• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.357-366
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• 2019

We first confirmed the involvement of MalQ (4-${\alpha}$-glucanotransferase) in Escherichia coli glycogen breakdown by both in vitro and in vivo assays. In vivo tests of the knock-out mutant, ${\Delta}malQ$, showed that glycogen slowly decreased after the stationary phase compared to the wild-type strain, indicating the involvement of MalQ in glycogen degradation. In vitro assays incubated glycogen-mimic substrate, branched cyclodextrin (maltotetraosyl-${\beta}$-CD: G4-${\beta}$-CD) and glycogen phosphorylase (GlgP)-limit dextrin with a set of variable combinations of E. coli enzymes, including GlgX (debranching enzyme), MalP (maltodextrin phosphorylase), GlgP and MalQ. In the absence of GlgP, the reaction of MalP, GlgX and MalQ on substrates produced glucose-1-P (glc-1-P) 3-fold faster than without MalQ. The results revealed that MalQ led to disproportionate G4 released from GlgP-limit dextrin to another acceptor, G4, which is phosphorylated by MalP. In contrast, in the absence of MalP, the reaction of GlgX, GlgP and MalQ resulted in a 1.6-fold increased production of glc-1-P than without MalQ. The result indicated that the G4-branch chains of GlgP-limit dextrin are released by GlgX hydrolysis, and then MalQ transfers the resultant G4 either to another branch chain or another G4 that can immediately be phosphorylated into glc-1-P by GlgP. Thus, we propose a model of two possible MalQ-involved pathways in glycogen degradation. The operon structure of MalP-defecting enterobacteria strongly supports the involvement of MalQ and GlgP as alternative pathways in glycogen degradation.

• Korean Journal of Veterinary Research
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• v.61 no.2
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• pp.13.1-13.8
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• 2021

Mammalian reovirus (MRV) causes respiratory and intestinal disease in mammals. Although MRV isolates have been reported to circulate in several animals, there are no reports on Korean MRV isolates from wildlife. We investigated the biological and molecular characteristics of Korean MRV isolates based on the nucleotide sequence of the segment 1 gene. In total, 144 swabs from wild animals were prepared for virus isolation. Based on virus isolation with specific cytopathic effects, indirect fluorescence assays, electron microscopy, and reverse transcription-polymerase chain reaction, only one isolate was confirmed to be MRV from a Korean roe deer (Capreolus pygargus). The isolate exhibited a hemagglutination activity level of 16 units with pig erythrocytes and had a maximum viral titer of 10^5.7 50% tissue culture infectious dose (TCID₅₀)/mL in Vero cells at 5 days after inoculation. The nucleotide and amino-acid sequences of the partial segment S1 of the MReo2045 isolate were determined and compared with those of other MRV strains. The MReo2045 isolate had nucleotide sequences similar to MRV-3 and was most similar (96.1%) to the T3/Bat/Germany/342/08 strain, which was isolated in Germany in 2008. The MReo2045 isolate will be useful as an antigen for sero-epidemiological studies and developing diagnostic tools.

• Journal of Microbiology and Biotechnology
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• v.32 no.9
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• pp.1134-1145
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• 2022

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993⁺. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

• Journal of Microbiology and Biotechnology
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• v.31 no.7
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• pp.912-920
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• 2021

SOS response is a conserved response to DNA damage in prokaryotes and is negatively regulated by LexA protein, which recognizes specifically an "SOS-box" motif present in the promoter region of SOS genes. Myxococcus xanthus DK1622 possesses a lexA gene, and while the deletion of lexA had no significant effect on either bacterial morphology, UV-C resistance, or sporulation, it did delay growth. UV-C radiation resulted in 651 upregulated genes in M. xanthus, including the typical SOS genes lexA, recA, uvrA, recN and so on, mostly enriched in the pathways of DNA replication and repair, secondary metabolism, and signal transduction. The UV-irradiated lexA mutant also showed the induced expression of SOS genes and these SOS genes enriched into a similar pathway profile to that of wild-type strain. Without irradiation treatment, the absence of LexA enhanced the expression of 122 genes that were not enriched in any pathway. Further analysis of the promoter sequence revealed that in the 122 genes, only the promoters of recA2, lexA and an operon composed of three genes (pafB, pafC and cyaA) had SOS box sequence to which the LexA protein is bound directly. These results update our current understanding of SOS response in M. xanthus and show that UV induces more genes involved in secondary metabolism and signal transduction in addition to DNA replication and repair; and while the canonical LexA-dependent regulation on SOS response has shrunk, only 5 SOS genes are directly repressed by LexA.

