• Journal of Life Science
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• v.24 no.10
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• pp.1046-1054
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• 2014

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with over-production required to enable versatile utilization. We examined the overexpression of ${\beta}$-1,3-glucanase (EXGA) from Aspergillus oryzae using ${\delta}$-sequence-mediated integration. We constructed $pRS{\delta}$-exgA and $pRS{\delta}K$-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence [INU1 s.s]) for secretory production, and a ${\delta}$-sequence for integration of ${\beta}$-1,3-glucanase. The $pRS{\delta}$-exgA plasmid was transformed into the S. cerevisiae $BY4742{\Delta}exg1$ strain, and ${\beta}$-1.3-glucanase was stably overexpressed and secreted. Another plasmid, $pRS{\delta}K$-exgA, was introduced into the S. cerevisiae $BY4742{\Delta}exg1$ (YKY082) strain, and overexpression of ${\beta}$-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of ${\beta}$-1,3-glucanase increased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of ${\beta}$-1,3-glucanase. Subsequently, $pRS{\delta}K$-exgA was repeatedly transformed for sequential ${\delta}$-integration. The activity of ${\beta}$-1,3-glucanase reached about 0.063 unit/ml/$OD_{600}$, 0.095 unit/ml/$OD_{600}$, 0.131 unit/ml/$OD_{600}$ and 0.165 unit/ml/$OD_{600}$ by the first, second, third, and fourth round of integration, respectively. According to the increase in the activity of ${\beta}$-1,3-glucanase by sequential ${\delta}$-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant ${\beta}$-1,3-glucanase activity can be sequentially increased by repeated ${\delta}$-sequence integration.

• Journal of Life Science
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• v.34 no.2
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• pp.86-93
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• 2024

Since replication of plasmids must be strictly controlled, plasmids that generally perform rolling circle replication generally maintain a constant copy number by strictly controlling the replication initiator Rep at the transcriptional and translational levels. Plasmid pJB01 contains three orfs (copA, repB, repC or repABC) consisting of a single operon. From analysis of amino acid sequence, pJB01 CopA was homologous to the Cops, as a copy number control protein, of other plasmids. When compared with a CopG of pMV158, CopA seems to form the RHH (ribbon-helix-helix) known as a motif of generalized repressor of plasmids. The result of gel mobility shift assay (EMSA) revealed that the purified fusion CopA protein binds to the operator region of the repABC operon. To examine the functional role of CopA on transcriptional level, 3 point mutants were constructed in coding frame of copA such as CopA R16M, K26R and E50V. The repABC mRNA levels of CopA R16M, K26R and E50V mutants increased 1.84, 1.78 and 2.86 folds more than that of CopA wt, respectively. Furthermore, copy numbers owing to mutations in three copA genes also increased 1.86, 1.68 and 2.89 folds more than that of copA wt, respectively. These results suggest that CopA is the transcriptional repressor, and lowers the copy number of pJB01 by reducing repABC mRNA and then RepB, as a replication initiator.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.479-485
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• 1986

A 5200 basepair DNA fragment containing the Bacillus amyloliquefaciens amyE gene, encoding liquefying $\alpha$-amylase (1,4-$\alpha$-1)-glucan glucanohydrolase, EC 3.2.1.1), has been inserted into BamHI site of the pUB110 and the hybrid plasmid was designated as pSKS3. The pSKS3 was transformed into the Bacillus subtilis KM2l3 as a host which is a saccharifying $\alpha$-amylase deficient mutant of Bacillus subtilis NA64, and the plasmid in the transformed cell was expressed $\alpha$-amylase production and kanamycin resistance. The $\alpha$-amylase production of the transformed cell was reduced to one fifth of that of the donor strain. The Bacillus subtilis KM2l3 tarring pSKS3 indicated that the amyE gene product is a polypeptide which has the same electrophoretic mobility with that of the Bacillus amyloliquefaciens, but different from the saccharifying $\alpha$-amylase of Bacillus subtilis NA64. It means that the amyE gene of pSKS3 originales from the Bacillus amyloliquefaciens.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.88-93
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• 2016

Dirofilaria immitis is a filarial nematode parasite that causes cardiopulmonary dirofilariasis in dogs. The purpose of this study was the development of real-time PCR assays for efficient detection of D. immitis. The D. immitis-specific primers confirmed in our previous study and a newly designed TaqMan probe were used for quantitative diagnostics. First, SYBR Green real-time PCR was performed using the specific primers and serially diluted genomic DNA or plasmid DNA, and melting curve analyses were performed after amplification. The melting curve showed one specific peak in each of the genomic and plasmid DNA reactions, suggesting that the primers specifically amplify the D. immitis cytochrome c oxidase subunit I gene. Comparison of SYBR Green and TaqMan real-time PCR using serially diluted plasmid DNA showed higher efficiency and specificity with TaqMan real-time PCR. The real-time PCR assays developed in this study will provide improved diagnostic methods to overcome the limitations of conventional diagnostic tools and facilitate more rapid and accurate diagnoses.

