• Journal of Microbiology
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• 제34권4호
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• pp.349-354
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• 1996

Pseudomonas sp. P20 is a natural isolate which is capable of degrading biphenyl and 4-chlorobiphenyl. From a clone of pCK1022 harboring pcbCD genes of Pseudomonas sp P20, a pcbD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA) hydrolase was subcloned in Escherichia coli XL-1-Blue by using pBluescript SK(+) vector. The 2.8-kb HindII fragment harboring the pcbD gene cloned in pCK 1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 carrying pCK0124 degraded HOPDA to benzoate and 2-hydroxypenta-2, 4-dienoate by HOPDA hydrolase encoded by pcbD gene as effectively as E coli CK 1022 HARBORING pcbCD genes.

• Journal of Microbiology and Biotechnology
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• 제10권2호
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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.

• Archives of Pharmacal Research
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• 제27권5호
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• pp.570-575
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• 2004

2-Hydroxymuconic semialdehyde (2-HMS) dehydrogenase catalyzes the conversion of 2-HMS to 4-oxalocrotonate, which is a step in the meta cleavage pathway of aromatic hydrocarbons in bacteria. A tomC gene that encodes 2-HMS dehydrogenase of Burkholderia cepacia G4, a soil bacterium that can grow on toluene, cresol, phenol, or benzene, was overexpressed into E. coli HB 101, and its gene product was characterized in this study. 2-HMS dehydrogenase from B. cepacia G4 has a high catalytic efficiency in terms of V$_{max}$K$_{max}$ towards 2-hydroxy-5-methyl-muconic semialdehyde followed by 2-HMS but has a very low efficiency for 5-chloro-2-hydroxymuconic semialdehyde. However, the enzyme did not utilize 2-hydroxy-6-oxo-hepta 2,4-dienoic acid and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid as substrates. The molecular weight of 2-HMS dehydrogenase from B. cepacia G4 was predicted to be 52 kDa containing 485 amino acid residues from the nucleotide sequence of the tomC gene, and it exhibited the highest identity of 78% with the amino acid sequence of 2-HMS dehydrogenase that is encoded in the aphC gene of Comamonas testosteroni TA441. 2-HMS dehydrogenase from B. cepacia G4 showed a significant phylogenetic relationship not only with other 2-HMS dehydrogenases, but also with different dehydrogenases from evolutionarily distant organisms.sms.

• Archives of Pharmacal Research
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• 제23권2호
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• pp.187-195
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• 2000

Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) of Pseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 genes. All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A). However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream of pcbC gene. Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. The orf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues. A deduced amino acid sequence of the orf1 gene product exhibited 21-33％ identity with those of indole dioxygenase and phenol hydroxylase components. The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. The orf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues. A deduced amino acid sequence of the orf2 gene product exhibited 31％ identity with that of a nitrilotriacetate monooxygenase component.

• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.668-676
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• 2001

Pseudomonas sp. strain SY5 is a PCB-degrading bacterium [24] that includes two different enzymes (BphC1 and BphC2) encoding 2,3-dihdroxybiphenyl 1,2-dioxygenase and BphD encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase. The bphC1 and bphC2 genes were found to consist of 897 based encoding 299 amino acids and 882 bases encoding 294 amino acids, respectively, whereas the bphD gene consisted of 861 bases encoding 287 amino acids. According to a homology search, a 50% and 39% similarity between the bphC1 and bphC2 genes at the nucleotide and amino acid level was shown, respectively. The bphC1 gene showed a 38% and 45% similarity at the amino acid level to Alcaligenes eutrophus A5 and Rhodococcus rhodochrous, respectively, whereas, bphC2 showed a 95% and 43% similarity, respectively. A comparison of the deduced amino acid sequence of the bphD product of Pseudomonas sp. SY5 with that of A. eutrophus A5, Pseudomons sp. KKS102, and LB400 showed a sequence identity of 92, 92, and 79%, respectively. Strain SY5 was originally isolated from municipal sewage containing recalcitrant organic compounds an found to have a high degradability of various aromatic compounds [23]. The current study found that strain SY5 had two extradiol-type dioxygenases, which did not hybridize with each other as they had a low similarity, yet a similar structure of evolutionarily conserved amino acids residues for catalytic activity between BphC1 and BphC2 was observed.

• Journal of Microbiology
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• 제35권1호
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• pp.53-60
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• 1997

Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 isolated from the polluted environment are capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce benzoic acid and 4-chlorobenzoic acid (4CBA) respectively, by pcbABCD-encoded enzymes. 4CBA can be further degraded by Pseudomonas sp. DJ-12, but not by Pseudomonas sp P20. However, the meta-cleavage activities of 2, 3-dihydroxybiphenyl (2, 3-DHBP) and 4-chloro-2, 3-DHBP dioxygenases (2, 3-DHBD) encoded by pcbC in Pseudomonas sp. P20 were stronger than Pseudomonas sp. DJ-12. In this study, the pcbC gene encoding 2, 3-DHBD was cloned from the genomic DNA of Pseudomonas sp. P20 by using pKT230. A hybrid plasmid pKK1 was constructed and E. coli KK1 transformant was selected by transforming the pKK1 hybrid plasmid carrying pcbC into E. coli XL1-Blue. By transferring the pKK1 plasmide of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation, a recombinant strain Pseudomonas sp. P20, Pseudomonas sp. DJ-12, and the recombinant cell assay methods. Pseudomonas sp. DJ12-C readily degraded 4CB and 2, 3-DHBP to produce 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA), and the resulting 4CBA and benzoic acid were continuously catabolized. Pseudomonas sp. DJ12-C degraded 1 mM 4CB completely after incubation for 20 h, but Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 showed only 90% and Pseudomonas sp. DJ-12 had, but its degradation activity to 2, 3-DHBP, 3-methylcatechol, and catechol was improved.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.