• Mycobiology
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• v.49 no.6
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• pp.582-588
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• 2021

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

• Journal of Life Science
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• v.26 no.10
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• pp.1121-1129
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• 2016

High-molecular-weight glutenin subunits (HMW-GSs) are extremely important determinants of the functional properties of wheat dough. Transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding a HMG-GS were produced from the Korean wheat cultivar ‘Jokyeong’ and used to enhance the bread-making quality of rice dough using the Agrobacterium-mediated co-transformation method. Two expression cassettes with separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately into the Agrobacterium tumefaciens EHA105 strain for co-infection. Rice calli were infected with each EHA105 strain harboring TaGlu-Ax1 or HPTII at a 3:1 ratio of TaGlu-Ax1 and HPTII. Among 210 hygromycin-resistant T₀ plants, 20 transgenic lines harboring both the TaGlu-Ax1 and HPTII genes in the rice genome were obtained. The integration of the TaGlu-Ax1 gene into the rice genome was reconfirmed by Southern blot analysis. The transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in rice T₁ seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened in the T₁ generation. There were no morphological differences between the wild-type and marker-free transgenic plants. The quality of only one HMW-GS (TaGlu-Ax1) was unsuitable for bread making using transgenic rice dough. Greater numbers and combinations of HMW and LMW-GSs and gliadins of wheat are required to further improve the processing qualities of rice dough. TaGlu-Ax1 marker-free transgenic plants could provide good materials to make transgenic rice with improved bread-making qualities.

• KSBB Journal
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• v.15 no.2
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• pp.125-133
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• 2000

Specific carotenoids and astaxanthin biosynthesis power of Phaffia rhodozyma mutant 876, which was obtained after NTG a and UV treatments, was higher than those of the wild type by 40% and 50%, respectively. The mutant strain did not show t the catabolite repression even at 22% (w/v) glucose concentration. The optimum C{N ratio was 2.0, and the optimum t temperature and initial pH were $22^{\circ}C$ and 6.0, respectively. 80th cell growth and astaxanthin formation decreased drastically a as the fermentation temperature was increased over $22^{\circ}C$, whereas they were comparable in the pH range between 5.0 and 7 7.0. Inoculum size did not affect the final cell density nor the carotenoids biosynthesis, and 3%(v/v) was selected as optimal. H Higher dissolved oxygen concentration facilitated astaxanthin biosynthesis, and aeration rate of 1.0 v/0/m and agitation speed of 400 rpm were selected as optimum. The final cell dens때 of 43.3 g/L and the volumetric astaxanthin and carotenoids concentrations of 110.6 mg/L and 149.4 mg/L, respectively, were obtained. The specific carotenoids concentration was 3.45 m mg{g-yeast(dry). Yx/s and Yp/s values of 0.37 and 1.08 were obtained. The result of this study will provide basic information u useful for mass production of astaxanthin from P. rhodozyma fermentation.

• Korean Journal of Microbiology
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• v.46 no.3
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• pp.233-239
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• 2010

Four genes (yqfR, yfmL, ydbR, deaD) were identified as putative DEAD-box RNA helicase genes in the genomic sequence of Bacillus subtilis by homology search. To understand the function of these genes, each of the genes was deleted and the constructed strains were tested for their growth charateristics at different temperatures. The growth rate of ydbR deletion mutant ($T_d$=53 min) was a little bit reduced at $37^{\circ}C$ as compared to that of wild type strain (CU1065). But the growth rate of other three (yqfR, yfmL, deaD) deletion mutants ($T_d$=30-40 min) is nearly equal to the growth rate of wild type ($T_d$=32 min). On the other hands, the growth rate of deletion mutants were reduced at $22^{\circ}C$ in order of yqfR ($T_d$=151 min), yfmL ($T_d$=214 min), ydbR ($T_d$=343 min), which showed cold-sensitive phenotype. The deletion mutant of deaD ($T_d$=109 min) grew equally as compared to the growth rate ($T_d$=102 min) of the wild type at $22^{\circ}C$ and did not show cold-sensitive growth. Double, triple and quadruple deletion mutants of these genes were constructed, and growth rate of these mutants were measured at various temperature conditions ($22^{\circ}C$, $37^{\circ}C$, $42^{\circ}C$) using LB broth. Multiple deletion mutations showed more severe cold-sensitive growth than single deletion mutations, and double deletion of ydbR and yfmL ($T_d$=984 min) showed most cold-sensitive growth than any other double mutants. Such a cold-sensitive growth of these mutations is quite similar to the result of csdA or srmB deletion in E. coli and suggested that physiological role of ydbR and yfmL is related with ribosome assembly.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.92-96
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• 2011

