• 미생물학회지
• /
• 제40권2호
• /
• pp.121-126
• /
• 2004

4-chlorobiphenyl (4CB)의 분해균주인 Pseudomonas sp. strain DJ-12의 16S rDNA의 염기서열을 분석한 결과 Comamonas sp. strain DJ-12로 재분류 되었다. Pseudomonas sp. strain DJ-12로부터 4CB의 분해결과 생성되는 2,3-dihydroxybiphenyl을 계속 분해하는데 관여하는 pcbC1Dl 유전자를 이미 보고된 바 있다. 이번 연구에서는 Comamonas sp. strain DJ-12로부터 4CB 분해에 관여하는 pcbABC2D2 유전자를 클로닝하여 염기서열을 분석하였다. PcbAB 및 pcbCD 유전자들의 염기서열은 48, 65％, 추정 아미노산 서열은 33, 42％의 낮은 유사도를 보였다. 본 연구에서 얻어진 pcbC2D2 유전자는 이미 보고만 pcbCIDl 유전자와 염기의 개수와 서열의 유사도가 서로 다름을 보여 주었다. Comamonas sp. strain DJ-12의 두 가지 pcbCD유전자들은 Southern hybridization 결과에서도 유사성을 보이지 않았으며, 서로 다른 위치에 존재함을 보여주었다. 그러나 2,3-dihydroxybiphenyl의 분해 특성은 동일하였다. 이와 같은 결과는 Comamonas sp. strain DJ-12 균주가 2조의 pcbCD 유전자를 가지고 있다는 것을 의미하는 것이다.

• 미생물학회지
• /
• 제31권2호
• /
• pp.129-134
• /
• 1993

4-Chlorobiphenyl(4CB) 과 biphenyl 을 분해하는 Pseudomoas sp. DJ-12 의 pcbAB 는 분해초기 단계에 작용하는 4-chlorobiphenyl dioxygenase 와 dihydrodiol dehydrogenase 효소를 생산하는 유전자들이다. 이 유전자를 E. coli XL1-Blue 에 플로닝하여 CU101 형질전환체를 얻었다. CU101 의 pCU101 재조합 plasmid 에 클로닝된 pcbAB 유전자는 크기가 약 2.2 kb 이고 3 개의 Hind III 제한효소 위치가 있었으며, 독자적인 promoter 를 가지고 있었다. CU101 에 대하여 biphenyl 을 기질로 하여 생성된 대사산물을 resting cell assay 를 한 결과 2, 3-dihydroxybiphenyl 이 검출되어 pcbAB 유전자들이 E. coli 에서 잔 발현된다는 것을 의미하였다. 그러나 dechlorination 작용은 pcbAB 유전자와 관계없이 4AB 의 개환과정 후 생성된 4-chlorobenzoate 에서 일어나는 것으로 해석된다.

• 한국미생물·생명공학회지
• /
• 제22권1호
• /
• pp.37-44
• /
• 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.

• 미생물학회지
• /
• 제32권1호
• /
• pp.40-46
• /
• 1994

Polychlorinaed biphenyls(PCBs) 와 biphenyl을 분해하는 Pseudomonas sp. DJ-12에서는 그 초기 분해과정에 pcb ABCD 유전자들이 관여하고 있음이 밝혀졌다. 그 중 pcbCD와 pcdD 유전자를 E. coli XL1-Blue에 클로닝하여 E. coli CU103 과 CU105 균주를 각각 제조하였다. E. coli CU103은 2,3-dehydroxybuphenyl dioxygenase(2,3-DHBP)와 meta-cleavage compound(MCP) hydrolase를 생성하여 2,3-dihydroxybiphenyl을 benzoate로 변환시켜 주었다. E. coli CU1 과 CU103 에서 pcbC 유전자의 산물인 2,3-DHBP dioxygense의 활성도는 Pseudomonas sp. DJ-12에서 보다 약 17배 높았으며, E. coli CU105에서 pcbD의 산물인 MCP hydrolase는 약 3배 더 높게 나타났다.

