• 한국식품저장유통학회지
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• 제3권2호
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• pp.187-193
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• 1996

To investigate the antimicrobial activities and effect of grapefruit seed extract(GFSE) on the physiological function of microorganism, antimicrobial activity, fatty acids of bacterial cell lipid and amino acids of bacterial cell protein were measured. The change of cell morphotype was observed by transmission electron microscope. GFSE was very stable on the wide range temperture and pH. The growth rate of E. coli and B. suvtilis were decreased above 40ppm GFSE There fore, minimum inhibitory concentration (MIC) of the E. coli and B. subtilis to GFSE were determined around 40ppm. In the change of fatty acids quantities, hexadecanoate was significantly decreased on the treatment compared with control in case of E. coli, whereas tridecanoate was not detected in case of B. subtilis. In the change of amino acids quantities, alanine, glutamic acid, glycine, lysine were decreased on the treatment compared with control in case of E. coli and B. subtilis Transmission electron microsgraphs(TEM) showed the microbial cells were destroyed by GFSE.

• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.982-995
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• 2020

A putative multidrug efflux gene, yddA, was cloned from the Escherichia coli K-12 strain. A drug-sensitive strain of E. coli missing the main multidrug efflux pump AcrB was constructed as a host and the yddA gene was knocked out in wild-type (WT) and drug-sensitive E. coliΔacrB to study the yddA function. Sensitivity to different substrates of WT E.coli, E. coliΔyddA, E. coliΔacrB and E. coliΔacrBΔyddA strains was compared with minimal inhibitory concentration (MIC) assays and fluorescence tests. MIC assay and fluorescence test results showed that YddA protein was a multidrug efflux pump that exported multiple substrates. Three inhibitors, ortho-vanadate, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and reserpine, were used in fluorescence tests. Ortho-vanadate and reserpine significantly inhibited the efflux and increased accumulation of ethidium bromide and norfloxacin, while CCCP had no significant effect on YddA-regulated efflux. The results indicated that YddA relies on energy released from ATP hydrolysis to transfer the substrates and YddA is an ABC-type multidrug exporter. Functional study of unknown ATP-binding cassette (ABC) superfamily transporters in the model organism E. coli is conducive to discovering new multidrug resistance-reversal targets and providing references for studying other ABC proteins of unknown function.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5287-5290
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• 2012

Throughout human history, plant products have been used for many purposes including as medicines. Herbal products and spices can be used as preventive agents against cancer due to their antimicrobial, antioxidant and antitumorigenic properties. This study was designed to evaluate the potential protective effect of curcum in rats administered nitrosamine precursors; dibutylamine (DBA) and sodium nitrate (NaNO3); and infected with Escherichia coli (E. coli) and also to monitor changes in nuclear factor the Kappa B p65 (NF-${\kappa}B$ p56) pathway and its downstream products, Bcl-2 and interleukin-6 (IL-6), in parallel with nitrosamine precursors, E. coli and curcum treatment. Rats were divided into three groups (n=25 each; except of control group, n+20). Group I a normal control group, group II administered DBA/NaNO3 in drinking water and infected with E. coli and group III was administered DBA/NaNO3 in drinking water, infected with E. coli and receiving standard diet containing 1% curcum powder. Histopathological examination reflected that the curcum treated group featured a lower incidence of urinary bladder lesions, and lower levels of NF-${\kappa}B$, Bcl-2 and IL-6, than the group receiving nitrosamine precursor and infected with E. coli. These findings suggested that curcum may have a protective role during the process of bladder carcinogenesis by inhibiting the NF-${\kappa}B$ pathway and its downstream products.

