• Title/Summary/Keyword: coli B

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In Vivo Analysis of fadB Homologous Enzymes Involved in Biosynthesis of Polyhydroxyalkanoates in Recombinant Escherichia coli (재조합 대장균에서 fadB 유사효소의 Polyhydroxyalkanoates 합성에 미치는 역할의 규명)

  • 최종일;박시재;이상엽
    • KSBB Journal
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    • v.19 no.4
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    • pp.331-334
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    • 2004
  • In vivo characterization of FadB homologous enzymes including PaaG, YdbU and YgfG for medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis was carried out in fadB mutant Escherichia coli. Previously, it was reported that amplification of FadB homologous enzymes such as PaaG and YdbU in fadB mutant E. coli resulted in enhanced biosynthesis of MCL-PHA by greater than two fold compared with control strain. In this study, we constructed paaG fadB double mutant E. coli WB114 and ydbU fadB double mutant E. coli WB115 to investigate the roles of PaaG and YdbU in biosynthesis of MCL-PHA. Inactivation of paaG and ydbU genes in fadB mutant E. coli harboring Pseudomonas sp. 61-3 phaC2 gene reduced the MCL-PHA production to 0.16 and 0.16 PHA g/L, respectively from 2 g/L of sodium decanoate, which are much lower than 0.43 PHA g/L obtained with fadB mutant E. coli WB101 harboring the phaC2 gene. Also, we identified new FadB homologous enzyme YgfG, and examined its roles by overexpression of ygfG and construction of ygfG fadB double mutant E. coli WB113.

Prophylactic Effects of Bifidobacterium longum HY8001 against Escherichia coli O157:H7 and Salmonella typhimurium DT104 Enteric Infection and Evaluation of Vero Cytotxin Neutralizing Effects (Bifidobacterium longum HY8001 균주의 Escherichia coli O157:H7과 Salmonella typhimurium DT104 장곤 내 감염 예방효과 및 Vero cytotoxin 중화효과)

  • 양수진;윤장원;서근석;구혜정;김소현;배형석;백영진;박용호
    • Microbiology and Biotechnology Letters
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    • v.27 no.5
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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.

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A gene responsible for ozone sensitivity (ozrB) in chromosome of escherichia coli B. MQ 1844 (E. coli B. MQ1844 균주의 오존감수성 유전자의 특성)

  • ;Hamelin C.
    • Korean Journal of Microbiology
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    • v.25 no.2
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    • pp.103-109
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    • 1987
  • An ozone-sensitive mutant of Escherichia coli strain B, MQ 1844 is described. Its properties, including high sensitivity to ozone and radiation, inducible filamentation, extensive DNA degradation and impaired DNA synthesis following ozonation, are attributable to a mutation in ozrB, a gene which is cotransducible with malB. Based on differences in phenotypic expression as well as on the particular location of this gene on the bacterial chromosome, ozrB appears as distinct from the other ozone-or radiation-sensitivity genes previously described.

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Optimized Culture Conditions for Production of the chimaeric protein, Uropathogenic Escherichia coli Adhesin - Cholera Toxin A2B Subunits, in Escherichia coli TB1

  • Lee, Yong-Hwa;Kim, Byung-Oh;Rhee, Dong-Kwon;Pyo, Suh-Kneung
    • Biomolecules & Therapeutics
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    • v.12 no.3
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    • pp.179-184
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    • 2004
  • The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause for urinary tract infections. In our previous study, the Adhesin/CTXA2B was expressed as soluble recombinant chimaeric protein derived from the uropathogenic Escherichia coli adhesin genetically coupled to cholera toxin A2B (CTXA2B) subunit in Escherichia coli. Since it is very important to optimize IPTG concentration and culture temperature to maximize cell growth and productivity, These optimal culture factors were determined to increase the productivity of the expressed Adhesin/CTXA2B chimaeric protein in Escherichia coli TB1 carrying pMALfimH/ctxa2b. Our data demonstrate that optimal concentration of IPTG for increased production of chimaeric protein was 0.5 mM. Additionally, culture time was 10 hours and temperature, 37${\circ}C$.

Cloning and Expression of the dapD Gene from Brevibacterium lactofermentum in E. coli (Brevibacterium lactofermentum의 dapD 유전자의 Cloning 및 E. coli에서의 발현)

  • 김옥미;박선희;박혜경;이승언;하대중;이갑랑
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.30 no.5
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    • pp.802-805
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    • 2001
  • The dapD gene of Brevibacterium lactofermentum encoding tetrahydrodipicolinate N-succinyl transferase, one of the enzymes involved in lysine biosynthesis, was cloned by complementation of Escherichia coli dapD mutnat. The recombinant plasmid pLS1 was found to contain a 3.6 kb DNA fragment. Southern hybridization analysis confirmed that the cloned DNA fragment originated from B. lactofermentum. The data of L-lysine production showed that the B. lactofermentum dapD gene was expressed in E. coli.

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Optimal culture conditions for production of Escherichia coli Adhesin protein coupled to Escherichia coli Heat Labile Enterotoxin A2B in Escherichia coli TB1.

