• 대한미생물학회지
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• 제35권2호
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• pp.109-115
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• 2000

FtsH is a membrane-bound, ATP-dependent metalloprotease that is involved in a variety of cellular functions including the regulation of responses to heat and stress shock. Previously, we had cloned and sequenced pneumococcal ftsH gene whose deduced amino acid sequence was very similar to those of several gram-positive bacteria and Escherichia coli, except for the N-terminal domain that was responsible for membrane anchoring. In order to better understand the role of Streptococcus pneumoniae FtsH, we expressed pneumococcal ftsH gene in Escherichia coli. When it was expressed from a strong promoter, $P_{tac}$, a considerable amount of the recombinant FtsH was produced, although the prolonged induction resulted in not only accumulation of breakdown products but also ceasing of the further growth of E. coli host. This indicated that the expression of the exogenous ftsH gene was tightly regulated since the excessive FtsH appeared detrimental to bacterial cells. In Western blotting, the pneumococcal FtsH protein, whether native or recombinant, was reactive to anti-E. coli FtsH serum. The observation that FtsH proteins were well conserved throughout the bacterial kingdom and its expression level was fine-tuned suggests an important role for this protein in the stress adaptation which may be related to infecting process by pneumococci.

• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.39-47
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• 2009

Gaegurin 4(GGN 4), an antimicrobial peptide isolated from a Korean frog, is five times more potent against Gram-positive than Gram-negative bacteria, but has little hemolytic activity. To understand the mechanism of such cell selectivity, we examined GGN4-induced $K^+$ efflux from target cells, and membrane conductances in planar lipid bilayers. The $K^+$ efflux from Gram-positive M. luteus(2.5 ${\mu}g/ml$) was faster and larger than that from Gram-negative E. coli(75 ${\mu}g/ml$), while that from RBC was negligible even at higher concentration(100 ${\mu}g/ml$). GGN4 induced larger conductances in the planar bilayers which were formed with lipids extracted from Gram-positive B. subtilis than in those from E. coli(p<0.01), however, the effects of GGN4 were not selective in the bilayers formed with lipids from E. coli and red blood cells. Addition of an acidic phospholipid, phosphatidylserine to planar bilayers increased the GGN4-induced membrane conductance(p<0.05), but addition of phosphatidylcholine or cholesterol reduced it(p<0.05). Transmission electron microscopy revealed that GGN4 induced pore-like damages in M. luteus and dis-layering damages on the outer wall of E. coli. Taken together, the present results indicate that the selectivity of GGN4 toward Gram-positive over Gram-negative bacteria is due to negative surface charges, and interaction of GGN4 with outer walls. The selectivity toward bacteria over RBC is due to the presence of phosphatidylcholine and cholesterol, and the trans-bilayer lipid asymmetry in RBC. The results suggest that design of selective antimicrobial peptides should be based on the composition and topology of membrane lipids in the target cells.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.209-212
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• 1985

산소 투과율이 좋은 silicone tube안쪽에 nutrient 공급을 위한 3개의 isotropic polypropylene hollow fiber 3개를 넣어 제작된 이중 실관 반응기에서 E. coli cell을 immobilization 하여 cell density와 packing characteristics를 조사해 보았다. E. coli cell들은 거의 100% Packing되어 동물조직에서 처럼 층을 이루면서 자랐고 ceil density를 측정해 본 결과 약 550g/$\ell$고농도 세포배양이 가능하였다.

• KSBB Journal
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• 제9권5호
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• pp.492-498
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• 1994

설관막 장치를 이용하여 대장균의 투석배양을 수 행했다. 투석액으로 배지 내의 초산을 제거함으로써 초산의 저해효과를 경감할 수 있었다. 초산 생성속도는 포도당과 용존산소농도에 대단히 민감하였고, 따라서 막을 통한 포도당의 투과속도는 산소공급속도와 균형을 유지해야 했다. 막을 통해 포도당이 천 천히 공급될 때, 대장균의 비성장속도는 포도당 투 과속도에 좌우되었고 초산의 생성은 억제되였다.

• Journal of Microbiology and Biotechnology
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• 제24권4호
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• pp.568-576
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• 2014

TIM-1 (also known as KIM-1 and HAVcr-1) is a type I transmembrane glycoprotein member of the TIM family that may play important roles in innate and adaptive immune responses. The overexpression of proteins associated with membrane proteins is a major obstacle to overcome in studies of membrane protein structures and functions. In this study, we successfully coupled the overexpression of the TIM-1 protein with a C-terminal enhanced green fluorescent protein (GFP) tag in Escherichia coli. To the best of our knowledge, this report is the first to describe the overexpression of human TIM-1 in E. coli. The purified TIM-1-EGFP fusion protein recognized and bound directly to apoptotic cells and did not to bind to viable cells. Furthermore, we confirmed that the interactions of TIM-1-EGFP with apoptotic cells were blocked by TIM-1-Fc fusion proteins. This fusion protein represents a readily obtainable source of biologically active TIM-1 that may prove useful in future studies of human TIM-1.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.2028-2036
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• 2017

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2008년도 International Meeting of the Microbiological Society of Korea
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• pp.135-135
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• 2008

