• The Plant Pathology Journal
• /
• 제33권3호
• /
• pp.318-328
• /
• 2017

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was $50^{\circ}C$. However, it was inactivated with time when it was incubated at $40^{\circ}C$ and $50^{\circ}C$. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

• KSBB Journal
• /
• 제8권2호
• /
• pp.133-142
• /
• 1993

효모균에서의 유전자 재조합에 의한 단백절 생산에 미치는 유전학적, 환경적인 요인의 영향을 연구하였. Plasmid 안정도와 개수는 $REP^+$ 체계 에서 대단히 높은 반면, rep 체계에서는 매우 낮았다. $2{\mu}m$ circle plasmid genome을 포함하는 plasmid의 경우에 있어서. $[cir^o]$ 형 세포에서의 plasmid 안정도와 개수가 $[cir^+]$형 세포에서보다 높기때문에 $[cir^+o]$형 세포가 더 선호되는 세포였다. 유전자 발현은 plasmid 개수와 안정도에 좌우 되었다. 촉진제의 양이 유전자 발현에 매우 중요 한 역할을 했다. 유전자 발현의 촉진에 필요한 g떠actose의 농도는 0.8% 이 변 충분했다. 높은 안정 도와 개수를 갖는 plasmid의 경우 촉진속도는 매우 빨랐다. Galactose가 배양의 시작부분부터 첨가 될 때가 mid-exponential ph잃e에 첨가될 때보다 유전자 발현의 극대점에 이르는 시간이 걸었다. 상대적 촉진제의 양이 증가함에 따라 glucc잉e억제 현상은 감소되었다.

• Journal of Microbiology and Biotechnology
• /
• 제22권4호
• /
• pp.510-515
• /
• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Molecules and Cells
• /
• 제28권4호
• /
• pp.321-329
• /
• 2009

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and $H_2O_2$ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.

• 생명과학회지
• /
• 제20권1호
• /
• pp.97-102
• /
• 2010

식품단백질의 물성과 기능성을 개선시켜 산업적인 가치가 매우 높은 TGase를 생산하는 S. platensis YK-2의 분자 유전학적인 연구를 위해 형질전환방법을 확립하였으며 본 연구를 위해 도입된 방법은 대장균을 plasmid DNA의 공여체로, S. platensis의 포자를 수용체로 사용하는 접합전달법이다. S. platensis를 위한 최적 접합전달조건은 50 mM의 $MgCl_2$를 첨가한 MS배지에 열처리를 하지 않은 포자와 $5{\times}10^7$의 plasmid DNA 공여체인 E. coli를 사용하는 것이다. 또 본 연구를 통해 얻어진 접합전달체의 attB site에 대한 분석을 통해 S. platensis chromosome의 pirin 상동체를 코드하는 ORF내에 attB site가 단일위치로 존재하고 있으며 이미 밝혀진 다른 방선균유래 attB site의 염기서열에 대해 77.8%~96.3%의 상동성을 나타냈다.

• KSBB Journal
• /
• 제9권5호
• /
• pp.532-540
• /
• 1994

재조합 플라스미드 함유 효모 h$\alpha$1Y2SUCSTA 의 glucoamylase의 분비효율은 배양 4연째에 약 74%이였으며 염색체 삽입 재조합효모 h$\alpha$1Y2S­U UCSTA-I의 경우에는 4일째에 약 65%로 나타났다. 높은 분비효율을 나타낸 것은 glucoamy­I lase의 발현수준이 숙주세포 분비기관의 능력에 미치지 못하기 때문으로 추정된다. 두 재조합 균주에셔 모두 대부분의 세포내 glucoamylase는 c cytoplasm에 존재하고 약간의 부분만이 (10% 이내) 전 배양기간을 통하여 peri plasm에 존재하였다. 재조합 효모로부터 분비되는 glucoamy­1 lase의 특 생 을 Western blot 분 석 을 통하여 조사하였다. 배양액으로 분비된 glucoamylase는 매우 h heterogeneous하였고 그 분자량은 약 200에서 3 300 kilodalton이였다. 분비된 glucoamylase의 당 잔기량은 약 80% 이상이였고 세포내 glucoamy­l lase의 endoplasmic reticulum 형태는 약 55 에서 65kilodalton 사이의 여러 가지의 밴드로 나타났 다.

• Journal of Microbiology and Biotechnology
• /
• 제26권2호
• /
• pp.337-346
• /
• 2016

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpE-GroEL-GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

• Journal of Microbiology and Biotechnology
• /
• 제25권4호
• /
• pp.511-520
• /
• 2015

Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX₍₈₎K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

• Journal of Microbiology
• /
• 제42권2호
• /
• pp.111-116
• /
• 2004

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli $tRNA_{1}$$^{Gln}$ with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-$tRNA_{1}$$^{Gln}$ formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also amelio-rated growth inhibition, presumably by competitively preventing $tRNA_{1}$$^{Gln}$ misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of $tRNA_{1}$$^{Gln}$, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-$tRNA^{Gln}$ amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mis-charging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli Glu-$tRNA_{1}$$^{Gln}$, and converts it to the cognate Gln-$tRNA_{1}$$^{Gln}$ species. B. subtilis GluRS-dependent Glu-$tRNA_{1}$$^{Gln}$ formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권6호
• /
• pp.510-515
• /
• 2006

The putative EPA synthesis gene cluster was mined from the entire genome sequence of Shewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO1600 (acyl transferase, AT), ORFSO1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO1597 (enoyl reductase, ER), ORFSO1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified from S. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genes orfSO1597 and orfSO1604. The DNA fragment was cloned into Escherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in four E. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in the E. coli clone was affected by cultivation temperature, showing maximum yield at $20^{\circ}C$ and no production at $30^{\circ}C$ or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.