  • Lee, Yong-Hwa;Rhee, Dong-Kwon;Pyo, Suhk-Neung
    • Proceedings of the PSK Conference
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    • 2003.04a
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    • pp.226.2-226.2
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    • 2003
  • The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically linked to the Itxa2b gene, which was then cloned into the pMAL -p2E expression vector. The chimaeric construction of pMALfimH/Itxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)

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Construction of Candida antarctica Lipase B Expression System in E. coli Coexpressing Chaperones (대장균에서의 Chaperone 동시 발현을 통한 Candida antarctica Lipase B 발현 시스템 구축)

  • Jung, Sang-Min;Lim, Ae-Kyung;Park, Kyung-Moon
    • KSBB Journal
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    • v.23 no.5
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    • pp.403-407
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    • 2008
  • Recently, Candida antarctica lipase B (CalB) draws attention from industries for various applications for food, detergent, fine chemical, and biodiesel, because of its characteristics as an efficient biocatalyst. Since many industrial processes carry out in organic solvent and at high temperature, CalB, which is stable under harsh condition, is in demand from many industries. In order to reform CalB promptly, the expression system which has advantages of ease to use and low cost for gene libraries screening was developed using E. coli. The E. coli strains, Rosettagami with competence for enhanced disulfide bond formation, Novablue, and $DH5{\alpha}$, were exploited in this study. To obtain the soluble CalB, the pCold I vector expressing the cloned gene at $15^{\circ}C$ and the chaperone plasmids containing groES/groEL, groES/groEL/tig, tig, dnaK/dnaJ/grpE, and dnaK/dnaJ/grpE/groES/groEL were used for coexpression of CalB and chaperones. The colonies expressing functional lipase were selected by employing the halo plate containing 1% tributyrin, and the CalB expression was confirmed by SDS-PAGE. E. coli Rosettagami and $DH5{\alpha}$ harbouring groES/groEL chaperones were able to express soluble CalB effectively. From a facilitative point of view, E. coli $DH5{\alpha}$ is more suitable for further mutation study.

Expression and Characterization of Uropathogenic Escherichia coli Adhesin Protein Linked to Cholera Toxin A2B Subunits in Escherichia coli TB1

  • Lee, Yong-Hwa;Ryu, Dong-Kyun;Kim, Byung-Oh;Pyo, Suhk-Neung
    • Journal of Microbiology and Biotechnology
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    • v.13 no.4
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    • pp.552-559
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    • 2003
  • The FimH subunit of type 1-fimbriated Escherichiu coli (E. coli) has been determined as a major cause for urinary tract infections. Thus, to produce a possible vaccine antigen against urinary tract infections, the fimIH gene was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. A fusion protein, based on fusing adhesin to the cholera toxin subunit A2B (CTXA2B), was induced with 0.01 mM isopropyl-${\beta}-D-thiogalactoside$ (IPTG) for 4 h at $37^{\circ}C$ to yield a soluble fusion protein. The fusion protein was then purified by affinity chromatography. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was also analyzed. The orderly-assembled fusion protein was confirmed by a modified $G_{Ml}-ganglioside$ ELISA, using antibodies to adhesin. The results indicated that the purified fusion protein was an adhesin/CTXA2B protein containing E. coli adhesin and the $G_{Ml}-ganglioside$ binding activity of CTXB. Accordingly, this adhesin/CTXA2B protein may be a potential antigen for oral immunization against uropathogenic E. coli.

Molecular Cloning of a Thermostable $\alpha$-Amylase Gene from Bacillus stearothermophilus and Its Expressions in E. coli (Bacillus stearothermophilus의 열안정성 $\alpha$-amylase 유전자의 E. coli내에서의 cloning과 발현)

  • Huh, Tae-Lin;Koh, Suk-Hoon;Lee, Se-Yong
    • Microbiology and Biotechnology Letters
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    • v.13 no.4
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    • pp.349-354
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    • 1985
  • A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75% of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.

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Phylogenetic Groups and Virulence Factors of Escherichia coli Causing Urinary Tract Infection in Children (소아 요로감염의 원인 Escherichia coli 균의 계통 분류와 독성인자 분석)

  • Kim, Ji Mok;Cho, Eun Young;Lee, Jae Ho
    • Pediatric Infection and Vaccine
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    • v.22 no.3
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    • pp.194-200
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    • 2015
  • Purpose: Urinary tract infection (UTI) is a common bacterial infection in children and Escherichia coli is a predominant pathogen. The purpose of this study is to evaluate phylogenetic groups and virulence factors of E. coli causing UTI in children in Korea. Methods: From October 2010 to April 2013, urinary E. coli strains were isolated from the 33 pediatric patients of UTI. Multiplex polymerase chain reactions were performed to evaluate the phylogenetic groups and 5 virulence factor genes (fimH, sfa, papA, hylA, and cnf1) of E. coli. Distribution of molecular characteristics of E. coli was analyzed by clinical diagnosis and accompanying vesicoureteral reflux (VUR). Results: Most (84.8%) uropathogenic E. coli were belonged to phylogenetics group B2 and the others (15.2%) were belonged to group D. The virulence factors were distributed as: fimH (100%), sfa (100%), hylA (63.6%), cnfI (63.6%), and papA (36.4%). According to clinical diagnosis, phylogenetic distribution of E. coli strain was 92.3% of B2 and 7.7% of D in acute pyelonephritis and 57.1% of B2 and 42.9% of D in cystitis. Distribution of virulence factors was similar in both groups. In patients with acute pyelonephritis, phylogenetic distribution was similar in VUR and non-VUR group, but proportion of papA genes were lower in VUR group than that of non-VUR group (43.8% vs. 20.0%, P=0.399). Conclusions: This study provides current epidemiologic molecular data of E. coli causing pediatric UTI in Korea and will be a fundamental for understanding the pathogenesis of pediatric UTI.