• 한국잠사곤충학회지
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• 제36권1호
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• pp.62-68
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• 1994

뽕나무 추기벌채 후 피해가 큰 가지썩음 증상에서 빙핵활성세균의 분리, 생물검정 및 동정과 밀도를 조사하여 본 결과 1. 총 36개의 분리세균 중 -1$0^{\circ}C$ 이상 온도에서 동결이 되는 세균은 12개였으며, -5$^{\circ}C$ 이상의 온도에서는 2개균주였다. 2. 공시세균의 생물검정에 있어서 토마토와 옥수수에서는 -1$0^{\circ}C$ 이상의 동결균주에서는 일정한 경향을 볼 수 없었으나 뽕나무에서는 -5$^{\circ}C$ 이상에서 동결이 되는 세균에서만 육안으로 확실한 동상해피해를 볼 수 있었다. 3. 뽕나무, 옥수수, 토마토유묘에 피해를 주며 -5$^{\circ}C$ 이상에서 영핵활성이 있는 균주 SE9316, SE9338을 동정해 본 결과 gram음성, 간상형, 운동성이 있고 극모를 가지고 호기성균인 Pseudomonas syringae로 동정되었다. 4. 5cm, 10cm 이병뽕나무가지에서 Pseudomonas 속 영핵활성세균의 밀도는 2월과 4월이 가장 높았고, 5월에는 감소하는 경향이었으며 병반에서 멀어질수록 밀도는 감소하였다. 또한 그 밀도는 105cfu/g 이상으로서 동상해를 유발시키기에 충분한 밀도였다.

• 한국식품과학회지
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• 제29권2호
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• pp.326-336
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• 1997

INP를 포함한 빙핵활성세균을 식품 가공 단계에 직접 이용하기 위해 빙핵활성을 최대한 유지시키면서 완전 살균하는 방법을 찾기 위해 가열, 고압, 방사선으로 처리한 후 생균수 및 빙핵활성을 측정하였다. 균주는 Pseudomonas syringae, Xanthomonas campestris와 INP유전자를 포함한 plasmid가 재조합된 Escherichia coli JM109/pEIN229, Gluconobacter oxydans/pKIN230를 사용하였다. 빙핵활성을 T90 (drop의 90%가 어는 온도)으로 나타내었을 때 방사선 조사시 $0.3^{\circ}C{\sim}0.7^{\circ}C$, 초고압 처리시 $1^{\circ}C{\sim}2^{\circ}C$, 가열 처리시 $4^{\circ}C{\sim}7^{\circ}C$가 감소하여 방사선 조사에 의한 살균시 빙핵활성의 감소가 가장 적은 것으로 나타났다. 각 살균점에서의 cumulative ice nucleation activity spectra를 측정한 결과 A부류의 빙핵($-5^{\circ}C$ 이상에서 빙핵으로 작용)의 수는 방사선처리시 거의 감소가 없었으며 초고압처리시 90% 이상 감소하였으며 가열처리시 존재하지 않았다. 증류수에 방사선조사로 살균된 빙핵활성세균을 첨가한 후 DSC를 이용해 thermogram을 얻었다. 빙핵활성세균을 첨가하지 않은 증류수보다 $11^{\circ}C{\sim}15^{\circ}C$ 정도 높은 온도에서 발열 피크가 형성되기 시작했으나 흡열 피크는 큰 차이를 보이지 않았으며, 발열량 및 흡열량은 감소했다. 한편, DSC를 이용해 측정된 빙핵활성은 drop freezing method에 의해 측정된 값과 높은 연관성$(R^{2}>0.993,\;p<0.0001)$을 보여 간편하고 정확한 빙핵활성 측정법으로의 사용 가능성을 제시했다.

• Journal of Microbiology and Biotechnology
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• 제22권2호
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• The Plant Pathology Journal
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• 제38권4호
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• pp.403-409
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• 2022

In biological particles such as Fusarium species, ice nucleation activity (INA) has been observed. Fusarium strains isolated from apple declined trees in Korea were identified with a multilocus sequence analysis using the tef1 and rpb1 genes. Droplet-freezing and tube-freezing assays were used to determine the INA of the strains, using Pseudomonas syringae pv. syringae KACC 21200 as a positive control and resulting in seven INA+ fungal strains that were identified as F. tricinctum (KNUF-21-F17, KNUF-21-F18, KNUF-21-F29, KNUF-21-F32, KNUF-21-F38, KNUF-21-F43, and KNUF-21-F44). The effect of Fusarium INA+ KNUF-21-F29 was compared to that of INA- strains on Chrysanthemum morifolium cv. Shinma explants. A higher callus formation and no-shoot formation were observed, suggesting that fungal INA could play a role in cold injuries and be a factor to consider in rapid apple decline. To the best of our knowledge, this is the first report of INA fungal strains isolated in Korea.

• 한국균학회지
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• 제50권4호
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• pp.255-262
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• 2022

In this study, the fungal strain KNUF-21-F39 was isolated from a declined apple tree (Malus domestica) in the Chungcheongbuk province in Korea. The strain KNUF-21-F39 presented a slow growth rate and a variety of macroconidia shapes and sizes ranging from ovoid to fusoid and 1- to 5-septate, primarily showing 3- and 4-septate, with "S" -shaped macroconidia rarely observed. The strain was identified based on morphological characteristics along with phylogenetic analysis performed using the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-α (tef1), RNA polymerase largest subunit (rpb1), and calmodulin (cal) genes. The fungal strain KNUF-21-F39 was identified as Fusarium diversisporum, which has not been previously reported in Korea. The ice nucleation activity (INA) of the strain was also evaluated, identifying the strain as positive for INA. This is the first report characterizing F. diversisporum as an IN-active fungal species.

