• Title/Summary/Keyword: Pseudomonas pseudoalcaligenes KF707

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Plant Terpenes Enhance Survivability of Polychlorinated Biphenyl (PCB) Degrading Pseudomonas pseudoalcaligenes KF707 Labeled with gfp in Microcosms Contaminated with PCB

  • Oh, Eun-Taex;Koh, Sung-Cheol;Kim, Eung-Bin;Ahn, Young-Hee;So, Jae-Seong
    • Journal of Microbiology and Biotechnology
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    • v.13 no.3
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    • pp.463-468
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    • 2003
  • Polychlorinated biphenyl are toxic pollutants and their degradation is quite slow in the environment. Recently, interest if bioremediation using PCB-degrading bacteria has increaset,. In a previous report, plant terpenes (p-cymene, (S)-(-)-limonene, ${\alpha}-pynene$, and ${\alpha}-terpinene$) have been found to be utilized by a PCB degrader and to induce the biphenyl dioxygenase gene in pure culture. In this study, Pseudomonas pseudoalcaligenes KF707, a PCB-degrading Gram-negative soil bacterium, was used to determine whether the terpene stimulation of PCB degrader occurred in the natural environment. First, P. pseudoalcaligenes KF707 was genetically tagged using a transposon with gfp (green fluorescent protein) as a reporter gone. The population dynamics of P. pseudoalcaligenes KF707 harboring gfp gene in a PCB-contaminated environment was examined with or without terpenoids added to the microcosm. About 10-100-fold increase was found in the population of PCB degraders when terpene was added, compared with control (non-terpenes samples and biphenyl added samples). It was proposed that the gfp-monitoring system is very useful and terpenes enhance the survivability of PCB degraders in PCB-contaminated environments.

Homology Analysis Among the Biphenyl and 4-Chlorobiphenyl Degrading Genes by Southern Hybridization (Southern Hybridization에 의한 Biphenyl 및 4-Chlorobiphenyl 분해유전자들의 상동성 분석)

  • 남정현;김치경;이재구;이길재
    • Microbiology and Biotechnology Letters
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    • v.22 no.1
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    • pp.37-44
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    • 1994
  • The homology among the genes coding for degradation of bipheny(BP) and 4-chlorobiphenyl(4CB) was comparatively analyzed by Southern hybridization in several BP/4CB degrading bacterial strains. As the hybridization results of their genomic DNAs with pcbABCD as the DNA probe, the group of Pseudomonas sp. DJ-12. P08 and P27 strain was separated by the group of P20 and P1242 strains. The P. pseudoalcaligenes KF707 showed the hybidization signal which was homologous to the group of DJ-12, but they had different restriction endonuclease sites. The pcbAB genes in pCUl recombinant plasmid from Pseudomonas sp. DJ-12 appeared to be homologous to pchAB genes in pKTF20 cloned from P. pseudoalcaligenes KF707, but the C genes in both strains were not homologous. The bphABC in pKTF20 showed the signals homologous to the cbp ACB in pAW6194 cloned from P. putida OU83, but homologous signal was not found botween the pcbABCD genes in pCUl and the cbpADCB genes in pAW6194 recombbinant plasmid.

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Inhibition and Chemical Mechanism of Protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Pseudomonas pseudoalcaligenes KF707에서 유래한 protocatechuate 3,4-dioxygenase 의 저해 및 화학적 메커니즘)

  • Kang, Taekyeong;Kim, Sang Ho;Jung, Mi Ja;Cho, Yong Kweon
    • Journal of Life Science
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    • v.25 no.5
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    • pp.487-495
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    • 2015
  • We carried out pH stability, chemical inhibition, chemical modification, and pH-dependent kinetic parameter assessments to further characterize protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. Protocatechuate 3,4-dioxygenase was stable in the pH range of 4.5~10.5. L-ascorbate and glutathione were competitive inhibitors with $K_{is}$ values of 0.17 mM and 0.86 mM, respectively. DL-dithiothreitol was a noncompetitive inhibitor with a $K_{is}$ value of 1.57 mM and a $K_{ii}$ value of 8.08 mM. Potassium cyanide, p-hydroxybenzoate, and sodium azide showed a noncompetitive inhibition pattern with $K_{is}$ values of 55.7 mM, 0.22 mM, and 15.64 mM, and $K_{ii}$ values of 94.1 mM, 8.08 mM, and 662.64 mM, respectively. $FeCl_{2}$ was the best competitive inhibitor with a $K_{is}$ value of $29{\mu}M$. $FeCl_{3}$, $MnCl_{2}$, $CoCl_{2}$, and $AlCl_{3}$ were also competitive inhibitors with $K_{is}$ values of 1.21 mM, 0.85 mM, 3.98 mM, and 0.21 mM, respectively. Other metal ions showed noncompetitive inhibition patterns. The pH-dependent kinetic parameter data showed that there may be at least two catalytic groups with pK values of 6.2 and 9.4 and two binding groups with pK values of 5.5 and 9.0. Lysine, cysteine, tyrosine, carboxyl, and histidine were modified by their own specific chemical modifiers, indicating that they are involved in substrate binding and catalysis.

