• Journal of Microbiology and Biotechnology
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• 제25권1호
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• pp.98-108
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• 2015

Staphylococcus aureus is an important foodborne pathogen that causes diverse diseases ranging from minor infections to life-threatening conditions in humans and animals. To further understand its pathogenesis, the genome of the strain S. aureus FORC_001 was isolated from a contaminated food. Its genome consists of 2,886,017 bp double-stranded DNA with a GC content of 32.8%. It is predicted to contain 2,728 open reading frames, 57 tRNAs, and 6 rRNA operons, including 1 additional 5S rRNA gene. Comparative phylogenetic tree analysis of 40 complete S. aureus genome sequences using average nucleotide identity (ANI) revealed that strain FORC_001 belonged to Group I. The closest phylogenetic match was S. aureus MRSA252, according to a whole-genome ANI (99.87%), suggesting that they might share a common ancestor. Comparative genome analysis of FORC_001 and MRSA252 revealed two non-homologous regions: Regions I and II. The presence of various antibiotic resistance genes, including the SCCmec cluster in Region I of MRSA252, suggests that this strain might have acquired the SCCmec cluster to adapt to specific environments containing methicillin. Region II of both genomes contains prophage regions but their DNA sequence identity is very low, suggesting that the prophages might differ. This is the first report of the complete genome sequence of S. aureus isolated from a real foodborne outbreak in South Korea. This report would be helpful to extend our understanding about the genome, general characteristics, and virulence factors of S. aureus for further studies of pathogenesis, rapid detection, and epidemiological investigation in foodborne outbreak.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.276-283
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• 2004

Screening was performed to isolate cellulose-producing microorganisms from the Korean traditional fermented persimmon vinegar. The resulting strain, KJ $145^{T}$, was then taxonomically investigated by phenotypic characterization, particularly chemotaxonomic, and by phylogenetic inference based on a 16S rDNA sequence analysis including other related taxa. Strain KJ $145^{T}$ was found to grow rapidly and form pale white colonies with smooth to rough surfaces on a GYC agar. Strain KJ $145^T$ also produced acetate from ethanol, and was tolerable to 10% ethanol in SM medium. In a static culture, a thick cellulose pellicle was produced, and in GYC broth, the strain grew at temperatures ranging from 28 to $40^\circ{C}$ with an optimum pH of 4.0. The genomic DNA G+C content of strain KJ $145^T$ was 61.9 mol%, and the predominant ubiquinone was Q 10 as the major quinone and Q9 as the minor quinone. The major cellular fatty acids were $C_{16:0}$ and the sum in feature 7 ($C_{18:1}$ w9c, w12t and/or w7c). A 16S rRNA-targeted oligonucleotide probe specific for strain KJ $145^T$was constructed, and the phylogenetic position of the new species was derived from a 16S rDNA-based tree. When comparing the 16S rDNA nucleotide sequences, strain KJ $145^T$ was found to be most closely related to G. hansenii LMG $1527^T$ (99.2%), although KJ $145^T$ was still distinct from G. hansenii LMG $l527^T$ and G. xylinus LMG $1515^T$ in certain phenotypic characteristics. Therefore, on the basis of 16S rDNA sequences and taxonomic characteristics, it is proposed that strain KJ $145^T$ should be placed in the genus Gluconacetobacter as a new species, Gluconacetobacter persimmonis sp. nov., under the type-strain KJ $145^T$ (=KCTC =$10175BP^T$=KCCM=$10354^T$).

• 대한유전성대사질환학회지
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• 제6권1호
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• pp.15-23
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• 2006