• 한국식물병리학회지
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• 제3권3호
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• pp.180-186
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• 1987

단감나무와 차나무의 표면에서 분리한 Pseudomonas syringae 2계통 PS8401, PS8402를 $2.5\%$ glycerol이 첨가된 nutrient agar에서 배양한 다음, 증류수로 세포현탁액($10^8$ colony forming unit/ml)을 조제하여 마이크로피펫법으로 동결온도를 측정하여 본 결과, 각각 -2.5와 $-3.8^{\circ}C$에서 동결하여 빙핵활성이 인정되었다. 한편 동일한 방법에 의한 증류수의 동결온도는 $-21.8^{\circ}C$였고 옥수수를 비롯한 8가지 작물즙액의 동결온도는 $-11.6^{\circ}C$ 이하였다. PS 8401의 nutrient broth 현탁액$(10^8\;cfu/ml)$을 살포한 옥수수유묘는 $-2^{\circ}C$에서부터 얼기 시작하여 $-4^{\circ}C$가 되면, 거의 전체 식물이 상해를 입었으나 nutrient broth만 살포한 대조구의 유묘는 온도가 $-9^{\circ}C$로 내려가기 전까지는 피해를 입지 않았다. PS8401 현탁액을 살포한 후 48시간에 측정한 유묘의 피해는 살포한 세균의 량이 증가함에 따라 심해지는 경향이었다. 상기 PS 8401의 빙핵활성은 현탁액내 세포의 수가 많아짐에 따라 증가하였으며 세균을 배양한 배지의 조성이 배양온도에 따라서도 빙핵활성이 변하였는데 $2.5\%$ glycerol 혹은 $2.5\%$ glucose를 첨가한 nutrient agar와 탄소원을 별도로 첨가하지 않은 nutrient agar에서 자란 세균현탁액$(10^2\;cfu/ml)$의 동결온도는 각각 -4.0, -4.4, $-7.2^{\circ}C$였고, 배지를 $2.5\%$ glycerol을 함유한 nutrient agar로 고정하고 온도를 달리하여 생육시킨 세균현탁액 $(10^2\;cfu/ml)$의 동결온도는 $-4.0^{\circ}C$(생육온도가 $15\~25^{\circ}C$인 경우)와 $-7.6^{\circ}C$(생육온도가 $30^{\circ}C$인 경우)였다.

• The Plant Pathology Journal
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• 제39권1호
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• pp.88-107
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• 2023

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of -0.7℃, indicating an INA⁺ bacterium. The ice-nucleating temperature was -4.7℃ for a non-treated control (sterilized distilled water), whereas it was -9.6℃ for an INA^- bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.673-679
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• 2004

A microbial cryoprotectant formulation using a W/O/W multiple emulsion system was developed. The psychrotolerant microorganism, B4, isolated from soil in South Korea, was observed by the drop freezing method, in which the microorganism sample inhibited ice nucleation activity. The antifreeze activity was eliminated when the microorganism sample was treated with protease, indicating that the antifreeze activity was due to the presence of antifreeze protein. The result of the l6S rDNA sequencing indicated the B4 strain was most closely related to a species of the genus Bacillus. Culture broth of B4 strain (Bacillus sp.) and rapeseed oil containing 1 % polyglycerine polyricinolate (PGPR) were used as core and wall material, respectively. The most stable W/O emulsion was prepared at a core/oil ratio of 1:2. The highest W/O/W emulsion stability was achieved when the primary emulsion to external aqueous phase containing 0.5% caster oil polyoxyethylene ether $(COG25^{TM})$ ratio was 1:1. Microcrystalline cellulose showed better W/O/W emulsion stability than other polymer types. The viability of cells in a W/O/W emulsion was higher than free cells during storage at $37^\circ{C}$. An acidic pH and UV exposure decreased the viability of free cells, but cells in W/O/W emulsion were more stable under these conditions.

• Journal of Microbiology and Biotechnology
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• 제24권3호
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• pp.379-385
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• 2014

The purpose of this study was to compare the ability of an engineered Escherichia coli to degrade chlorpyrifos (Cp) using an organophosphorus hydrolase enzyme, encoded in both Flavobacterium sp. ATCC 27551 or Pseudomonas diminuta, by employing the Lpp-OmpA chimera and the N-terminal domain of the ice nucleation protein as anchoring motifs. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by both anchors on the outer membrane. This is the first report on the presentation of OPH on the cell surface by Lpp-OmpA under the control of the T7 promoter. The results showed cell growth in the presence of Cp as the sole source of energy, without growth inhibition, and with higher whole-cell activity for both cells harboring plasmids pENVO and pELMO, at approximately 10,342.85 and 10,857.14 U/mg, respectively. Noticeably, the protein displayed by pELMO was lower than the protein displayed by pENVO. It can be concluded that Lpp-OmpA can display less protein, but more functional OPH protein. These results highlight the high potential, of both engineered bacteria, for use in the bioremediation of pesticide-contaminated sources in the environment.