• Title/Summary/Keyword: 16S rRNA gene-based sequencing

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Metagenomic SMRT Sequencing-Based Exploration of Novel Lignocellulose-Degrading Capability in Wood Detritus from Torreya nucifera in Bija Forest on Jeju Island

  • Oh, Han Na;Lee, Tae Kwon;Park, Jae Wan;No, Jee Hyun;Kim, Dockyu;Sul, Woo Jun
    • Journal of Microbiology and Biotechnology
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    • v.27 no.9
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    • pp.1670-1680
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    • 2017
  • Lignocellulose, composed mostly of cellulose, hemicellulose, and lignin generated through secondary growth of woody plant, is considered as promising resources for biofuel. In order to use lignocellulose as a biofuel, biodegradation besides high-cost chemical treatments were applied, but knowledge on the decomposition of lignocellulose occurring in a natural environment is insufficient. We analyzed the 16S rRNA gene and metagenome to understand how the lignocellulose is decomposed naturally in decayed Torreya nucifera (L) of Bija forest (Bijarim) in Gotjawal, an ecologically distinct environment. A total of 464,360 reads were obtained from 16S rRNA gene sequencing, representing diverse phyla; Proteobacteria (51%), Bacteroidetes (11%) and Actinobacteria (10%). The metagenome analysis using single molecules real-time sequencing revealed that the assembled contigs determined originated from Proteobacteria (58%) and Actinobacteria (10.3%). Carbohydrate Active enZYmes (CAZy)- and Protein families (Pfam)-based analysis showed that Proteobacteria was involved in degrading whole lignocellulose, and Actinobacteria played a role only in a part of hemicellulose degradation. Combining these results, it suggested that Proteobacteria and Actinobacteria had selective biodegradation potential for different lignocellulose substrates. Thus, it is considered that understanding of the systemic microbial degradation pathways may be a useful strategy for recycle of lignocellulosic biomass, and the microbial enzymes in Bija forest can be useful natural resources in industrial processes.

16S rRNA Gene Sequence-based Microbial Diversity Analyses of the Geothermal Areas of Cisolok, Kamojang, and Likupang in Indonesia (16S rRNA 분석을 통한 인도네시아의 Cisolok, Kamojang, Likupang 지열지대 내 미생물 다양성 분석)

  • Seo, Myung-Ji;Kim, Jeong-Nyeo;Pyun, Yu-Ryang
    • Microbiology and Biotechnology Letters
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    • v.40 no.3
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    • pp.268-273
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    • 2012
  • Microbial diversity analyses were performed in several geothermal areas in Indonesia using a culture-independent approach with 16S rRNA gene sequencing. All areas and the majority of samples were noted as being affiliated with Proteobacteria. In addition, unclassified bacteria with no phylum affiliation were detected at an incidence rate of 20.0-26.5% in every location. The majority groupings in the geothermal hot stream in Cisolok belonged to ${\beta}$-Proteobacteria (27.1%) and Cyanobacteria (11.0%), whereas the majority from the volcanic area in Kamojang was ${\gamma}$-Proteobacteria (51.5%) followed by Aquificales (12.9%). The predominant groups around an underwater thermal vent in the sea at Likupang were ${\gamma}$-Proteobacteria (33.3%) and then Bacteroidetes (27.6%). This detailed microbial community analyses of each area strongly support a possible association with plausible community groups and environmental habitats, such as extremely geothermal or marine habitats. This study has significantly contributed to the expansion of scientific knowledge of the microbial community in Indonesia.

Analysis of the Microbiota on Lettuce (Lactuca sativa L.) Cultivated in South Korea to Identify Foodborne Pathogens

  • Yu, Yeon-Cheol;Yum, Su-Jin;Jeon, Da-Young;Jeong, Hee-Gon
    • Journal of Microbiology and Biotechnology
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    • v.28 no.8
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    • pp.1318-1331
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    • 2018
  • Lettuce (Lactuca sativa L.) is a major ingredient used in many food recipes in South Korea. Lettuce samples were collected during their maximum production period between April and July in order to investigate the microbiota of lettuce during different seasons. 16S rRNA gene-based sequencing was conducted using Illumina MiSeq, and real-time PCR was performed for quantification. The number of total bacterial was greater in lettuce collected in July than in that collected in April, albeit with reduced diversity. The bacterial compositions varied according to the site and season of sample collection. Potential pathogenic species such as Bacillus spp., Enterococcus casseliflavus, Klebsiella pneumoniae, and Pseudomonas aeruginosa showed season-specific differences. Results of the network co-occurrence analysis with core genera correlations showed characteristics of bacterial species in lettuce, and provided clues regarding the role of different microbes, including potential pathogens, in this microbiota. Although further studies are needed to determine the specific effects of regional and seasonal characteristics on the lettuce microbiota, our results imply that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria in lettuce.