Purpose: Glycogen storage disease type III (GSD-III), is a rare autosomal recessive disorder of glycogen metabolism. The affected enzyme is amylo-1,6-glucosidase, 4-alpha-glucanotransferase (AGL, glycogen debranching enzyme), which is responsible for the debranching of the glycogen molecule during catabolism. The disease has been demonstrated to show clinical and biochemical heterogeneity, reflecting the genotype-phenotype heterogeneity among different patients. In this study, we analyzed mutations of the AGL gene in three unrelated Korean GSD-III patients and discussed their clinical and laboratory implications. Methods: We studied three GSD-III patients and the clinical features were characterized. Sequence analysis of 35exons and part exon-intron boundaries of the AGLgene in patients were carried out by direct DNA sequencing method using genomic DNA isolated from patients' peripheral leukocytes. Results: The clinical features included hepatomegaly (in all patients), seizures (in patient 2), growth failure (in patients 1), hyperlipidemia (in patients 1 and 3), raised transaminases and creatinine kinase concentrations (in all patients) and mild EKG abnormalities (in patients 2). Liver transplantation was performed in patient 2due to progressive hepatic fibrosis. Administration of raw-corn-starch could maintain normoglycemia and improve the condition. DNA sequence analysis revealed mutations in 5 out of 6 alleles. Patient 1 was a compound heterozygote of c.1282 G>A (p.R428K) and c.1306delA (p.S603PfsX6), patient 2 with c.1510_1511insT (p.Y504LfsX10), and patient 3 with c.3416 T>C (p.L1139P) and c.l735+1 G>T (Y538_R578delfsX4) mutations. Except R428K mutation, 4 other mutations identified in3 patients were novel. Conclusion: GSD-III patients have variable phenotypic characteristics resembling GSD-Ia. The molecular defects in the AGL gene of Korean GSD-III patients were genetically heterogeneous.

• 식물조직배양학회지
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• 제25권5호
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• pp.341-346
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• 1998

색소유전자의 전이를 통하여 새로운 색소발현체계를 가진 품종을 육종하기 위한 기초연구로 stock (Matthiola incana R. Br.)의 꽃봉오리로부터 cDNA-library를 합성하였고 screening을 통하여 anthocyanin 합성경로의 중요효소의 하나인 DFR (dihydroflavonol 4-reductase) 유전자를 분리하였다. 염기서열분석을 수행하여 분리유전자의 크기가 1450bp 이며 이중 coding region은 1029 bp 임을 확인하였다. 이미 밝혀진 다른 식물체의 DFR 유전자와 서로 염기서열의 일치성을 비교해 본 결과 외자엽식물인 옥수수와 보리와는 각각 61%를 보였으며, 쌍자엽식물인 페튜니아, 금어초, 거베라, 과꽃 그리고 카네이션 등 과는 66%-67%의 일치성을 나타내었다. 아울러 염기서열의 G/C 함량분석을 통하여 쌍자엽식물의 G/C 함량은 외자엽식물의 그것에 비해 매우 낮은 수치를 나타내었다. 분리유전자의 발현을 확인하기 위하여 인위적으로 기내에서의 전사와 해석을 수행한 결과 42-44 kd 크기의 단백질을 확인하였다. Southern blot 분석의 결과 DFR 유전자는 stock의 genome에 다른 대부분의 식물체와 유사하게 한 개가 존재하며 야생종과 돌연변이종의 stock을 분리 DFR 유전자를 probe 로 Northern blot 분석을 수행하여 돌연변이종인 lineK17b가 DFR 돌연변이임을 확인하였다.

• 한국미생물·생명공학회지
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• 제33권2호
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• pp.84-89
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• 2005

Phosphomannomutase는 진핵 생물과 원핵 생물에 있어서 중요한 효소로, ${\alpha}$-D-mannose 6-phosphate를 ${\alpha}$-D-mannose 1-phosphate로 전환시켜 GDP-mannose를 생산한다. 이 기질은 여러 대사 경로에서 중요하게 작용하는 mannosyl기를 제공하도록 돕는다. 본 논문에서는 Sphingomonas chungbukensis DJ77에서 phosphomannomutase를 암호화하는 유전자를 유전체 library로부터 동정하고 이를 pmmC로 명명하였으며, 이를 발현 vector에 클로닝하고 염기서열을 분석하였다. 유전자 pmmC는 ATG를 개시 코돈으로 사용하고, TAG를 종결 코돈으로 사용하는 750 bp의 open reading frame임을 확인하였고, 이 ORF의 5 bp앞쪽으로 리보좀 결합 부위가 존재한다. 이 ORF로부터 유추되는 아미노산은 249개이며, 단백질 분자량은 약 27.4 kDa이다. 이 유전자를 구성하는 아미노산 서열은 NCBI의 conserved domain search를 통한 분석으로 eukaryotic phosphomannomutase와 약 $86.9\%$ 유사성이 있음을 나타냈고, 기질에 대한 활성을 측정한 결과 pmmC 유전자가 암호화하는 단백질이 phosphomannomutase임을 확인할 수 있었다.

