• Genomics & Informatics
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• 제10권4호
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• pp.249-255
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• 2012

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular- weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.744-750
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• 1999

Forty alcohol yeast strains were isolated from the main mashes (10 strains from each mash) for brewing of 4 different kinds of Korean traditional liquor (3 different types of Yakju and 1 Takju). Thirty-eight out of 40 strains were identified to be the same strain, Saccharomyces boulardii, by the Automated Bacteria, Yeast, and Fungi Identification System (Biolog Co., U.S.A.) based on the metabolic fingerprints. One strain that showed the highest ethanol production among the 38 strains in YPD medium, designated SHY 111, was selected and used for differentiating from other yeast type strains using the polymerase chain reaction (PCR). Amplified DNA, from transcribed internal spacers of SHY 111 chromosomal DNA, was found to be the same in both size and sequence as those of S. cerevisiae KCCM 11215 (formerly S. coreanus) and S. boulardii along with that of S. cerevisiae AB 972, which was used as a type strain for the yeast genome project. However, when PCR was carried out with the intron splice site primer, it resulted in the amplification of the SHY 111-specific DNA fragment which was about 200 bp in size. When PCR was carried out using the primer to test diversity of 40 isolated yeast strains, it was found that the PCR patterns were similar to each other except for the 200 bp bands derived from all the 10 strains from one Yakju, and 2 strains from another Yakju. These results suggest the strain identified as S. boulardii by the Automated Identification System to be a dominant strain for the fermentation of Korean traditional liquors.

• Journal of Plant Biotechnology
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• 제46권1호
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• pp.1-8
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• 2019

The main goal of this study was to determine the genetic diversity among 36 grape cultivars grown in Palestine by using ISSR-polymerase chain reaction (PCR) fingerprints. Among the tested primers, 17 produced reasonable amplification products with high intensity and pattern stability. A total of 57 DNA fragments (loci) separated by electrophoresis on agarose gels were detected and they ranged in size, from 150 to 900 bp. Out of these fragments, 55 (88%) were polymorphic and 2 (3.5%) monomorphic. Our results also revealed an average of 3.1 loci per primer. A minimum of 1 and maximum of 10 DNA fragments were obtained (S-17, #820 and #841) and (S-31) primers, respectively. Therefore, the later primer (S-31) is considered to be the most powerful primer among the tested ones. The genetic distance matrix showed an average distance range of between 0.05 and 0.76. The maximum genetic distance value of 0.76 (24% similarity) was exhibited between the (Shami and Marawi.Hamadani.Adi) as well as (Bairuti and Marawi.Hamadani.Adi) genotypes. On the other hand, the lowest genetic distance of 0.05 (95% similarity) was exhibited between (Jandali.Tawel.Mofarad and Jandali. Kurawi.Mlzlz) along with (Shami.Aswad and Shami.mtartash. mlwn) genotypes. Furthermore, the UPGMA dendrogram generally clusters the grape cultivars into eight major clusters in addition to an isolated genotype. Based on these figures, the cultivars tested in this study could be characterized by large divergence at the DNA level. This is taking the assumption that our region has a very rich and varied clonal grape genetic structure.

• 한국균학회지
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• 제24권2호통권77호
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• pp.111-126
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• 1996

