• Korean Journal of Microbiology
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• v.29 no.1
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• pp.49-55
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• 1991

The heat shock responses of Campylobacter jejuni were studied by examination of their survival rates and synthesis of heat shocd proteins. When C. jejuni cells were treated at the sublethal temperatures of 48.deg.C for 30 minutes, most of the cells maintained their viabilities and synthesized the heat shock proteins of 90, 73, and 66 kD in molecular weight. By the method of two-dimensional electrophoresis, the heat shock proteins of C. jejuni were identified to be Hsp90, Hsp73, and Hsp66. During the heat shock at 48.deg.C, the heat shock proteins were induced from about 5 minutes after the heat shock treatment. Their synthesis was continued upto 30 minutes, but remarkably retarded after 50 minutes. When C. jejune cells were heat shocked at 51.deg.C for 30 minutes, the survival rates of the cells were decreased by about $10^{3}$ fold and synthesis of heat shock proteins and normal proteins was also generally retarded. The cells exposed to 55.deg.C for 30 minutes died off by more than $10^{5}$ cells and the new protein synthesis was not observed. But when C. jejuni cells were heat-shocked at the sublethal temperature of 48.deg.C for 15 to 20 minutes and then were exposed at the lethal temperature of 55.deg.C for 30 minutes, their viabilities were higher than those exposed at 55.deg.C for 30 minutes without pre-heat shock at 48.deg.C. Therefore, the heat shock proteins synthesized at the sublethal temperature of 48.deg.C in C. jejuni were thought to be responsible for thermotolerance. However, when C. jejuni cells heat-shocked at various ranges of sublethal and lethal temperatures were placed back to the optimum temperature of 42.deg.C, the multiplication patterns of the cells pretreated at different temperatures were not much different each other.

• Journal of Life Science
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• v.23 no.10
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• pp.1192-1198
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• 2013

To improve yeast strains for bioethanol production, yeasts with ethanol tolerance, thermotolerance, and ${\beta}$-1,3-glucanase activity were bred using yeast genome shuffling. Saccharomyces cerevisiae $BY4742{\Delta}exg1$/pAInu-exgA, which has extracellular ${\beta}$-1,3-glucanase activity, and the Aspergillus oryzae and S. cerevisiae YKY020 strains, which exhibit ethanol tolerance and thermotolerance, were fused by yeast protoplast fusion. Following cell fusion, four candidate cells (No. 3, 9, 11, and 12 strains) showing thermotolerance at $40^{\circ}C$ were selected, and their ethanol tolerance (7% ethanol concentration) and ${\beta}$-1,3-glucanase activity were subsequently analyzed. All the phenotypes of the two parent cells were simultaneously expressed in one (No. 11) of the four candidate cells, and this strain was called BYK-F11. The BYK-F11 fused cell showed enhanced cell growth, ethanol tolerance, ${\beta}$-1,3-glucanase activity, and ethanol productivity compared with the $BY4742{\Delta}exg1$/pAInu-exgA and YKY020 strains. The results prove that a new yeast strain with different characters and the same mating type can be easily bred by protoplast fusion of yeasts.

• Molecules and Cells
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• v.37 no.11
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• pp.833-840
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• 2014

Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.

• Journal of Life Science
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• v.29 no.3
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• pp.376-381
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• 2019

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.

• Proceedings of the Zoological Society Korea Conference
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• 1996.10a
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• pp.215.2-215
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• 1996

No Abstract, See Full Text

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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• pp.76-78
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• 2002

Heat shock proteins (HSPs) are induced in most living cells by mild heat treatment, ethanol, heavy metal ions and hypoxia. In yeast Saccharomyces cerevisiae, mild heat pretreatment strongly induces Hsp104 and thus provide acquired thermotolerance. The ability of hsp104 deleted mutant $({\triangle}hsp104)$ to acquire tolerance to extreme temperature is severely impaired. In providing thermotolerance, two ATP binding domains are indispensible, as demonstrated in ClpA and ClpB proteases of E. coli. The mechanisms by which Hsp104 protects cells from severe heat stress are not yet completely elucidated. We have investigated regulation of mitochondrial metabolic pathways controlled by the functional Hsp104 protein using $^{13}C_NMR$ spectroscopy and observed that the turnover rate of TCA cycle was enhanced in the absence of Hsp104. Production of ROS, which are toxic to kill cells radiply via oxidative stress, was also examined by fluorescence assay. Mitochondrial dysfunction was manifested in increased ROS levels and higher sensitivity for oxidative stress in the absence of Hsp104 protein expressed. Finally, we have identified mitochondrial complex I and Ferritin as binding protein(s) of Hsp104 by yeast two hybrid experiment. Based on these observations, we suggest that Hsp104 protein functions as a protector of oxidative stress via either keeping mitochondrial integrity, direct binding to mitochonrial components or regulating metal-catalyzed redox chemistry.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.771-781
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• 2015

