• Korean Journal of Microbiology
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• v.38 no.4
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• pp.270-270
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• 2002

Pathogenic Salmonella typhimurium can invade the intestinal epithelium and cause a wide range of diseases including gastroenteritis and bacteremia in human and animals. To identify the genes involved in the infection, the invasion determinant was obtained from S. typhimurium 82/6915 and was subcloned into pGEM-7Z. A subclone DHl (pSV6235) invaded HEp-2 and Chinese hamster ovary epithelial cells and contained a 4.4 kb fragment of S. typhimurium genomic region. Compared with the host strain E. coli DHl, the subclone DHl (pSV6235) invaded cultured HEp-2 and Chinese hamster ovary cells at least 75- and 68-fold higher, respectively. The invasion rate of E. coli DHl for the cells significantly increased by harbouring the genomic region derived from pathogenic S. typhimurium 82/6915.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.6
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• pp.510-515
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• 2006

The putative EPA synthesis gene cluster was mined from the entire genome sequence of Shewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO1600 (acyl transferase, AT), ORFSO1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO1597 (enoyl reductase, ER), ORFSO1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified from S. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genes orfSO1597 and orfSO1604. The DNA fragment was cloned into Escherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in four E. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in the E. coli clone was affected by cultivation temperature, showing maximum yield at $20^{\circ}C$ and no production at $30^{\circ}C$ or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.292-300
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• 2003

Pseudomonas fluorescens B16 is a plant glowth-prornoting rhizobacterium, which produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacrerium tumefaciens and Raistonia solanacearum. We mutagenized the strain B16 with Omegon-Km and isolated six antibacterial-activity-deficient mutants. Two cosmid clones that hybridized with the mutant clones also were isolated from a genomic library of tile parent strain. Using deletion and complementation analyses, it was found that the biosynthesis genes resided in a 4.3-kb SalI-NarI fragment. When a plasmid clone carrying the fragment was introduced into P. fluorescens strain 1855.344, which does not exhibit any antibacterial activity, the transconjugants exhibited antibacterial activity, indicating that the plasmid clone carried all the genes essential for production of the antibacterial compound. DNA sequence analysis of the fragment identified four putative open reading frames (ORFs): orf1 through orf4 The deduced amino acid sequences of ORF1, ORF2, and ORF4 were similar to cystathionine gamma lyase, pyruvate formate-lyase activating enzyme, and transcriptional regulator, respectively, yet the amino acid sequence of ORF3 showed no similarities to any known proteins. It was also demonstrated that the antibacterial activity was responsible for biological control of the bacterial wilt caused by R. solanacearum.

• Journal of Life Science
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• v.13 no.4
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• pp.522-528
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• 2003

The RAD4 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. A yeast RAD4 gene has been previously isolated by functional complementation. In order to identify the RAD4 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD4 DNA, and then isolated homologous C. cinereus chromosome. The insert DNA of the RAD4 homolog was contained 3.2 kb. Here, we report the characterization of fungus C. cinereus homolog of yeast RAD4 gene. Southern blot analysis confirmed that C. cinereus contains the RAD4 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from C. cinereus cells was hybridized with the 1.2 kb PvuII DNA fragment of the S. cerevisiae RAD4 gene, a 2.5 kb of transcript was detected. In order to investigation whether the increase of transcripts by DNA damaging agent, transcripts levels were examined after treating the cells. The level of transcript did not increase by untraviolet light (UV). This result indicated that the RAD4 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the RAD4 homologous gene is essential for cell viability.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1023-1027
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• 2004

The RAD3 gene of Saccharomyces cerevisiae is essential for the incision step of UV-induced excision repair. An yeast RAD3 gene has been previously isolated by functional complementation. In order to identify the RAD3 homologous gene from fungus Coprinus cinereus, we have constructed cosmid libraries from electrophoretically separated chromosomes of the C. cinereus. The 13 C. cinereus chromosomes were resolved by pulse-field gel electrophoresis, hybridized with S. cerevisiae RAD3 DNA, and then isolated RAD3 homologous DNA from C. cinereus chromosome. The RAD3 homolog DNA was contained in 3.2 kb DNA fragment. Here, we report the results of characterization of a fungus C. cinereus homolog to the yeast RAD3 gene. Southern blot analysis confirmed that the C. cinereus chromosome contains the RAD3 homolog gene and this gene exists as a single copy in C. cinereus genome. When total RNA isolated from the C. cinereus cells were hybridized with the 3.4 kb PvuII DNA fragment of the S. cerevisiae RAD3 gene, transcripts size of 2.8 kb were detected. In order to investigate whether the increase of the amount of transcripts by DNA damaging agent, transcript levels were examined after treating agents to the cells. The level of transcripts were not increased by untraviolet light (UV). This result indicated that the RAD3 homologous gene is not UV inducible gene. Gene deletion experiments indicate that the HRD3 gene is essential for viability of the cells and DNA repair function. These observations suggest an evolutionary conservation of other protein components with which HRD3 interacts in mediating its DNA repair and viability functions.

