• 대한미생물학회지
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• 제17권1호
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• pp.35-41
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• 1982

Enterotoxigenk E. coli is one of the major causative agents of the infantile diarrhea and traveler's diarrhea. The heat-labile enterotoxin is thought to be a virulence factor in the pathogenesis of the diarrhea and to be a marker for identification of the enterotoxigenic E. coli from non pathogenic E. coli. Therefore knowledge about the heat-labile enterotoxin is essential not only for understanding the pathogenesis but also for the diagnosis of the diarrhea. However the in-vitro heat-labile enterotoxin production is reported to be greatly affected by the cultural condition. In this regards, this study was designed to know the optimal conditions for the production of the heat-labile enterotoxin by assaying the permeability factor in the 18 hours culture supernatant of E. coli 08K25(B2) H9 and of E. coli 015 H11. Results obtained were summerized as follows: 1. Amounts of heat-labile enterotoxin produced were greater at initial pH 8.5 than at 7.0 of CYES-2 broth culture. However, the bacterial growth itself was more abundant at 7.0 than at 8.5. 2. Heat-labile enterotoxin per unit volume of culture supernatant was greater at shaking culture than at standing culture condition, but ratio of the enterotoxin produced over the unit mass of E. coli calculated was greater at standing culture than shaking culture condition, indicating that the greater yields of the toxin produced at shaking culture was due to increase in E. coli cell mass compared to the standing culture condition: 3. The enterotoxin produced in the lincomycin(128 microgram/ml) supplemented media was 5 or 11 times greater on the basis of enterotoxin per unit mass of E. coli, compared to the lincomycin-non-supplemented media, indicating that lincomycin itself increases the enterotoxin production. 4. Treatment of 18 hours culture of E. coli with polymyxin B(0.2 mg/ml) for 1 hour increased the yields of enterotoxin amounting to 2 or 5 times of the non-treated control cultures.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.314-319
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• 2008

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

• Pediatric Infection and Vaccine
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• 제17권2호
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• pp.67-73
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• 2010

목적:소아 UTI에서 non-E. coli UTI 의 빈도를 확인하고 원인균주별(E. coli vs. non-E. coli) 임상양상과 항생제 내성률을 비교하고자 하였다. 방 법: 2008년 9월부터 2009년 8월까지 UTI 입원 환아 중에서 원인균주가 배양 증명된 환아들을 대상으로 하였다. 인구학적 특징, 임상양상, 검사소견, 원인균주와 항생제 내성률을 전향적으로 조사하였다. 결과:전체 84명 중 E. coli UTI 은 63명(75.0%)이었고 non-E. coli UTI은 21명(25.0%)이었다. 성별 연령군에 따라서 non-E. coli의 분포는 차이를 보였다 : 6개월 미만 남아군, 10.5%; 6개월 이상 남아군, 50.0%; 6개월 미만 여아군, 43.7%; 6개월 이상 여아군, 25.0% (P =0.014). E. coli UTI군에 비해, non-E. coli UTI 군에는 입원 전 항생제를 치료받은 환아의 비율이 높았고(P=0.017), 혈뇨가 동반된 환아의 비율은 낮았다(P=0.014). E. coli 보다 non-E. coli는 cefazolin과 cefotaxime, imipenem, TMP/SMX, tetracycline에 대해 높은 내성률을 보였다(P<0.05). 결론:소아 UTI 치료과정에서, 증가 추세의 non-E. coli UTI 빈도와 성별 연령군별 원인균주의 차이, E. coli 보다 높은 non-E. coli의 항생제 내성률은 고려되어야 할 사항이다.

• 한국미생물·생명공학회지
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• 제29권3호
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• pp.169-175
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• 2001