Various biosensors were evaluated for identifying and detecting foodborne pathogens in a rapid and effective manner. First, five strains of Escherichia coli and six strains of Salmonella were identified using Fourier transform infrared spectroscopy and a statistical program. For doing this, lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) were extracted from a cell wall of each bacterial strain. As a result, each strain was identifed at the level of 97% for E. coli and 100% for Salmonella. Second, E. coli O157:H7, S. Enteritidis, and Listeria monocytogenes were identified by multiplex PCR products from four specific genes of each bacteria using a capillary electrophoresis (CE). Also, ground beef for E. coli O157:H7, lettuce for S. Enteritidis, and hot dog for L. monocytogenes were used to determine the possibility of detecting pathogens in foods. Foods inoculated with respective pathogen were cultivated for six hours and multiplex PCR products were obtained and assessed. The minimum detection levels of tested bacteria were <10 cells/g, <10 cells/g, and $10^4$ cells/g for E. coli O157:H7, S. Enteritidis, and L. monocytogenes, respectively. Third, it was possible to detect S. Typhimurium in a pure culture and lettuce by a bioluminescence-based detection assay using both recombinant bacteriophage P22::luxI and a bioluminescent bioreporter. In addition, bacteriophage T4 was quantitatively monitored using E. coli including luxCDABE genes.

• 한국식품과학회지
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• 제31권2호
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• pp.368-374
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• 1999

식품공정에 있어서 비가열 살균 기술로 개발되고 있는 고전압 펄스 전기장(pulsed electric fields, PEF)을 과실주스의 품질 향상에 응용하고자 pilot-scale의 고전압 펄스 전기장 살균장치를 개발하였으며, 이것을 과실 주스의 모델로 선정한 사과주스의 대표적 오염지표균 또는 품질을 저하시키는 미생물 중 Escherichia coli, Bacillus subtilis, Rhodotorula minuta에 적용시켜 세포의 생리특성을 조사하였다. 내염성, 자외선 흡수 물질, 세포염색, 세포의 회복도, 세포의 표면구조 등을 조사함으로서 PEF의 미생물 생리특성에 대한 영향을 규명하고자 하였다. E. coli, B. subtilis, R. minuta를 40 kV/cm, 84 pulse, $10{\mu}s$ pulse duration의 PEF 조건에서 처리하여 내염성을 조사한 결과 1 log cycle 정도의 생존율 감소를 나타내었다. PEF 처리에 의한 세포 내부 성분의 유출 여부를 살펴본 자외선 흡수물질 측정결과 260 nm, 280 nm에서 모두 PEF 처리 세포가 무처리구에 비하여 현저하게 증가된 흡수물질 농도를 나타내었다. PEF 처리된 R. minuta를 세포 염색하여 관찰한 결과 세포막이 터져서 개열됨에 따라 염색시약이 세포 내, 외부로 고루 염색되어 세포 본래의 형태를 관찰할 수가 없었다. PEF 처리후 세포의 회복도는 무처리구에 비하여 E. coli와 B. subtilis가 약 5시간, R. minuta가 약 8시간 정도 지연된 lag time을 보였다. 그리고 PEF 처리후의 E. coli, B. subtilis, R. minuta는 정상적인 세포와는 달리 세포막이 상당한 손상을 입어 허물어진 상태를 관찰할 수 있었다.

• 미생물학회지
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• 제47권1호
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• pp.7-13
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• 2011

일반적으로 대장균을 숙주로 이용하여 고등생물 유래 막단백질을 발현시킬 경우 발현된 막단백질은 숙주 세포에 치명적인 독성을 보인다. 우리가 발현을 시도한 15개의 인간 막단백질 중에서 특히 퓨린수용체 $P2X_4$ 발현은 대장균에 강한 독성을 보였다. 이러한 독성의 원인을 알아보기 위해 hydroxylamine을 사용하여 하여 인간 $P2X_4$ 유전자를 돌연변이 시키고 독성이 약해진 돌연변이체를 선별하였다. 돌연변이체 단백질을 면역블랏으로 분석한 결과 야생형에 비해 모두 단백질의 크기가 작았다. 크기가 제법 큰 돌연변이 두 개를 골라 DNA 서열분석을 해보니 130번째, 또는 194번째 Trp 코돈이 종결코돈으로 바뀜으로써 두 번째 막통과 도메인이 사라진 truncated protein이라는 사실을 알았다. 이들 돌연변이체의 세포내 위치를 추적해보니 둘 다 세포막에 삽입되어 있지는 않았다. 이런 결과를 종합해 볼 때 $P2X_4$의 발현이 대장균에 독성을 보이기 위해서는 전체 단백질의 올바른 세포막 삽입이 중요함을 시사한다.

• 한국미생물·생명공학회지
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• 제28권1호
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• pp.1-7
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• 2000

The bacterial strain JW-2 which conferred resistance against paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) was isolated from soil. The strain was identified as an Ochrobactrum anthropi based on its morphological, physiological, biological and fatty acid composition, and was designated as Ochrobactrum anthropi JW-2. We compard paraquat resistance of O. anthropi JW-2 with Escherichia coli J105. In the presence of 100mM paraquat, E. coli JM105 was not grown whereas the growth rate of O. anthropi was about 70% of control. We compared the sensitivity of O. anthropi JW-2 and E. coli J105 to redox-cycling compounds such as paraquat, plumbagin or menadione, which are known to exacebate wuperoxide generation. O. anthropi JW-2 did not show cross-resistance to plumbagin or menadione. superoxide dismutase activity was increased in paraqunt-treated E. coli JM105 while it was not increased in O.anthropi JW-2. These results suggest that the mechanism of paraquat resistance in O.anthropi JW-2 is probably due to selectively decreased permeability toward paraquat by membrane protein.