• KSBB Journal
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• v.31 no.2
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• pp.105-112
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• 2016

Ferritin is known to regulate iron metabolism and maintain iron in a variety of the eukaryotic organisms. The region encoding the mature ferritin (0.47 kb, H-type) of Periserrula leucophryna was amplified using the designed primers including restriction enzyme site and termination codon and subcloned in frame to the pRBAS secretion vector containing the signal sequence, RBS, and promoter of amylase gene (E. coli-Bacillus shuttle vector), resulting in recombinant pRBAS-PLF vector. Recombinant ferritin (18 kDa) was correctly processed and secreted from Bacillus subtilis LKS strain harboring the pRBAS-PLF vector and quantitatively analyzed by SDS-PAGE and western blot, respectively. Secretion of the ferritin was optimized by culture conditions (host, medium, temperature, nitrogen source) in 3 L batch culture and 5 L jar fermenter. Finally. the ferritin was largely produced using 50 L fermenter as the following conditions; at $30^{\circ}C$, 150 rpm, 1 vvm in Bacillus subtilis LKS using PY medium. The secreted ferritin was maximally measured (approximately 177.6 ug/ml) when the cell density reached to 14.4 at $OD_{600}$ (20 h incubation). The iron binding activity was confirmed by Perls' staining in 7.5% non-denaturing gel, indicating that the multimeric ferritin (composed of 24 subunits) was formed in the culture broth after secretion. Biologically, the culture broth and powder type containing ferritin were tested for possibility as feed additive in chicken broiler. As a result, the ferritin stimulated the growth of chick broil and improved feed efficiency and production index.

• Journal of Microbiology and Biotechnology
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• v.19 no.11
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• pp.1306-1318
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• 2009

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two $\alpha$-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same $\alpha$-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during S. sclerotiorum cultivation. The structural gene of Scamy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal $\alpha$-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known $(\beta/\alpha)_8$ barrel motif in domain A, have a typical structure of $\alpha$-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal $\alpha$-amylase described until now with the smallest catalytic domain.

• Molecules and Cells
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• v.42 no.12
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• pp.850-857
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• 2019

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.67-71
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• 2015

During late August and early September 2011, stem rot symptoms were observed on adzuki bean plants (Vigna angularis) growing in fields located in Beijing and Hebei Province, China, respectively. In this study, four isolates were obtained from infected stems of adzuki bean plants. Based on their morphology, and sequence and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of the ribosomal DNA internal transcribed spacers (rDNA-ITS) region, the four isolates were identified as Rhizoctonia solani in anastomosis group (AG) 4 HGI. Pathogenicity tests showed that all isolates were strongly pathogenic to adzuki bean and resulted in serious wilt symptoms which was similar to observations in the fields. Additionally, the isolates infected several other crops and induced related rot on the roots and basal stems. To our knowledge, this is the first report of Rhizoctonia solani AG 4 HGI causing stem rot on adzuki bean.

• Research in Plant Disease
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• v.15 no.2
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• pp.88-93
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• 2009

In 2008, leaf rot and blight on pepper seeding ("Dokya-chungchung") occurred in a pepper farm at Hwaseong-si, Gyeonggi-do, Korea. The typical symptom is water-soaking and dark brown leaf blight at edges and tips of leaves. The fungal colonies isolated from infected tissues were pinkish at first and turned gradually to gray. Conidia were fusiform, non-septum, and $8.1-17.0{\times}2.0-3.8{\mu}m$ in size. Several specific PCR primers derived from the sequence of the internal transcribed spacer (ITS) region of the rDNA, such as CaINT, CgINT and CcINT were used for the identification of the fungal pathogen. The C. acutatum-specific primer CaINT was amplified single fragment of 496 bp that discriminated C. acutatum from the other species. The pathogenicity test was performed on seedlings and fruits of red pepper. On the basis of the morphological, molecular characteristics and pathogenicity test, we identified as Colletotrichum acutatum. This is the first report on leaf rot and blight on pepper seedling caused by C. acutatum in Korea.

• Korean Journal of Ecology and Environment
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• v.37 no.2 s.107
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• pp.241-247
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• 2004

Isolation and identification of alga-lytic bacteria were carried out. Fifteen isolates of alga-lytic bacteria were screened by the double layer method using A. cylindrica NIES-19 as a sole nutrient and four isolates among them were compared with their alga-lytic activity. The isolate AK-05 exhibiting the highest alga-lytic activity was identified as Acidovorax temperans base on its 16S rDNA sequence. The culture supernatant of the isolate AK-05 was reliable for the alga-lytic. Alga-lytic activity assays of culture supernatant revealed that the major substances for alga-lytic activity were non-proteins and heat stable. The highest alga-Iytic activity was practical under alkaline conditions and at 25${\sim}$$30^{\circ}C$. It is indicating an advantage for the application of water blooms by cyanobacteria in eutrophic lakes where the pH is generally in alkaline region.