E coli O157:H7은 식품과 물에 의해서 전염되는 식중독균으로서 이들의 생성하는 vero toxin 이 장관의 상피세포내로 침입되어 질병을 유발시키게 된다. 또한 E coli O157:H7 이 체내에 감염되기 위해서는 유산균과 마찬가지로 장상피세포에 정착하여야 한다. 따라서 본 연구에서는 비피더스균이 E. coli O157:H7의 생육 및 정착에 대해서 억제효과가 있는지를 파악하기 위해서 비피더스균과 E. coli O157:H7을 혼합 배양했을 때의 억제 효과 비피더스균의 Caco-2 세포 정착에 따른 억제 효과 등을 조사하였다. B infantis K9 균주와 E. coli O157:H7을 단독 또는 혼합배양하면서 배양시간에따른 pH 균수 암모니아 함량을 측정하였다. 그결과 배양시간이 결과되어 B infantis K9 균주에 의해서 산성물질이 생성되면서 E. coli O157:H7의 균수는 급격히 감소되는것으로 나타났다. 한편 암모니아 함량은 E. coli O157:H7 단독배양 시료에 비해서 혼합배양 시료에서 8시간 이후부터 감소되는 것으로 볼때 비피더스 균 E. coli O157:H7이 생성하는 암모니아를 이용하는것으로 확인되었다.B infantis K9 균주와 E. coli O.157:H7 P4 균주를 정착시기를 달리하여 Caco-2 세포에 혼합 정착시켰을 때 B. onfantis K9 균준느 정착시기에 상관없이 유사한 정착율을 나타냈다. 반면에 B infantis K9 균주가 2시간 전에 미리 정착되어 있을 때에는 E. coli O157: HP P4 균주의 정착율이 2.6%에서 1.86%로 감소되는 경향을 보였다. 결론적으로 비피더스균의 E coli O157:H7에 대한 생육 및 정착 억제를 검토한 결과 생육억제효과는 비피더스균에 의해서 생성된 산성물질 에 의해서 pH4.40 이하 일때 저해 효과를 보였으며 비피더스균과 E. coli O157:H7 이 정착 부위를 동일하게 이용하지만 비피더스균이 우선적으로 정착이 되었을 때 E. coli O157:H7의 정착율이 저하되는 결과를 나타냈다.

• 한국미생물·생명공학회지
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• 제27권5호
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• pp.419-425
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• 1999

Prophylactic effects of Bifidobacterium longum HY8001, Korean isolate, against Escherichia coli O157:H7 and Salmonella typhimurium DT104 enteric infection were examined at four groups of specific pathogen free(SPF)-ICR mouse for each pathogen. B. longum HY8001+B. typhimurium DT104+B. longum HY8001(BL+ST+BL) group and B. longum HY8001+E. coli O157:H7+B. longum HY8001(BL+E+BL) group were fed with B. longum HY8001 before and after E. coli O157:H7 or s. typhimurium DT104 challenge, while B. longum HY8001+S. typhimurium DT104(BL+ST) and B. longum HY8001+e. coli O157:H7(BL+E) groups were fed with B. longum HY8001 only before E. coli O157:H7 or S. typhimurium DT104 challenge. E. coli O157:H7(E) and S. typhimurium DT104(ST) groups were challenged with each pathogen without B. longum HY8001 administration and control groups were administered with phosphate buffered solution(PBS). After the oral administration with B. longum HY8001(109cfu), th emice were challenged with E. coli O157:H7(2$\times$1010cfu) or S. typhimurium DT104(108cfu) and the mortality rate and the fecal shedding of challenged pathogen were also examined define the reactivity of the B. longum HY8001. Production of toxin neutralizing substance(s) of B. longum HY8001 was determined by cell cytotoxicity assay using Vero cells. Fecal shedding of th eS. typhimurium DT104 was significantly decreased in BL+ST+BL group fed with B. longum HY8--1 before and after challenge(p<0.05), while the fecal shedding s of S. typhimurium DT104 in BL+ST and St groups remained more than 106cfu. the protective effect of the B. longum HY8001 against E. coli O157:H7 was significantly high only in BL+E+BL group fed with b. longum Hy8001 before and after E. coli O157:H7 challenge from the result of fecal E. coli O157:H7 isolation rate, mortality rate, and intestinal contents culture to detect E. coli O157:H7. the mortality rate of the BL+e and E groups. The cytopathic effect (CPE) of the Vero cytotoxin (Shiga like toxin I & II) in Vero cell was neutralized in B. longum HY8001 culture supernatant added wells which indicate the presence of soluble Vero cytotxin neutralizing substance(s) in B. longum HY8001 culture suprnatant.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1422-1428
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• 2006

An ABC transporter apparatus of the Gram-negative bacterial type I secretion pathway can be used as a secretory protein expression system in Escherichia coli. Four types of coexpression systems for the Pseudomonas fluorescens lipase gene, tliA, and its cognate ABC transporter gene cluster, tliDEF, were constructed. When the relative expression levels were changed by adding different concentrations of IPTG, the secretion (16.9 U/ml of culture) of TliA in E. coli [pTliDEFA-223+pACYC184] was significantly higher than E. coli [pKK223-3+pTliDEFA-184] secreting the lowest level of TliA (5.2 U/ml of culture). Maximal accumulation of the lipase secreted occurred in the mid-exponential phase, implying that the efficient protein secretion via an ABC transporter was restricted only to actively growing cells. Finally, the secretion level of TliA in E. coli [pTliDEFA-223+pACYC184] was increased to 26.4 U/ml by inducing gene expression at the culture initiation time. These results indicate that a significant increase in the ABC transporter-dependent protein secretion can be achieved by simply controlling the relative expression levels between the ABC transporter and its passenger protein, even in the recombinant E. coli cells.