Cloning and Sequence Analysis of the xyIL Gene Responsible for 4CBA-Dihydrodiol Dehydrogenase from Pseudomonas sp. S-47

  • 박동우;이상만;가종옥;김지경
    • Korean Journal of Microbiology
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    • v.38 no.4
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    • pp.275-275
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    • 2002
  • Pseudomonas sp. S-47 is capable of catabolizing 4-chlorobenzoate (4CBA) as carbon and energy sources under aerobic conditions via the mesa-cleavage pathway. 4CBA-dioxygenase and 4CBA-dihydrodiol dehydrogenase (4CBA-DD) catalyzed the degradation af 4CBA to produce 4-chlorocatechol in the pathway. In this study, the xylL gene encoding 4CBA-DD was cloned from the chromosomal DNA of Pseudomonas sp. S-47 and its nucleotide sequence was analyzed. The xylL gene was found to be composed of 777 nucleotide pairs and to encode a polypeptide of 28 kDa with 258 amino acid residues. The deduced amino acid sequence of the dehydrogenase (XylL) from strain S-47 exhibited 98% and 60% homologies with these of the corresponding enzymes, Pseudomonas putida mt-2 (XyIL) and Acinetobacter calcoaceticus (BenD), respectively. However, the amino arid sequences show 30% or less homology with those of Pseudomonas putida (BnzE), Pseudomonas putida Fl (TodD), Pseudomonas pseudoalcaligenes KF707 (BphB), and Pseudomonas sp. C18 (NahB). Therefore, the 4CBA-dihydrodiol dehdrogenase of strain S-47 belongs to the group I dehydrogenase involved in the degradation of mono-aryls with a carboxyl group.

Characterization of the pcbD Gene Encoding 2-Hydroxy-6-Ox0-6-Phenylgexa-2,4-Dienoate Hydrolase from Pseudomonas sp. P20

  • Lim, Jong-Chul;Lee, Jeong-Rai;Lim, Jai-Yun;Min, Kyung-Rak;Kim, Chi-Kyung;Ki, Young-Soo
    • Journal of Microbiology and Biotechnology
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    • v.10 no.2
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    • pp.258-263
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    • 2000
  • 2-Hydroxy-6-oxo-6phenylhexa-2,4-dienoate (HOPDA) hydrolase catalyzes the hydrolytic cleavage of HOPDA to bemzpate and 2-hydroxypenta-2, 4-dienoate (HPD) during microbial catabolism of biphenyl and polychlorinated biphenyls. A HOPDA hydrolase gene (pcbD) was isolated from the genomic library of Pseudomonas sp. P20 and designated as pCNUO1201; a 7.5-kb XbaI DNA fragment from Pseudomonas sp. P20 was inserted into the pBluescript SK(+) XbaI site. E. coli HB101 harboring pCNU1201 exhibited HOPDA hydrolase activity. The open reading frame (ORF) corresponding to the pcbD gene consisted of 855 base pairs with an ATG initiation codon and a TGA termination codon. The ORF was preceded by a rebosome-binding sequence of 5'-TGGAGC-3' and its G+C content was 55 mol%. The pcbD gene of Pseudomonas sp. P20 was located immedeately downstream of the pcbC gene encoding 2,3- dihydroxybiphenyl 1,2-dioxygenase, and approximately 4-kb upstream of the pcbE gene encoding HPD hydratase. The pcbK gene was able to encode a polypeptide with a molecular weight of 31,732 containing 284 amino acid residues. The deduced amino acid sequence of the HOPDA hydrolase of Pseudomonas sp. P20 exhibited high identity (62%) with those of the HOPDA hydrolases of P. putida KF715, P. pseudoalcaligenes KF707, and Burkholderia cepacia LB400, and also significant homology with those of other hydrolytic enzymes including esterase, transferase, and peptidase.

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Cloning and Sequence Analysis of the xyIL Gene Responsible for 4CBA-Dihydrodiol Dehydrogenase from Pseudomonas sp. S-47

  • Park, Dong-Woo;Kim, Youngsoo;Lee, Sang-Mahn;Ka, Jong-Ok;Kim, Chi-Kyung
    • Journal of Microbiology
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    • v.38 no.4
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    • pp.275-280
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    • 2000
  • Pseudomonas sp. S-47 is capable of catabolizing 4-chlorobenzoate (4CBA) as rarbon and energy sources under aerobic conditions via the mesa-cleavage pathway. 4CBA-dioxygenase and 4CBA-dihydrodiol dehydrogenase (4CBA-DD) catalyzed the degradation af 4CBA to produce 4-chlorocatechol in the pathway. In this study, the xylL gene encoding 4CBA-DD was cloned from the chromosomal DNA of Pseudomonas sp. S-47 and its nucleotide sequence was analyzed. The xylL gene was found to be composed of 777 nucleotide pairs and to encode a polypeptide of 28 kDa with 258 amino acid residues. The deduced amino acid sequence of the dehydrogenase (XylL) from strain S-47 exhibited 98% and 60% homologies with these of the corresponding enzymes, Pseudomonas putida mt-2 (XyIL) and Acinetobacter calcoaceticus (BenD), respectively. However, the amino arid sequences show 30% or less homology with those of Pseudomonas putida (BnzE), Pseudomonas putida Fl (TodD), Pseudomonas pseudoalcaligenes KF707 (BphB), and Pseudomonas sp. C18 (NahB). Therefore, the 4CBA-dihydrodiol dehdrogenase of strain S-47 belongs to the group I dehydrogenase involved in the degradation of mono-aryls with a carboxyl group.

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