• Molecules and Cells
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• 제40권12호
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• pp.935-944
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• 2017

More than 50% of sepsis cases are associated with pneumonia. Sepsis is caused by infiltration of bacteria into the blood via inflammation, which is triggered by the release of cell wall components following lysis. However, the regulatory mechanism of lysis during infection is not well defined. Mice were infected with Streptococcus pneumoniae D39 wild-type (WT) and lipase mutant (${\Delta}lipA$) intranasally (pneumonia model) or intraperitoneally (sepsis model), and survival rate and pneumococcal colonization were determined. LipA and autolysin (LytA) levels were determined by qPCR and western blotting. S. pneumoniae Spd_1447 in the D39 (type 2) strain was identified as a lipase (LipA). In the sepsis model, but not in the pneumonia model, mice infected with the ${\Delta}lipA$ displayed higher mortality rates than did the D39 WT-infected mice. Treatment of pneumococci with serum induced LipA expression at both the mRNA and protein levels. In the presence of serum, the ${\Delta}lipA$ displayed faster lysis rates and higher LytA expression than the WT, both in vitro and in vivo. These results indicate that a pneumococcal lipase (LipA) represses autolysis via inhibition of LytA in a sepsis model.

• Journal of Plant Biotechnology
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• 제38권4호
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• pp.272-277
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• 2011

Plants expressing Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) are known to be resistant to glyphosate, a potent herbicide that inhibits the activity of the endogenous plant EPSPS. In order to develop herbicide-resistant rice, we prepared transgenic rice plants with CP4 EPSPS gene under the control of CaMV 35S promoter for over-expression. A recombinant plasmid was transformed into rice via Agrobacterium-mediated transformation. A large number of transgenic rice plants were obtained with glyphosate and most of the transformants showed fertile. The integration and expression of CP4 EPSPS gene from regenerated plants was analyzed by Southern and northern blot analysis. The transgenic rice plants had CP4 EPSPS enzyme activity levels more than 15-fold higher than the wild-type plants. EPSPS enzyme activity of transgenic rice plants was also identified by strip-test method. Field trial of transgenic rice plants further confirmed that they can be selectively survived at 100% by spay of glyphosate (Roundup$^{(R)}$) at a regular dose used for conventional rice weed control.

• Plant Biotechnology Reports
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• 제5권1호
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• pp.53-60
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• 2011

Artemisinin is effective against both chloroquine-resistant and -sensitive strains of Plasmodium species. However, the low yield of artemisinin from cultivated and wild plants is a serious limitation to the commercialization of this drug. Optimization of artemisinin yield either in vivo or in vitro is therefore highly desirable. To this end, we have overexpressed the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) gene (hmgr) from Catharanthus roseus L. in Artemisia annua L. and analyzed its influence on artemisinin content. PCR and Southern blot analyses revealed that the transgenic plants showed stable integration of the foreign hmgr gene. The reverse transcriptase-PCR results suggested that the hmgr was expressed at the transcriptional level in transgenic lines of Artemisia annua L., while the high-performance liquid chromatography analysis showed that artemisinin content was significantly increased in a number of the transgenic lines. Artemisinin content in one of the A. annua transgenic lines was 38.9% higher than that in non-transgenic plants, and HMGR enzyme activity in transgenic A. annua L. was also higher than that in the non-transgenic lines.

• 한국미생물·생명공학회지
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• 제24권1호
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• pp.59-66
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• 1996

Virginiae butanolide C(VB-C) is one of the butyrolactone autoregulators, which triggers the productin of virginiamycin in Streptomyces virginiae. To further understand the mechanism of virginiamycin induction, we isolated three mutants from S. virginiae by N-methyl-N'-nitrosoguanidine (NTG) treatment. The characteristics of the three mutants were confirmed as follows: the mutant No. 1 delayed the production of the VB-C, receptor and antibiotics; the mutant No.3 hyperproduced receptor; the mutant No.4 failed to produce the VB-C. The addition of synthetic VB-C couldn't induce the production of antibiotics in the mutant No.1 due to delayed production of receptor, could provoke the production of larger amount of antibiotics than parental wild type strain in the mutant No.3 due to the presence of large amount of receptor, and could induce production of very small amount of antibiotics in the mutant No.4 due to the absence of VB-C. Antimicrobial spectrum and HPLC analysis of the mutant No.1 and No.3 suggested that the VB-C might have a specific ability to induce the production of virginiamycin M and S. These results imply that the VB-C has an ability to trigger the production of virginiamycin under receptor existence in S. virginiae.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1538-1545
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• 2007

Clavulanic acid (CA) is an inhibitor of ${\beta}$-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHLl, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHLl3, and a 23.8-fold increase with pNQ1. The integrative pNQl strain has been successfully applied to enhance production.

