• Journal of Life Science
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• v.21 no.10
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• pp.1487-1493
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• 2011

E. tarda, a fish pathogen, can survive in seawater under relatively high salt conditions as well as in fish under physiological salt conditions. Bacterial growth under different salt concentrations may influence the expression of genes involved in bacterial structure and physiology. The growth rate of E. tarda culture in high salt (3.5% NaCl) was similar to that in low salt (1.0% NaCl, physiological salt concentration). Interestingly, the strain moved much faster in low salt conditions than in high salt conditions. Electron microscopic observation demonstrated that the bacterial cells grown in high salt had less or no flagellation. Obvious flagellation was observed in the parental strain E. tarda CK41 grown in low-salt condition. Two putative genes coding flagellin were identified in the E. tarda genome sequences. The amino acid sequence comparison of each gene revealed 93% identities. A flagellin gene was PCR amplified and cloned into a cloning vector. Using an E. coli protein expression system, a part of flagellin protein was overexpressed. Using the purified protein, an anti-flagellin antibody was raised in the rabbit. Immunoblot analyses with flagellin specific antibody demonstrated that E. tarda CK41 expressed falgellin in low salt conditions, which is consistent with the results seen in motility assay and microscopic observation. This is the first report of salt regulated flagella expression in E. tarda.

• The Korean Journal of Mycology
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• v.39 no.1
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• pp.39-43
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• 2011

A new cultivar "Dan Bi" of Pleurotus eryngii was developed by the method of mono-mono crossing between monokaryotic strains derived from KNR2312 and KNR2596. The parental strains, KNR2312 and KNR2596, are characterized by the property of high quality and a small number of primordia formation, respectively. The optimum temperature of mycelial growth was 25 and that of fruiting body development was $15{\sim}16^{\circ}C$. The period of harvesting including primordia formation was 0.7~1.3 days later than that of control strain Knneutari No. 3 in the culling cultivation. The color of pileus and stipe surface was neutral-brown and pure white, respectively. The shape of pileus was dome and has a scale like as cobweb. The yield was $93{\pm}9.7$ g per 850 cc of plastic bottle. Analysis of the genetic characteristics of the new commercial variety "Dan Bi" showed a different profile as that of the control strain, Knneutari No. 3, when RAPD (Random Amplified Polymorphic DNA) primer #8005 was used. This new variety "Dan Bi" of Pleurotus eryngii is characterized by a small number of primordia formation after scratching.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.4
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• pp.576-584
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• 2017

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ${\beta}-defensin-2$ (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.114-120
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• 2002

A 1,989-bp genomic region encoding nickel resistance genes was isolated from Legionella pneumophila, a pathogen for legionellosis. From a sequencing and computer analysis, the region was found to harbor two structural genes, a nreB-like protein gene (1,149 bp) and a nreA-like protein gene (270 bp), in a row. Both genes exhibited a significant degree of similarity to the corresponding genes from Synechocystis sp. PCC6803 ($54\%$ amino acid sequence identity) and Achromobacter xylosoxidans 31A ($76\%$). The gene was successfully expressed in E. coli MG1655 and conferred a nickel resistance of up to 5 mM in an LB medium and 3 mM in a TMS medium including gluconate as the sole carbon source. E. coli harboring the nickel resistance gene also exhibited a substantial resistance to cobalt, yet no resistance to cadmium or zinc. Since the extracellular concentration of nickel remained constant during the whole period of cultivation, it was confirmed that the nickel resistance was provided by an efflux system like the $Ni^2＋$permease (nrsD) of Synechocystis sp. strain PCC6803. Since polyphosphate (poly-P) is known as a global regulator for gene expression as well as a potential virulence factor in E. coli, the nickel resistance of a ppk mutant of E. coli MG 1655 harboring the nickel resistance gene from L. pneumophila was compared with that of its parental strain. The nickel resistance was significantly attenuated by ppk inactivation, which was more pronounced in an LB medium than in a TMS medium.

• Korean Chemical Engineering Research
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• v.55 no.3
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• pp.363-368
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• 2017

Methylotrophy is able to use reduced one-carbon compound, such as methanol and methylamine, as a sole carbon source. Methylobacterium extorquens AM1 is the most extensively studied methylotroph utilizing serine-isocitrate lyase cycle. Because the Poly 3-hydroxybutyrate (PHB) synthesis pathway in M. extorquens AM1 is likely to interlink with EMCP (ethylmalonyl-CoA pathway), glyoxylate, and TCA cycles, regulation of PHB production is needed to produce EMCP-derived acid or TCA acids. To adjust carbon flux to PHB production, PhaR, which seems to have function of regulator of PHB synthesis and acetyl-CoA flux, was knocked out in M. extorquens AM1 by using markerless gene deletion methods. As a result, PHB granules were remarkably reduced in the knockout strain ${\Delta}phaR$ compared to parental strain. Although lag phase was extended for 12h, ${\Delta}phaR$ showed similar cell growth and methanol consumption rate compared to wild type.