• 생명과학회지
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• 제14권2호
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• pp.325-330
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• 2004

효모에 있어 자외선에 의한 절제회복 관여 DNA회복유전자가 많이 알려져 있으나, 이들이 어떤 기능을 하는지는 아직 잘 알려져 있지 않다. 본 연구에서는 자외선 조사 시 절제회복의 초기 단계에 절대적으로 필요한 RAD3 유전자와 유사한 유전자인 HRD3 유전자를 분열형 효모인 Schizosaccharomyces pombe에서 분리하여 그 특성을 연구하였다. 이 결과 분리한 유전자는 효모 RAD3 유전자와 염기서열에서 약 70%이상의 유사성을 보였다. 이 유전자의 염기서열 결과 유전자 산물의 분자량은 75 kDa였다. 2-D gel 결과 과잉발현 시 HRD3 단백질은 숙주 단백질의 합성 억제 또는 분해 촉진을 유발하여 숙주세포인 대장균에 독성초과를 나타내었다. HRD3 유전자와 lacZ 유전자를 융합시킨 여러 가지 재조합 vector를 만들어 이들 융합단백질을 분리, 연구 한 결과 HRD3 단백질의 카르복실 말단부분이 효모에 있어서 DNA회복기능과 대장균에서의 독성효과를 나타내는 중요부위임이 확인되었다.

• 한국미생물·생명공학회지
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• 제38권4호
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• pp.373-378
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• 2010

우리술의 주질개선 및 현장 실용화기술을 개발하기 위해 술 담금의 기본재료인 누룩의 규격화 및 품질향상 연구가 필요하다. 이와 같은 요구에 부응하기 위해, 누룩에 생육하는 우수 양조미생물의 분리 발굴, 균주개량 및 보존관리 등 우리 고유의 양조미생물 자원을 확보하고 활용함으로써 다양한 종류의 누룩 제조가 가능할 것으로 여겨진다. 따라서, 본 연구는 충청지역에서 시판되고 있는 누룩을 수집하여 희석평판 배양법으로 174개 균주를 분리하여 효소활성 검정을 통하여 효소 활성이 높은 균주를 다수 분리하였다. 분리된 곰팡이를 대상으로 액화력 및 당화력 등의 효소학적 특성을 조사하였고, 선발된 활성균주의 동정을 위해 genomic DNA상의 ITS(Internal Transcribed Spacer) 부위의 염기서열을 분석한 결과 Aspergillus sp.(4균주), Rhizopus sp.(4균주), Rhizomucor sp.(2균주), Mycocladus sp.(2균주)으로 동정되었다. 밀기울을 사용한 고체배양에서 액화력, 당화력 및 산 생성능을 검토한 결과, Rhizopus sp. CN084, CN174, Aspergillus sp. CN161와 Mycocladus sp. CN042 균주가 효소활성이 높은 것으로 나타났으며 Aspergillus sp. CN010, CN161, Rhizopus sp. CN105, CN168 균주와 Rhizomucor sp. CN088 균주는 산 생성능이 우수한 것으로 나타났다. 따라서 이들 균주가 양조를 위한 누룩의 원료 균주로서 사용될 경우 단일 누룩제조가 가능할 것으로 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.444-450
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• 2013

A Gram-negative, strictly aerobic, non-motile, non-spore-forming, and rod-shaped bacterial strain designated FW-$6^T$ was isolated from a freshwater sample and its taxonomic position was investigated by using a polyphasic approach. Strain FW-$6^T$ grew optimally at $10-42^{\circ}C$ and at pH 7.0 on nutrient and R2A agar. Strain FW-$6^T$ displayed ${\beta}$-glucosidase activity that was responsible for its ability to transform ginsenoside $Rb_1$ (one of the dominant active components of ginseng) to Rd. On the basis of 16S rRNA gene sequence similarity, strain FW-$6^T$ was shown to belong to the family Sphingomonadaceae and was related to Novosphingobium aromaticivorans DSM $12444^T$ (98.1% sequence similarity) and N. subterraneum IFO $16086^T$ (98.0%). The G+C content of the genomic DNA was 64.4%. The major menaquinone was Q-10 and the major fatty acids were summed feature 7 (comprising $C_{18:1}{\omega}9c/{\omega}12t/{\omega}7c$), summed feature 4 (comprising $C_{16:1}{\omega}7c/iso-C_{15:0}2OH$), $C_{16:0}$, and $C_{14:0}$ 2OH. DNA and chemotaxonomic data supported the affiliation of strain FW-$6^T$ to the genus Novosphingobium. Strain FW-$6^T$ could be differentiated genotypically and phenotypically from the recognized species of the genus Novosphingobium. The isolate that has ginsenoside converting ability therefore represents a novel species, for which the name Novosphingobium ginsenosidimutans sp. nov. is proposed, with the type strain FW-$6^T$ (= KACC $16615^T$ = JCM $18202^T$).