• Journal of Microbiology and Biotechnology
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• 제12권5호
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• pp.753-759
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• 2002

$\beta$-Cyclodextrin glucanotransferase ($\beta$-CGTase) was overproduced extracellularly using recombinant E. coli by transforming the plasmid pECGT harboring a secretive signal peptide. The $\beta$-CGTase gene of alkalophilic Bacillus firmus var alkalophilus was inserted into the high expression vector pET20b(+) containing a secretive pelB signal peptide, and then transformed into E. coli BL2l(DE3)pLysS. The optimum culture conditions fer the overproduction of $\beta$-CGTase were determined to be TB medium containing 0.5% (w/v) soluble starch at post-induction temperature of $25^{\circ}C$. A significant amount of $\beta$-CGTase, up to 5.83 U/ml, which was nine times higher than that in the parent strain B. firmus var. alkalophilus, was overproduced in the extracellular compartment. A pH-stat fed-batch cultivation of the recombinant E. coli was also performed to achieve the secretive overproduction of $\beta$-CGTase at a high cell density, resulting in production of up to 21.6 U/ml of $\beta$-CGTase.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Food Science and Biotechnology
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• 제17권1호
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• pp.191-194
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• 2008

The antagonistic activity of probiotic strains (Bifidobacterium animalis BB-12, Bifidobacterium bifidum A, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus paracasei ATCC 25598, and Lactobacillus rhamnosus GG) against nalidixic acid resistant ($NA^R$) Escherichia coli O157:H7 MF1847, E. coli O157:H7 H2439, E. coli O157:H7 ATCC 43894, and E. coli O157:H7 C7927 was investigated using the agar-overlay, well diffusion, and broth culture tests. L. paracasei ATCC 25598 was the most effective probiotic strain in terms of in vitro antagonistic activity against $NA^R$ E. coli O157:H7, followed by L. rhamnosus GG, B. longum B6, and L. acidophilus ADH. The use of selected probiotic strains could be an effective pre-harvest intervention strategy to reduce the risk of $NA^R$ E. coli O157:H7 by maintaining a balanced microflora in animals and might provide many potential benefits in lieu of using antimicrobials.

• 대한의생명과학회지
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• 제10권2호
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• pp.153-161
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• 2004

Antibiotic resistance is often associated with the production of inner membrane proteins (for example, AcrAB/TolC efflux pump) that are capable to extrude antibiotics, detergents, dyes and organic solvents. In order to evaluate the unknown MarB function of Escherichia coli, especially focused on the function of OmpF porin, several mutants were construted by T4GT7 transduction. MarA plays a major roles in mar (multiple antibiotic resistance) phenotype with AcrAB/TolC efflux pump in E. coli K-12. Futhermore, MarA decreases OmpF porin expression via micF antisense RNA. Expression of acrAB is increased in strains containing mutation in marR, and in those carrying multicopy plasmid expressing marA. MarB protein of E. coli K-12 showed its activity at OmpF porin & TolC protein as target molecule. Some paper reported MarB positively regulates OmpF function. MarA shows mar phenotype, and MarB along with MarA show decreased MIC through OmpF function. By this experiment, MarB could decrease MIC through the OmpF porin & TolC protein as target.

• 한국미생물·생명공학회지
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• 제31권2호
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• pp.111-116
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• 2003

Xylan 분해 균주인 Bacillus stearothermophilus No. 236 분리균의 $\beta$-xylosidase 생산 유전자(xylA)의 염기 서열 및 transcription start site를 결정한 이전 연구 결과에 의하면 xylA 유전자는 매우 특이하게 UUG codon에서 translation이 시작되며 initiation codon 15dp 윗쪽에는 promoter로 추정되는 염기 서열을 가지고 있는 것으로 분석되었다. 이와 같은 xylA 유전자 promoter region의 구조는 E. coli에 클로닝된 xalA 유전자를 이용한 실험 결과로도 확인되었다. xalA promoter의 -10 element는 CATAAT로서 6개의 염기 중 5개가 그리고 -35 element의 경우는 TTGTTA로서 6개의 염기 중 4개가 consensus sequence와 일치되었으나 두 hexamer 사이의 거리가 최적 거리에서 크게 벗어난 12 bp인 것으로 분석되었다. 본 연구에서는 $\beta$-xylosidase의 대량 생산을 위한 연구의 일환으로 xalA promoter sequence의 체계적 구조 변화에 의한 promoter strength에 미치는 효과를 E. coli와 B. subtilis두 숙주 세포에서 조사 분석해 본 결과, 첫째로 두 promoter elements사이의 거리를 최적거리인 17 bp로 바꾸었을 때 xalA의 발현율은 E. coli에서는 1.6배, B. subtilis에서는 2.5배 정도 증가함을 보여주었다. 그리고 -35 element는 consensus sequence와 같이 5'쪽에서 네번째 위치에 있는 T$\longrightarrow$A로 변이 시켰을 때 E. coli경우 2.3배, 특히 B. subtilis에서는 35배나 되는 가장 높은 promoter 활성의 증가를 보였다. 그러나 -10 sequence의 경우 consensus sequence와 같이 5' 쪽에서 첫번째 위치에 있는 C$\longrightarrow$T로 transition시켰을 때 예상외로 오히려 발현율이 5~15배까지 낮아지는 특이한 결과를 얻었다. 따라서 본 연구 결과 xalA promoter의 경우 -10 sequence인 CATAAT의 C와 -35 element의 두 염기가 promoter활성에 있어 가장 중요한 염기임을 알 수 있었다.