• The Plant Pathology Journal
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• 제18권3호
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• pp.115-120
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• 2002

Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean, induces hypersensitive response (HR) in a non-host plant, hot pepper (Capsicum annuum). A wild-type strain (8ra) and its non-patho-genic mutant (8-13) of X. axonopodis pv. glycines were inoculated into the pepper leaf tissues and their subcellular responses to the bacterial infections were examined by electron microscopy. Intrastructural changes related to HR were found in the leaf tissues infected with 8ra from 8 h after inoculation, characterized by separation of plasmalemma from the cell wall, formation of small vacuoles and vesicles, formation of cell wall apposition, and cellular necrosis. No such responses were observed in the tissues infected with the mutant. In 8ra, the bacterial cells were attached to the cell walls, with the cell wall material dissolved into and appearing to encapsulate the bacterial cells. The bacterial cells later became entirely embedded in the cell wall material. On the other hand, in 8-13, the bacterial cells were usually not attached tightly to the plant cell wall, and no or poor encapsulation of the bacteria by the wall material occurred, although these were encircled by rather loose wall materials at the later stages.

• Molecules and Cells
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• 제21권3호
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• pp.381-388
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• 2006

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

• 공업화학
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• 제23권5호
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• pp.496-500
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• 2012

Arthrospira platensis (A. platensis)는 상업적으로 중요한 사상형 미세조류이다. 화학적 혹은 물리적 돌연변이원에 의해 유도된 변이주 분리는 균주 개량에 중요하게 작용한다. 본 연구에서, A. platensis에서 자외선(UV-B)조사에 따른 영향을 살펴보았고, 그에 따른 변이주를 확보하였다. A. platensis를 자외선(15 Watt, 254 nm)을 이용하여 1, 3, 5, 10 min 동안 처리 한 후, 얻어진 변이주를 각각 UM1, UM3, UM5, UM10으로 명명하였다. 특히, UM5 변이주는 야생균주와 비교하여 지질의 함량이 8~11배 가량 크게 증가하였다. 또한, 카로티노이드 함량과 항산화 효소(peroxidase, superoxide dismutase) 활성이 증가하였다. 이 결과, 자외선에 의해 유도된 변이주는 생리활성 물질을 축적하였으며, 이러한 유용성분을 생산하는 미세조류의 균주 개발은 미세조류의 산업화를 촉진시킬 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.330-336
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• 2005

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human $\beta$-defensin-l (hBD-l), which kills both Gram-positive and Gram-negative bacteria in L. lactis AI64. When the hBD-l cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-l against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-l gene was fused to the DsbC- Tag of pET -40b(+), then introduced to L. lactis A 164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-l fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-l, the DsbC-hBD-l fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-l exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A 164 produced a biologically active hBD-I. In addition, the recombinant L. lactis A 164 was also found to produce the same level of nisin Z as the wild-type.

• 한국미생물·생명공학회지
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• 제31권1호
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• pp.36-41
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• 2003

Saccharomyces cerevisiae로부터 외래 $\alpha$-amylase의 발현 및 분비를 증진시키기 위하여 여러 실험이 수행되었다. ADC1 promoter와 mouse salivary $\alpha$-amylase cDNA gene의 native signal sequence를 효모의 PRB1 promoter와 invertase leader sequence로 대치한 plasmid vector pCNN(AMY)를 제작하였다. 효모세포에서 생성된 $\alpha$-amylase의 세포외로의 분비율은 mouse o-amylase의 native signal sequence인 경우는 약 89.4%이었으며 invertase leader sequence로 치환된 경우는 96.3%로 분비효율이 증진되었다. 야생주인 K8l/pCNN(AMY)와 호흡결여변이주인 K81/pCNN(AMY)p-의 혐기적 조건하에서의 배양 결과 $\alpha$-amylase 생산량이 K8l/pCNN(AMY)보다 K81/pCNN(AMY)p-가 약 5~8배 정도 증가하였다. $\alpha$-Amylase의 생산에 있어서 배지조성에 따른 K81/pCNN(AMY)의 생산증진의 비교는 배지성분인 yeast extract와 peptone의 구성비율을 비교하였을 때 yeast extract 1%와 peptone 2%, NaCl의 경우 100 mM, 2-mercaptoethanol인 경우에는 0.015%(w/v)을 첨가하였을 때 최대 효소 활성을 나타내었고, 특히 2-mercaptoethanol인 경우에는 대조구에 비해 효소 생산량이 약 3배 정도 증진되었다.