Rhodococcus sp. strain DK17 possesses three megaplasmids (380 kb pDK1, 330 kb pDK2, and 750 kb pDK3). The alkylbenzene-degrading genes (akbABCDEF) are present on pDK2 while the phthalate operons which are duplicated are present on both pDK2 (ophA'B'C'R') and pDK3 (ophABCR). DK17 with an optimal temperature of $30^{\circ}C$ showed no growth at $37^{\circ}C$. When transferred to $30^{\circ}C$, however, the $37^{\circ}C$ culture began to grow immediately, indicating that $37^{\circ}C$ is not lethal but stressful for DK17 growth. In addition, when exposed to $37^{\circ}C$ even for a short time, a part of DK17 cells lost the ability to degrade o-xylene (a model compound of alkylbenzenes). When two hundred colonies were randomly selected for colony PCR for pDK2-specific akbC, ophC', or pDK3-specific ophC, a total of 29 colonies were found to have lost at least one of the three genes. PFGE analysis clearly showed that all the mutants have different megaplasmid profiles from that of DK17 wild type, which are divided into five different cases: Type I (10 mutants, pDK2 loss and acquisition of a new ~700 kb plasmid), Type II (9 mutants, pDK2 loss), Type III (8 mutants, pDK3 loss and acquisition of a new ~400 kb plasmid), Type IV (1 mutant, pDK3 loss), and Type V (1 mutant, pDK2 and pDK3 loss and acquisition of the ~400 kb and ~700 kb plasmids). The above results showing that growth temperature changes can induce physical changes in bacterial genomes suggest that environmental changes in habitats including temperature fluctuations affect significantly the evolution of bacteria.

• Journal of Practical Agriculture & Fisheries Research
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• v.20 no.1
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• pp.35-47
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• 2018

A edible mushroom, Clitocybe maxima (Lentinus giganteusis) commercially cultivated in China and Taiwan. However, the researches of cultivation and cultural characteristics were not reported in Korea. In this study, we conducted on cultural characteristics and artificial cultivation of C. maxima. Six isolates were collected from China(3 isolates, commercial strain), Taiwan(1 isolate, commercial strain) and Korea(2 isolates, wild type). C. maxima and L. giganteus collected in China and Taiwan, respectively, are the same in China and are estimated to be of the same species as cultured characteristics. The mycelial growth of the collected strains was not significantly different in agar medium but it showed the best growth in YPMG in liquid culture. Optimum temperature for mycelial growth and induction of fruit body were 25℃ and 30℃, respectively. In order to artificial cultivation of C. maxima, cultural characteristics and artificial cultivation were carried out using agricultural by-products and forestry by-products materials. Mycelial growth was suitable in rice straw, cottonwood sawdust, corncob and rice seed medium, and it was selected as a cultivation medium. The suitable medium for artificial cultivation of C. maxima was selected to mixed medium 2(compounding ratio(v/v): 55% of hardwood sawdust, 5% of cottonseed pellets, 10% of cottonseed, 15% of beet pulp, 15% of swollen rice husks). It took about 30 days to be able to harvest, it was faster than oyster mushrooms. The cultivation period was about 30days. A isolate, CMA-002 was not initiation to fruit body primordiuma on the used cultivation substrate. Other 5 isolates were initiate and development to fruit body on the substrate used in this study. The strain CMA-003 was initiated to be fruiting body by 8~10 days after induction of fruiting body in all of the substrates. Isolate CMA-003 was generate to a bundle fruit body. Other isolates, however, were form fruit body individually. The CMA-003 strain was likely highly recommendable strains for farming. The optimum conditions for the induction and growth of C. maxima fruit body were 25~30℃, 8 hr illumination per day with white fluorescent lamp, 90~95% relative humidity, and 1,500 ppm of CO₂ concentration in a cultivation room.