• Journal of Life Science
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• v.20 no.9
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• pp.1306-1313
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• 2010

The gene encoding esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est1R) was 2,465 bp in length, encoding a protein of 366 amino acid residues, and the molecular weight of the enzyme was 61,166 Da. Est1R of rumen cosmid library shared 5.9% amino acid identity with Est1R (P37967) of PNB carboxylesterase, 6.1% with Est1R (1EEAA) of acetylcholinesterase and 6.1% with Est1R (1H23A) of chain A. BlastP in NCBI database analysis of Est1R revealed that it was not homologous to previous known lipases and esterases. Est1R showed optimum activity at pH 7.0 and $40^{\circ}C$. On the other hand, the enzyme was found to be most active without organic solvent, followed by 95% activity with methanol, and the enzyme activity was highly affected by hexane (lost 51% activity). Therefore, the novel esterase gene est1R is likely obtainable from cow rumen metagenome and may be utilized for industrial purposes.

• The Korean Journal of Mycology
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• v.28 no.1
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• pp.22-25
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• 2000

This study was carried out to compare DNA sequence of mating type locus concerning with direct formation of fruiting body in Schizophyllum commune which is growing in North America with that of same species growing in South America. The nucleotide sequence appeared to have about 96% homology to 1-71 $A{\alpha}3$ allele from South America strain, showing a conservative feature. The polypeptide sequence showed about 82% homology when compared partially with mating activity region of 1-71 $A{\alpha}3$ allele. In addition, this polypeptide sequence indicated 74% and 82% identity in homeodomain and acidic-rich regions known as a transcription factor respectively.

• Journal of Life Science
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• v.12 no.2
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• pp.173-181
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• 2002

This study was conducted to do the comparative analysis of mating type locus controlling the direct formation of fruiting body in Schizophyllum commune which is indigenous to North America with that of other identified mating locus. The 3120 bp Y-region nucleotide of A $\alpha$ 3 mating locus activating a developmental pathway in S. commune was determined, and appeared to have about 96% homology to S. commune 1-71 $A\alpha$3 allele indigenous to South America, showing strongly a conservative feature. This nucleotide analysis also showed above 96% homology highly in the seven presumed exons, and about 97% in the acidic rich region (AR), about 99% in homeodomain (H7), about 97% in the basic rich region (BR), about 95% in the serine rich region (Ser) respectively. In the comparative analysis to the translated polypeptide sequence, S. commune A $\alpha$ 3 mating locus containing Y-region also showed about 97% homology to the region of S. commune indigenous to North America, but the identity ratio to Y1 including Y4, Y5, Y6 different allele types was declined to about 41~49%. In the analysis of functional loci controlling mating activity, it is assumed to have a highly conservative feature showing about 98% homology in homeodomain polypeptide. Especially, it is notable that the homology ratio of above 85% in homeodomain motif between mating type alleles was higher than in the AR, BR, Ser showing about 10~50% homology.

• Journal of Microbiology
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• v.44 no.2
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• pp.192-199
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• 2006

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

• Journal of Life Science
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• v.18 no.11
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• pp.1592-1599
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• 2008

The $\beta$-lactamase gene was cloned into E. coli DH5$\alpha$ from Bacillus sp. J105 with strong resistance against $\beta$-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. $\beta$-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-${\Delta}4.6$ with $\beta$-lactamase activity was obtained by subcloning of the recombinant plasmid ($\beta$-lac +). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the $\beta$-lactamase gene encodes 309 amino acids. The 0.17 kb upstream region was similar to those of B. thuringinesis and B. cereus with 97% identity. The deduced amino acids sequence was also similar to those of $\beta$-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the $\beta$-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-${\Delta}4.6$ in E. coli, revealed that the secretion efficiency of $\beta$-lactamase was $4{\sim}5%$ and the molecular weight was as same as that of original $\beta$-lactamase (31 kDa) from Bacillus sp. J105.