표고 Lentinus edodes의 균사체로부터 핵을 분리하여 사철느타리 Pleurotus florida 원형질체에 폴리에틸렌 글라이콜로 전이하여 속간(屬間) 핵전이체(核轉移體)를 16균주 얻었다. 주된 핵전이주는 합핵체(合核體) synkaryon 또는 이질핵체로 꺽쇠연결체 clamp connections가 없었다. 그러나 톱밥배지를 이용하여 일정한 저온상태에서 광을 조사하여 꺽쇠연결체를 가진 균사가 다시 생장하여 사철느타리와 유사한 자실체를 형성하였으나 갓색택은 다소 달랐다. 꺽쇠연결체 형성에 영향을 주는 주요한 요인으로는 광, 온도, 배지의 영양, 배지의 생리적 상태로 나타났다. 핵치환체(核置換體) reconstituted cell는 꺽쇠연결체를 가진 표고와 유사한 균총형태로 자실체도 표고와 거의 유사하였다. 핵융합체(核融合體) nuclear hybrid는 이질배수체(異質倍數體) heteroploid로 균총생장이 빠르고 benormyl 첨가배지에서 균총분리가 나타나며 꺽쇠연결체와 자실체를 형성하지 않았다. 동위효소 esterase, leucine aminopeptidase 분석결과 핵융합체는 전혀 새로운 벤드양상이었으나 합핵체는 사철느타리의 주된 밴드를 형성하면서 양친에 없는 다소의 새로운 밴드를 형성하여 구분되었다. PCR에 의한 RAPD 벤드의 크기는 0.25-4.0 Kb였으며 유전유사도 분석으로 5군으로 구분할 수 있었는데 표고와 핵치환체, 핵합체 p669, 사철느타리와 12 핵합체, 핵융합체 p674, 핵융합체 p678이었다. 합핵체의 담자포자로 2 핵전이체를 유전분석한 결과 원영양형(原營養型) prototroph과 영양요구형(營養要求型) riboflavine으로 분리되었는데 그 비는 1 : 1, 1 : 2였다.

• 한국미생물·생명공학회지
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• 제35권2호
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• pp.150-157
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• 2007

디젤 오염 토양 정화를 위해 식물과 미생물의 상호관계를 활용하는 생물복원에 관한 연구를 수행하였다. 디젤을 분해하는 근권 세균인 Rhodococcus sp. 412와 디젤에 내성을 가지고 있는 식물인 옥수수(Zea mays)를 이용하여 디젤로 오염되어진 토양의 디젤 제거능과 미생물 군집변화를 조사하였다. 실험 개시 30일 후, 디젤 오염 토양에서 Rhodococcus sp. 412를 접종한 토양의 옥수수의 성장이 412균주를 접종하지 않은 토양에서의 옥수수 성장보다 약간 우수하였다. 또한 식물을 식재하거나 412균주를 접종한 토양에서 존재하는 토양에서 디젤의 잔류농도도 낮게 나타났다. 이러한 결과를 디젤 오염 토양 정화를 위해 옥수수와 Rhodococcus sp. 412를 동시에 활용하는 것이 유리함을 의미한다. 토양세균 군집 변화를 16S rDNA-PCR과 DGGE(denaturing gradient gel electrophoresis) fingerprinting 방법을 이용하여 분석하였다. 비오염 토양 시료와 디젤 오염토양 시료의 DGGE fingerprint의 유사도는 $20.8{\sim}39.3%$이었다. 또한, 비오염 토양 시료 사이의 DGGE fingerprint의 유사도는 $21.9{\sim}53.6%$, 그리고 디젤 오염 토양 시료 사이의 유사도는 $31.6{\sim}50.0%$이었다. 이러한 결과는 디젤 오염으로 인해 토양 세균 군집구조가 영향을 받았음을 시사한다.

• 원예과학기술지
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• 제32권6호
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• pp.853-863
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• 2014

국내외에서 재배되고 있는 딸기 100품종의 DNA profile 데이터베이스를 구축하기 위하여 다형성이 높은 microsatellite 마커의 선정과 유전적 유사도 분석을 통한 품종식별력 검정 등에 대한 연구를 수행하였다. 딸기 21품종을 274개의 microsatellite 마커로 검정하여 반복 재현성이 높은 25개의 다형성이 높은 마커를 선정하였다. 이들 마커와 국내외에서 재배되고 있는 딸기 100품종을 검정하였을 때 마커당 평균 대립유전자수는 7.50개로 나타났고, 3-13개까지 다양한 분포를 나타내었다. PIC 값은 마커의 유전자형에 따라 0.333-0.841 범위에 속하였으며 평균값도 0.706으로 높게 나타났다. Microsatellite 마커의 대립유전자를 이용하여 딸기 100 품종에 대한 계통도를 작성하였을 때 품종 육성의 계보 및 육성 지역에 따라 7개의 그룹으로 크게 나누어졌으며 2품종을 제외한 98품종이 microsatellite 마커의 유전자형에 의해 식별이 되는 것으로 나타났다. 본 연구에서 얻어진 딸기 품종별 DNA profile 데이터베이스는 품종보호 출원 품종의 재배심사 및 품종진위성과 관련된 종자분쟁을 해결하는 수단으로 유용하게 활용될 수 있을 것이다.