• 한국미생물·생명공학회지
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• 제17권4호
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• pp.334-342
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• 1989

Bacillus stearothermophilus의 cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase：EC 3.5.4.5)를 코딩하는 cdd 유전자를 E. coli cdd$^-$ 결손변이주를 cloning host로 하여 3-10Kbp의 B. stearothermophilus DNA 단편으로부터 shot gun 법으로 클로닝하였다. 고 복제수 플라스미드 pBR322 의 PstI 부위에 3.0Kb의 B. stearothermophilus DNA 단편을 함유한 pJSC101이 cdd$^+$와 tetracy-line 내성으로서 cloning되었으며, 이어서, 결실 및 subcloning을 연속 수행한 결과 약 1.35kbp의 Eco RI$_1$/PstI$_2$단편이 동일 부위의 pBR322에 삽입된 cdd 양성의 pJSC201을 얻었다. Mini 세포 실험결과, 이 단편에서 합성되는 polypeptide는 약 33 KDa이었기에 이 polypeptide가 cytidine deaminase 로 추정되었다. 또한 이 단편에 함유한 550bp의 EcoRI/AvaI 부분을 lacZ 프로모터 영역에 삽입한 경우 프로모터 활성을 나타내었기에 이 단편의 Eco RI 부위에서 PstI부위로 cdd 유전자가 전사됨을 알 수 있었다. B. subilis와 E. coli에서 발현이 가능한 shuttle vector에 cdd가 함유된 단편을 삽입한 후 이를 양세포에서 동시 발현시켰을 때 B. subtilis에서 발현시킨 경우가 E. coli에서 보다높은 cytidine deaminase 활성을 나타내었으며 이 유전자는 B. subtilis 에서도 E. coli에서와 같이 안정하게 유지됨을 알 수 있었다.

• KSBB Journal
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• 제16권5호
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• pp.486-492
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• 2001

숙지황 물추출물로부터 분리된 fraction(RG-III) 의 항돌연변이원성의 기작을 E. coli GW, B/r 균주를 이용하여 조사하였다. SOS 유도를 반영하는 $\beta$-galactiosidase 활성이 E. coli GW 1060, 1103, 1107, 1105에서 증가되지 않았다. RG-III는 RecA는 단백질의 합성을 증폭시키거나 LexA 산물의 분해를 저해하지 않았으므로 SOS en 기능의 발현이 영향을 미치지 못했다. 따라서 DNA 수복의 경로가 다른 E. coli B.r 변이주를 사용하여 4NQO와 MNNG에 대한 세포내 항돌연변이원성과 생존효과를 조사하였다. ZA159(uvrB, 최)를 제외한 WP2, WP2s, WP67, CM561, CM611에서 RG-III는 4NQO에 대한 생존력을 미약하게나마 증가시켰으나, 이러한 생존력 재활성을 수복모드에 의해 설명할 수 없었다. WP2, WP2s, WP67, CM561, CM611에서 RG-III는 MNNG로 유도된 돌연변이원성과 치사력을 증가시킴에도 불구하고 ZA159(uvr B, chl)에서는 감소시켰다. 4NQO의 변이원성을 두드러지게 억제하였으나 ZA159(uvr B, chl)에서 상승효과가 상대적으로 감소되었다. 이러한 결과들은 RG-III가 4NQO의 변이원성을 방어하는 차단제임을 시시하여, chl 산물의 기능과 유사한 작용을 하는 것으로 사료된다.