• Journal of Mushroom
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• v.16 no.3
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• pp.198-202
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• 2018

'Yong-A' was bred by Jeollanamdo Agricultural Research and Extension Services in 2015. It was primarily selected through monospore crossing between JNM21008 and JNM21013 in 2009. The strain is named as JNM-Mi-194 and 'Yong-A', showing major characteristics with good production yield. The cultivation characteristics, yield potential, and fruiting body characteristics of 'Yong-A' are as follows; The wrinkle of the fruiting body was of branching type its shape was wave type. MCM, YM, Malt, and PDA media were suitable for growth of this cultivar. The number of effective stipes was 13 ea/0.9 kg. The minor axis of pileus was 5 cm and the major axis was 9 cm. The yield was 291 g per plastic bag (0.9 kg). Yong-A required 60 days for mycelial running at $20^{\circ}C$. The growth and primordial period required 24 days, which was shorter than that of JNM-Mi-21002 (the conventional cultivar). Somatic incompatibility was observed between parental lines and Yong-A. Analysis of genetic diversity in the new variety 'Yong-A' showed a different profile compared to that of the parent strain when Random Amplified Polymorphic DNA primers were used.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.142-149
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• 1988

This experiment was carried out to obtain a hybrid with potent ethanol fermenting ability, by means of protoplast fusion between a thermophilic strain (D-71) of Saccharomyces cerevisiae and an osmotolerant strain (SR-S) of Zygosaccharomyces rouxii. The conditions for formation of protoplasts from both strains and for their fusion and regeneration were studied. Favorable conditions for formation of protoplasts from Saccharomyces cerevisiae D-71 were : treatment of the cells at late-exponential phase with 2-mercaptoethanol (l% v/v) for 10 minutes in the presence of 0.5M sorbitol, then incubation for 60 minutes in the set medium containing Zymolyase-20T (4mg/$m\ell$) ; and from Zygosaccharomyces rouxii SR-S were : treatment of the cells at mid-exponential phase with 2-mercaptoethanol (1% v/v) for 10 minutes in the presence of 0.5M or 1M mannitol, then incubation for 120 minutes in the set medium containing Zymolyase-20T(4mg/$m\ell$). The protoplasts of parental cells were fused in the presence of 20mM CaCl$_2$, 0.5M sorbitol and 40% of polyethyleneglycol (M.W 4000), then fusants obtained were selected as regenerated colonies which embedded and grown in the minimal medium containing 3% of agar. The frequencies of fusant formation were 1.2$\times$10$^{-6}$ to 9.1$\times$10$^{-6}$ for the regenerated protoplast.

• Journal of Mushroom
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• v.15 no.3
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• pp.129-133
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• 2017

Oyster mushroom is a type of mushroom that is commonly cultivated and consumed in Korea. P. ostreatus 'Suhan' is a preferred cultivar for many mushroom farmers because it has a dark pileus and thick stipe. However, as it is very sensitive to environmental conditions, farmers consistently demand an alternative cultivar. To develop a new cultivar, the parental strains KMCC01680 ('Suhan') and KMCC00478 ('Gosol') were selected from various collected P. ostreatus strains by cultivating genetic resources. P. ostreatus 'Heuksol' was developed by the method of Mon-Mon crossing between monokaryotic strains derived from 'Suhan' and 'Gosol'. Thirty strains of 174 crossed strains were initially selected by cultivation experiments. After bulk cultivation tests, 'Heuksol' was selected. The nuclear DNA profile of 'Heuksol' was similar to those of the parental strains, 'Suhan' and 'Gosol', when RAPD (random amplified polymorphic DNA) primers and UPF (Universal PCR Fingerprinting) 2, 3, and 4 were used. The optimum temperature for mycelial growth was $30^{\circ}C$ for 'Heuksol', but medium-high temperatures were also appropriate, especially $13-20^{\circ}C$. The fruiting body production per bottle (1,100 mL) was approximately 140.8 g. When compared to the control strain 'Suhan', the thickness of the stipe of 'Heuksol' was greater than that of 'Suhan' (13.5 mm vs 9.4 mm). The pileus diameter of 'Heuksol' was similar to that of 'Suhan' and the pileus thickness of 'Heuksol' and 'Suhan' was 19.7 mm and 21.8 mm, respectively. 'Heuksol' had more a productive stipe number than 'Suhan' and the pileus of 'Heuksol' was dark gray, even at high temperatures. Therefore, it was suggested that this new cultivar, 'Heuksol', could provide an alternative to 'Suhan' and contribute to the profit of oyster mushroom farms.

• Journal of Mushroom
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• v.11 no.3
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• pp.154-158
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• 2013

To develop a new variety of oyster mushroom(Pleurotus ostreatus), parental strains was selected by the method of Mon-Mon crossing between monokaryotic strains derived from ASI 2596(Suhan No.3) and ASI 2782(Black pileus mutant). The SB-73(ASI 2596-11 x 2782-8) was shown the best cultural characteristics, selected to be a new variety and named as 'Dagul'. The 'Dagul' was formed incompatibility line distinctly in the confrontation growth of parental strains Suhan No.3 and ASI 2782. The optimum temperature for mycelial growth, fruiting body development and pH arrange were $25{\sim}30^{\circ}C$, $14{\sim}17^{\circ}C$ and pH5~8, respectively. Fruiting body production per bottle was about $68.0{\pm}24.1$ g which is almost 115% quantity compared to that of other variety Suhan No.3. And also the stipe is long and individual generation is multiple. Analysis of the genetic characteristics of the new variety 'Dagul' showed different DNA bands as that of the control strains, Suhan No.3 and ASI 2782, when RAPD(Random Amplified Polymorphic DNA) primers URP7 and Rcb1 were used. This new variety 'Dagul' of oyster mushroom is characterized by multiple of individual generation and the stipe is long. We therefore expect that this new strain will increase of the income by cultivation of field.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.143-151
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• 1997

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.