• 원예과학기술지
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• 제32권2호
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• pp.210-216
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• 2014

The distribution of S-haplotypes and genetic relationships were evaluated for 47 accessions of 'Danji' radish (Raphanus sativus L. var. hortensis Baker f. gigantissimus Makino) originating from Jeju Island in South Korea. A total of 22 S-haplotype-specific SCAR markers for the S locus glycoprotein (SLG) and S receptor kinase (SRK) loci were tested, and six primer sets amplified locus-specific PCR fragments from at least one 'Danji' radish accession. S5 and S21 alleles atthe SLG locus were the most frequently distributed, and detected from 87.5% and 64.6% of the accessions, respectively. The frequency of the class-II haplotype at the SLG locus was 75%, more frequent than the class-I haplotype. The S23 allele at the SRK locus was detected from 7 accessions. Grouping of the accessions based on S-allele composition revealed three major groups, while 8 accessions showed a unique allelic composition. The genetic diversity of 47 'Danji' radishes and 1 'Gwandong' radish were also evaluated with 38 RAPD primers. A total of 312 bands were scored, and showed that 138 bands (44.2%) were monomorphic among the accessions, whereas 174 (55.8%) bands were polymorphic. Polymorphism rates ranged from 0.2 to 1.0, indicating significant variations in detecting polymorphism across RAPD primers. The genetic similarity coefficients among all pairs of the 48accessions varied from 0.62 to 0.93, and 42% of the comparisons exhibited values higher than 0.85. All the cultivars could be distinguished based on the DNA fingerprints revealed by RAPD. The comparisons between the dendrograms based on S-haplotypes and RAPDs indicate an unrelated and sporadic distribution for several accessions; however, there was a tendency for accessions with the same S-allelic composition to group into the same cluster.

• Journal of Microbiology and Biotechnology
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• 제20권1호
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• pp.169-178
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• 2010

The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis. Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthrobacter koreensis and $\beta$-Proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic-contaminated soils. The majority of the As-resistant isolates were Gramnegative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. The PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using the Pearson product-moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. Although many isolates were genetically diverse, others were clonal in nature. Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.614-622
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• 2013

Daqu, a traditional fermentation starter, has been used to produce attractively flavored foods such as vinegar and Chinese liquor for thousands of years. Although Bacillus spp. are one of the dominant microorganisms in Daqu, more precise information is needed to reveal why and how Bacillus became dominant in Daqu, and next, to assess the impact of Bacillus sp. on Daqu and its derived products. We combined culture-dependent and culture-independent methods to study the ecology of Bacillus during Daqu incubation. Throughout the incubation, 67 presumptive Bacillus spp. isolates were obtained, 52 of which were confirmed by 16S rDNA sequencing. The identified organisms belonged to 8 Bacillus species: B. licheniformis, B. subtilis, B. amyloliquefaciens, B. cereus, B. circulans, B. megaterium, B. pumilus, and B. anthracis. A primer set specific for Bacillus and related genera was used in a selective PCR study, followed by a nested DGGE PCR targeting the V9 region of the 16S rDNA. Species identified from the PCR-DGGE fingerprints were related to B. licheniformis, B. subtilis, B. amyloliquefaciens, B. pumilus, B. benzoevorans, and B. foraminis. The predominant species was found to be B. licheniformis. Certain B. licheniformis strains exhibited potent antimicrobial activities. The greatest species diversity occurred at the Liangmei stage of Daqu incubation. To date, we lack sufficient knowledge of Bacillus distribution in Daqu. Elucidating the ecology of Bacillus during Daqu incubation would enable the impact of Bacillus on Daqu to be accessed, and the quality and stabilization of Daqu-derived products to be optimized.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1966-1974
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• 2008

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.