II in Escherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13-15 kb in size. The bphC genes responsible for degradation of aromatic hydrocarbons via mets-cleavage were identified from the metagenomic libraries by colony hybridization using the bphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2, 3-DHBP) dioxygenase gene (bphC ), capable of degradation of 2,3-DHBP, was cloned and its nucleotide Sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and Showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results in-dicated that the bphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.

• BMB Reports
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• v.35 no.4
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• pp.432-436
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• 2002

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

• Journal of Microbiology and Biotechnology
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• v.33 no.7
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• pp.875-885
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• 2023

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

• Journal of Microbiology
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• v.35 no.4
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• pp.295-299
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• 1997

Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

• BMB Reports
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• v.32 no.4
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• pp.399-404
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• 1999

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), which catalyzes the ring meta-cleavage of 2,3-dihydroxybiphenyl, is encoded by the phnQ gene of biphenyl- and phenanthrene-degrading Pseudomonas sp. strain DJ77. We determined the nucleotide sequence of a DNA fragment of 1497 base pairs which included the phnQ gene. The fragment lncluded an open reading frame of 903 base pairs to accommodate the enzyme. The predicted amino acid sequence of the enzyme subunit consisted of 300 residues. In front of the gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli. The deduced amino acid sequence of the PhnQ enzyme exhibited 85.6% identity with that of the corresponding enzyme in Sphingomonas yanoikuyae Q1 (formerly S. paucimobilis Q1) and 22.1% identity with that of catechol 1,2,3-dioxygenase from the same DJ77 strain. PhnQ showed broader substrate preference than previously-cloned PhnE, catechol 2,3-dioxygenase. Ten amino acid residues, considered to be important for the role of extradiol dioxygenases, were conserved.