• Journal of Microbiology and Biotechnology
• /
• 제11권5호
• /
• pp.812-818
• /
• 2001

Alginate and alginate-derived oligomannuronate enhanced penicillin production in shake flask and fermentor cultures of Penicillium chrysogenum Wis 54-1255 (containing a single copy of the penicillin gene cluster) and in the high producter strain P. chrysogenum AS-P-99 (containing multiple copies of the penicillin gene cluster). Alginate was not used as a single carbon source by P. chryogenum. The stimulatory effect on penicillin production was observed in a defined medium and, to a lower extent, in a complex production medium containing corn steep liquor. Alginate-supplemented cells showed higher transcript levels of the three penicillin biosynthetic genes, pcbAB, pcbC, and penDE, than cells grown in the absence of alginate. The promoters of the pcbAB, pcbC, and penDE genes were coupled to the reporter lacZ gene and introduced as monocopy constructions in P. chrysogenum Wis 54-1225 npe10 by targeted integration in the pyrG locus; the reporter ${\beta}$-galactosidase activity expressed from the three promoters was stimulated by alginate added to the culture medium of the transformants. These results indicate that the stimulation of penicillin production by alginate was derived from an increase in the transcriptional activity of the penicillin biosynthesis genes. The induction by alginate of the transcription of the three penicillin biosynthetic genes is good example of the coordinated induction of secondary metabolism genes by elicitors of plant (or microbial) origin.

• 한국미생물·생명공학회지
• /
• 제22권4호
• /
• pp.353-359
• /
• 1994

Pseudomonas sp. P20 was a bacterial isolate which has the ability to degrade 4-chlorobi- phenyl(4CB) to 4-chlorobenzoic acid via the process of meta-cleavage. The recombinant plasmid pCK1 was constructed by insetting the 14-kb EcoRI fragment of the chromosomal DNA containing the 4CB-degrading genes into the vector pBluescript SK(+). Subsequently, E. coli XL1-Blue was transformed with the hybrid plasmid producing the recombinant E. coli CK1. The recombinant cells degraded 4CB and 2,3-dihydroxybiphenyl(2,3-DHBP) by the pcbAB and pcbCD gene products, respectively. The pcbC gene was expressed most abundantly at the late exponential phase in E. coli CK1 as well as in Pseudomonas sp. P20, and the level of the pcbC gene product, 2,3-DHBP dioxygenase, expressed in E. coli CK1 was about two-times higher than in Pseudomonas sp. P20. The activities of 2,3-DHBP dioxygenase on catechol and 3-methylcatechol were about 26 to 31% of its activity on 2,3-DHBP, but the enzyme did not reveal any activities on 4-methylcatechol and 4-chlorocatechol.

• 한국발생생물학회:학술대회논문집
• /
• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
• /
• pp.63-63
• /
• 2003

In an effort to uncover the spermatogenic impairment by the polychlorinated biphenyls (PCBs), the expression of tight junctions (TJs) genes important for the formation of the blood testis barrier (BTB) were examined following the 3,3',4,4',5-pentachloro biphenyl (PCB126) treatment in cultured neonatal testis in mice. At 4 days (D4) after 10 and 100 nM PCB126 treatment the expression of claudin-11 was significantly increased when compared with vehicle control. In contrast no difference in occludin and claudin-1 expression was found among the experimental group. On D8, 100 nM PCB126 significantly increased the expression of claudin-11 but not occludin and claudin-1. 1 uM PCB126 treatment significantly decreased expressions of occludin and ciaudin -1, suggesting the general toxic effect on the Sertoli cell. Because PCB126 does not alter the proliferative activity of spermatogenic cells and Sertoli cells in neonatal testis, it is likely that increase in the expression of claudin-11 by low dose of PCB126 may attribute to the alteration of the Sertoli cells differentiation in testis. It also emphasized that PCB126 might have differentially affected the transcription of TJ genes in Sertoli cells. In conclusion, this result suggests that the structure of TJ may be targeted by PCB126 in neonatal testis in mice and that co-PCB is potentially harmful to spermatogenesis by alteration of the development of BTB.

• Journal of Microbiology and Biotechnology
• /
• 제5권2호
• /
• pp.68-73
• /
• 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.

• Archives of Pharmacal Research
• /
• 제23권2호
• /
• pp.187-195
• /
• 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
• /
• 제34권4호
• /
• pp.349-354
• /
• 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.