• BMB Reports
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• 제30권1호
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• pp.80-84
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• 1997

The synthesis and secretion of human angiogenin in E. coli by the natural leader sequence has been studied. We constructed a recombinant plasmid containing human angiogenin cDNA which encompassed all the coding region including leader sequence required for secretion. The recombinant plasmid was introduced into a suitable E. coli host. The angiogenin was detected in the culture medium and periplasm upon the induction of gene expression. The molecular weight of the secreted angiogenin was identical to that of authentic angiogenin purfied from human plasma when estimated by SDS-PAGE and immunoblotting. showing that the natural leader sequence was recognized and processed by the secretion machinery of E. coli. The angiogenin concentration in the culture medium reached a maximum within 2 h when expressed at $37^{\circ}C$ with 0.02~2 mM IPTG. In contrast, the expression level increased gradually over time up to 11 h at $23^{\circ}C$ with 0.002~2 mM IPTG and at $37^{\circ}C$ with 0.002 mM IPTG.

• 한국미생물·생명공학회지
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• 제26권1호
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• pp.61-67
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• 1998

재조합 인간상피세포 성장인자(rhEGF)가 E. coli BL21(pYHB101) 균주를 사용하여 발현되었다. 10g/L glucose를 첨가한 변형된 MBL 배지를 사용하여 10 $\mu\textrm{m}$ IPTG/lactose로 2시간 동안 유도배양한 후 27$^{\circ}C$에서 48시간 동안 배양하였을 때 44.5 mg/L의 rhEGF가 발현되었다. 상기의 결과는 E. coli BL2l(pYHB101)를 사용하여 rhEGF를 발현시 lactose를 IPTG와 동일한 유도 물질로 사용 가능하다는 것을 시사하는 것이다. 회분식 배양에서 glucose를 10 g/L 첨가한 변형된 MBL 배지에 유도물질로 10 $\mu\textrm{m}$ lactose를 사용하였으며 28시간 동안 배양하였을 때 최대 45 mg/L의 rhEGF가 발현되었다. 유가식 배양에서 정지기에 0.5%(w/v) lactose와 0.25%(w/v) yeast extract를 첨가하였을 때 160mg/L의 rhEGF가 발현되었으며 94.3%가 분비되었다. 이에 비하여 유도기에 lactose를 첨가한 경우는 120 mg/L의 rhEGF가 발현되었으며 cytoplasm으로 발현된 불용성 봉입체의 비율은 20.9%에 달하였다. 이것은 lactose의 첨가시기가 E. coli BL2l(pYHB101)로부터 soluble rhEGF의 생성에 중요하다는 것을 확인한 결과이다.

• 미생물학회지
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• 제11권4호
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• pp.175-180
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• 1973

This study was attempted to see more clear relationships among the enterobacteria, especially between the intestinal normal flora and pathogenic bacteria. It has been known that some intestinal normal flora produce the bactrial metabolites that are harmful to other enteric bacteria. One of the metabolites is known as colicin, the protein fraction, which possesses certain degree of inhibitory effect against other bacterial growth fraction, whih possesses certain degree of inhibitory effect against other bacterial growth. As a preliminary study for a colicin purification, the antagonistic effect of E, coli to groups of Salmonella and Shigella has been studied by means of in vitro quantitative culture method. 1. E.coli showed definite inhibitory effects aganist both Salmonella and Shigella groups in the mixture of two organisms. 2. The inhibitory effects of E.coli in the E.coli-Salmonella and the E.coli-Shigella mixture occurred from 4 hours incubation following the inoculation. 3. Even the complete inhibition of pathogenic enteric bacterial growth was noticed in the E.coli-Salmonella mixture at overnight incubation. 4. Among the diluted mixtures, 1:100, 1:1,000, and 1:10,000, survival rate of pathogenic enteric bacteria in the mixtures with E.coli showed least affected at the 1:1,000 dilution. 5. It was found that the antagonistic effect aganist groups of Salmonella-shigella was depending upon the groups of the genera.