• Mycobiology
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• v.30 no.2
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• pp.82-87
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• 2002

Species of Phellinus were known to harmful fungi causing white pocket rot and severe plant disease such as canker or heartrot in living trees in the West, but some species have been used to traditional medicines in the Orient for a long time. In this study the partial D1-D2 nucleotide sequences of 28S ribosomal DNA from 13 Phellinus strains were determined and compared with the sequences of 21 strains obtained from GenBank database. According to the neighbor-joining(NJ) method comparing the sequence data the phylogenetic tree was constructed. The phylogenetic tree displayed the presence of four groups. Group I includes P. ferreus, P. gilvus and P. johnsonianus, Group II contains P. laevigatus, P. conchatus and P. tremulae, Group III possesses P. linteus, P. weirianus, P. baumii, P. rhabarbarinus and P. igniarius, and Group IV comprises P. pini, P. chrysoloma. P. linteus and P. baumii, which were used mainly in traditional medicine, belong to the same group, but exactly speaking both were split into two different subgroups. To detect P. linteus only, we developed the PCR primer, D12HR. The primer showed the specific amplification of P linteus, which is permitted to medicinal mushroom in the East. The results make a potential to be incorporated in a PCR identification system that could be used for the rapid identification of this species from its related species, P. linteus especially.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.49-49
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• 2014

In order to find indigenous beneficial fungal species from crop field soils of Nigeria, 23 soil samples were collected from various places of Nigeria in June, 2013 and fungi were isolated through serial dilution technique. Isolated fungi were purified and differentiated according to their morphological and microscopic characteristics. In total, 38 different representative isolates were recovered and the genomic DNA of each isolates was extracted using QIAGEN$^{(R)}$ Plasmid Mini Kit (QIAGEN Sciences, USA) and the identification of fungi was carried out by sequence analysis of internal transcribed spacer (ITS) region of the 18S ribosomal DNA (18S rDNA). Recovered isolates belonged to 9 fungal genera comprising Fusarium, Aspergillus, Chaetomium, Coniothyrium, Dipodascaceae, Myrothecium, Neosartorya, Penicillium and Trichoderma. Aspergillus spp., Penicillium spp. and Trichoderma spp. were the most dominant taxa in this study. The antagonistic potentiality of species belonged to Trichoderma against 10 phytopathogenic fungi (F. oxysporum, C. gloesporoides, P. cytrophthora, A. alternata, A. solani, S. rolfsii, F. solani, R. solani, S. sclerotiorum and P. nicotiana) was assessed in vitro using dual culture assay. The dual culture assay results showed varied degree of antagonism against the tested phytopathogens. The potential Trichoderma spp. will be further evaluated for their antagonistic and plant growth promotion potentiality under in vivo conditions.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.53-63
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• 1998

Total of 1085 swine sera (1996-1997) from nation-wide were tested for the presence of antibodies to influenza A virus. Fifty nine percent of the tested sera showed seropositive by HI test. Positive sera consisted of 24--- of H3, 15--- of H1, and 20--- of the sample had both antibodies, respectively. Sera collected from various region represented 7~27--- seropositivity to H1N1, 15~25--- to H3N2, respectively. Swine influenza field isolate from nasal swab was characterized antigenically and genetically to elucidate its relatedness with other known strains of influenza A virus. The study was focused on the HA gene which is related to pathogenecity and antigenic variability of the influenza virus. By RT-PCR using influenza A/H1N1 specific primers, influenza virus H1N1 specific DNA fragment was amplified from A/Swine/Iowa/15/30(H1N1), US field isolate but not in H3N2 strain. PCR products were sequenced by dideoxy chain termination method to determine nucleotide homology with other strains of influenza A virus. The US field isolate and A/Swine/Indiana/1726/88 strain had 97--- of nucleotide homology and 98--- of amino acid homology. Based on the results obtained from this experiment, the field isolate was genetically related to A/Swine/Indiana/1726/88 and had higher homology with A/Swine/Indiana/1726/88 than with classical swine influenza virus, A/Swine/Iowa/15/30. The field isolate had no amino acid changes at the antigenic site compare to that of the A/Swine/Indiana/1726/88. The proteolytic enzyme cleavage site between HA1 and HA2 had no alteration and the amino acid arginine was intact. There is no evidence has been found that the field isolate has genetic shift or genetic drift which might altered antigenic determinant.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.85-91
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• 2001

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.