• Journal of Veterinary Clinics
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• v.32 no.2
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• pp.191-195
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• 2015

A 7-year-old castrated male Korean Shorthair cat was referred with lethargy and anorexia. Laboratory examination revealed moderate degenerative changes of peripheral neutrophils on blood smear examination and decreased levels of free and total thyroxine ($T_4$) as well as bacterial growth on blood culture. Molecular analyses of the 16S ribosomal RNA gene and heat shock protein 60 gene confirmed the bacterium as Enterobacter cloacae. A minimal inhibitory concentration test showed multidrug resistance of the bacterium against 16 antibiotics. Polymerase chain reaction (PCR) and subsequent sequencing specifically for $bla_{TEM}$, $bla_{SHV}$, $bla_{CTX-M}$, and plasmid-mediated ampC genes revealed positive results to $bla_{TEM-1}$, $bla_{CTX-M-15}$, and plasmid-mediated $bla_{ACT-1}$ genes, indicating that the isolated bacterium contains plasmids containing genes encoding extended-spectrum beta-lactamase and plasmid-mediated ampC beta-lactamase. After 1 month of treatment with antibiotics and levothyroxine, the cat's condition improved; both the thyroid function test and the blood culture showed no abnormalities. This is the first report of community-acquired multidrug-resistant E. cloacae-induced euthyroid sick syndrome in a cat. By the prompt diagnostic procedures and properly selected antibiotic therapy, the cat was recovered from the multidrug-resistant bacterium-induced septicaemia.

• 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.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.419-424
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• 2009

To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.309-319
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• 2018

Previously, six Schizosaccharomyce pombe mutants that induce pheromone even in the presence of nitrogen source were isolated from a bank of temperature sensitive mutants. In this report, one of these mutants, pws6 was further characterized. The pheromone induction in pws6 mutant cells was specific to nutrient: the M-factor pheromone was induced without nitrogen starvation but not without glucose starvation. This result suggests that the pws6 mutant might have a specific defect in the pathway for nitrogen starvation. The pws6 mutant induces P-factor pheromone as well as M-factor without starvation of nitrogen in temperature sensitive mode, suggesting that the pheromone induction phenotype of pws6 mutation is not cell-type specific. From cloning of the $pws6^+$ gene by complementation of the temperature sensitive growth defect, three plasmids containing 8.1 kb, 3.3 kb, and 4.8 kb yeast DNA were recovered. These plasmids complement the growth defect of the pws6 mutant by 100%, 70%, and 10~20%, respectively. The abilities of these plasmids to complement pheromone induction phenotype of pws6 mutant cells were correlated well with the efficiencies of complementation of the growth defect. With comparison of their open reading frames to the complementation efficiencies, it is concluded that the open reading frame, SPBC19C7.06 is responsible for the complementation of temperature sensitive phenotype of the pws6 mutant. This open reading frame, named prs1, contains one long exon with no intron and encodes a putative prolyl tRNA synthetase. The putative Prs1 protein exhibits significant similarities to the prolyl tRNA synthetases of other species.

• Korean Journal of Microbiology
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• v.18 no.1
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• pp.29-34
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• 1980

As preliminaries for the study of Plasmid pKM101 functions nad their interaction with the host DNA repair genes, seven mutants of pKM101, visualized on tetrazolium-galactose plates, deficient in their abitity to enhance mutagenesis were isolated and paritally characterizee. They all have altered functions not only for mutagenesis against MMS and 4-NQO but for the spontaneous reversion of host cell.

• YAKHAK HOEJI
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• v.43 no.6
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• pp.789-792
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• 1999

The complete nucleotide sequence of pKH4, a small multidrug resistance (smr) plasmid isolated from multidrug resistant Staphylococcus aureus SA5, was determined. Sequence analysis has revealed that pKH4 has two open reading frames for Rep and Smr proteins. The comparison of the amino acid sequence of Smr protein of pKH4 with those of other Smr proteins of various Staphylococcus showed that Smr protein of pKH4 is a new member of the SMR family.