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.584-592
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• 2004

A thermostable $\beta$-glycosidase gene, tfi $\beta$-gly, was cloned from the genomic library of Thermus filiformis Wai33 A1. ifi $\beta$-gly consists of 1,296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the $\beta$-glycosidases from other Thermus spp. belonging to the glycosyl hydrolase family 1. In the present study, the enzyme was overexpressed in Escherichia coli BL21 (DE3) using the pET21b(+) vector system. The recombinant enzyme was purified to homogeneity by heat treatment and a $Ni^{2+}$-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi $\beta$-glycosidase was a monomeric form with molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80- $90^{\circ}C$ and 5.0-6.0, respectively. Ninety-three percent of the enzyme activity was remained at $70^{\circ}C$ after 12 h, and its half-life at $80^{\circ}C$ was 6 h, indicating that Tfi $\beta$-glycosidase is highly thermostable. Based on its K_m$, or $K_{cat}K_m$, ratio, Tfi $\beta$-glycosidase appeared to have higher affinity for $\beta$-D-glucoside than for $\beta$-D-galactoside, however, $K_{cat} for \beta$-D-galactoside was much higher than that for $\beta$-D-glucoside. The activity for lactose hydrolysis was proportionally increased at $70^{\circ}C$ and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi TEX>$\beta$-glycosidase on 138 mM lactose at $70{^\circ}C$ and pH 7.0 was 134.9 U/mg. Consequently, this newly cloned enzyme appears to have a valuable advantage of conducting biotechnological processes at elevated temperature during milk pasteurization in the production of low-lactose milk.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1554-1560
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• 2006

A Gram-positive, aerobic or facultative anaerobic, non motile, endospore-forming bacterial strain, designated Gsoil $114^T$, was isolated from a soil sample of a ginseng field in Pocheon Province (South Korea), and was characterized taxonomically by using a polyphasic approach. It grew well on nutrient agar medium and utilized a limited number of organic substrates as sole carbon sources, including D-xylose and some other carbohydrates, but did not utilize L-amino acids and organic acids. The isolate was positive for oxidase test but negative for catalase, and negative for degradation of macromolecules such as starch, cellulose, xylan, casein, chitin, and DNA. The G+C content of the genomic DNA was 41.8 mol%. The predominant isoprenoid quinone was menaquinone 7 (MK-7). The major fatty acids were $anteiso-C_{15:0}$ (32.1%), $iso-C_{15:0}$ (30.5%), and $anteiso-C_{17:0}$ (30.2%). Comparative 16S rRNA gene sequence analysis showed that strain Gsoil $114^T$ fell within the radiation of the cluster comprising Bacillus species and joined Bacillus shackletonii LMG $18435^T$ with a bootstrap value of 95%. The highest 16S rRNA gene sequence similarities were found with Bacillus shackletonii LMG $18435^T$ (97.6%), Bacillus acidicola DSM $14745^T$ (96.9%), Bacillus sporothermodurans DSM $10599^T$ (96.5%), and Bacillus oleronius DSM $9356^T$ (96.5%). The phylogenetic distance from any other validly described species within the genus Bacillus was less than 96%. DNA-DNA hybridization experiments showed that the DNA-similarities between strain Gsoil $114^T$ and closest phylogenetic neighbors were less than 39%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil $114^T$ (=KCTC $13944^T$=DSMZ $18134^T$) was classified in the genus Bacillus as the type strain of a novel species, for which the name Bacillus ginsengihumi sp. nov. is proposed.