To examine if the molecular chaperone DnaK operon proteins of Streptococcus suis type 2 (SS2) are involved in adhesion to host cells, the abundance values of these proteins from the surface of two SS2 strains of different adhesion capability were compared. Their roles in growth and adhesion to human laryngeal epithelial cell line HEp-2 cells were investigated on SS2 strain HA9801 and its mutants with DnaK operon genes partially knocked-out (PKO mutant) under heat stress. The major difference was that DnaJ was more abundant in strain HA9801 than in strain JX0811. Pretreatment of the bacteria with hyperimmune sera to DnaJ, but not with those to other proteins, could significantly reduce SS2 adhesion to HEp-2 cells. PKO of dnaJ g ene resulted in decreased SS2 growth at 37℃ and 42℃, and reduced its adhesion to HEp-2 cells. The wild-type strain stressed at 42℃ had increased expression of DnaJ on its surface and elevated adhesion to HEp-2 cells, which was also inhibitable by DnaJ specific antiserum. These results indicate that the DnaJ of S. suis type 2 is important not only for thermotolerance but also for adhesion to host cells. Because DnaJ expression is increased upon temperature upshift with increased exposure on the bacterial surface, the febrile conditions of the cases with systemic infections might help facilitate bacterial adhesion to host cells. DnaJ could be one of the potential candidates as a subunit vaccine because of its good immunogenicity.

• Journal of The Korean Society of Grassland and Forage Science
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• v.17 no.4
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• pp.379-386
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• 1997

Recombinant plasmid, pBKH4, containing NPT II and P35S-BcHSP17.6 was constructed by ligation of Bum H I -digested pBKSl-l and BcHSP 17.6 (thermotolerance gene) 6om pBLH4. The tobacco leaf disc was cocultivated with transformed Agmbacterium tumefaciens bearing pBKH4 for 24 hours and transformed shoots were selected on MS-n/B medium containing $100\;{\mu\textrm{g}}/ml$ of kanamycin. Heat-killing temperature of Nicotima tabacum was $50^{\circ}$ for >15min, and transformed tobacco plants with BcHSP17.6 cDNA exhibited thermotolerance at the heat-killing temperature. The transgenic plants were analyzed by Southern blot hybridization with the probe of ${\alpha}^{_32}P$ labelled BcHSP17.6 cDNA. Transcription and expression level of BcHSP17.6 cDNA were also continued by Northern blot analysis and Ouchterlony double immunodiffusion assay. In this study, we suggest that the BcHSP17.6 cDNA introduced to tobacco plant is related to thenuoto-lerance and 17.6-kD LMW HSP acts as a protector from heat damage in plants.

• Journal of Life Science
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• v.29 no.8
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• pp.916-921
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• 2019

In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase ${\delta}$ and ${\varepsilon}$ encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures ($38^{\circ}C$ and $40^{\circ}C$), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than $38^{\circ}C$. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126-Ht strains that proliferated at $40^{\circ}C$ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.

• Molecules and Cells
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• v.20 no.1
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• pp.142-150
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• 2005

Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, $H_6GjCyp-1$ maintained the stability of E. coli proteins up to $50^{\circ}C$. For a functional bioassay for recombinant $H_6GjCyp-1$, the viability of E. coli cells overexpressing $H_6GjCyp-1$ was compared with that of cells not expressing $H_6GjCyp-1$ at $50^{\circ}C$. After high temperature treatment for 1 h, E. coli overexpressing $H_6GjCyp-1$ survived about three times longer than E. coli lacking $H_6GjCyp-1$. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.