Diversity Analysis of Lactic Acid Bacteria in Takju, Korean Rice Wine

  • Jin, Jianbo;Kim, So-Young;Jin, Qing;Eom, Hyun-Ju;Han, Nam-Soo
    • Journal of Microbiology and Biotechnology
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    • v.18 no.10
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    • pp.1678-1682
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    • 2008
  • To investigate lactic acid bacterial population in Korean traditional rice wines, biotyping was performed using cell morphology and whole-cell protein pattern analysis by SDS-PAGE, and then the isolates were identified by 16S rRNA sequencing analysis. Based on the morphological characteristics, 103 LAB isolates were detected in wine samples, characterized by whole-cell protein pattern analysis, and they were then divided into 18 patterns. By 16S rRNA gene sequencing, the isolates were identified as Lactobacillus paracasei, Lb. arizonensis, Lb. plantarum, Lb. harbinensis, Lb. parabuchneri, Lb. brevis, and Lb. hilgardii when listed by their frequency of occurrence. It was found that the difference in bacterial diversity between rice and grape wines depends on the raw materials, especially the com position of starch and glucose.

Phylogeny of the subfamily Salmoninae distributed in Korea based upon nucleotide sequences of mitochondrial ribosomal RNA genes (미토콘드리아 ribosomal RNA 유전자 염기서열분석에 의한 한국산 연어아과 어류의 유전적 계통도)

  • LEE Heui-Jung;PARK Jung-Youn;LEE Jeong-Ho;MIN Kwang-Sik;JEON Im Gi;YOO Mi-Ae;LEE Won-Ho
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.33 no.2
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    • pp.103-109
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    • 2000
  • Complete senuences of the mitochondrial rRNA Benes were determined among six salmonines in Korean Waters (Brachpmystax lenok, Onoorhpchus keta, O. masou mason, O. mason ishikawae, O. mykiss, and albino mutant of O. mykiss). The purposes of this study were to provide the basic information on levels of mtDNA polymorphism among these species for genetic characterization; discuss phylogentic relationships among three Oncorhynchus sepecies; demonstrate the utility of rRNA gene sequence data as a genetic marker for disringuishinf among Korean salmonines. PCR/direct sequencing data indicated the following consistent results; 1) 12S rRNA genes was 945 bases long in Oncorhynchus species, and 946 bases in B. lenot including one insertion. 2) Of sequence variation in mitochondrial rRNA regions, transitional substitutions were superior to transversion. 3) The significant differences were not shown in the intraspecific variation values in these gene regions. The percentage sequence divergence values were ranged from $0.066 to 0.212{\%}$. 4) The interspecific divergences were greater than the intraspecific variation. Nevertheless, ribosomal RMh genes were more conserved among species than the other mitochondrial genes, and they showed potentiality as an intergenic marker for systematics. In addition, phylogenetic trees, constructed from this data, supported that cherry salmon was closer to chum salmon than to rainbow trout, and that lenok was most distantly related species in six salmonid species.

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Microbiological Analysis of Dongchimi, Korean Watery Radish Kimchi, at the Early and Mid-phase Fermentation

  • Park, Sun-Jung;Chang, Jin-Hee;Cha, Seong-Kwan;Moon, Gi-Seong
    • Food Science and Biotechnology
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    • v.17 no.4
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    • pp.892-894
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    • 2008
  • During dongchimi fermentation at 5 and $25^{\circ}C$, the pH lowered slowly and reached 4.03 at $5^{\circ}C$ after 30 days, whereas it lowered dramatically and reached 3.59 at $25^{\circ}C$ after 2 days. The predominant bacteria were Leuconostoc (Leu.) mesenteroides at $25^{\circ}C$ until day 2 which changed into Lactobacillus (Lb.) plantarum at day 3, analyzed by a culture dependent method with partial 16S rRNA gene sequencing, whereas Leu. mesenteroides occupied predominantly at $5^{\circ}C$ until day 7. In a culture-independent method using a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) with partial 16S rRNA gene sequencing, Lb. algidus was predominant at $5^{\circ}C$ until day 7 and Lb. plantarum occupied predominantly at $25^{\circ}C$ until day 3, which is different from the results of the culture based method, indicating the both methods need to be combined for accuracy. Based on the culture-dependent method, Leu. mesenteroides might be responsible for the early and mid-phase of dongchimi fermentation.

Community Structure, Diversity, and Vertical Distribution of Archaea Revealed by 16S rRNA Gene Analysis in the Deep Sea Sediment of the Ulleung Basin, East Sea (16S rRNA 유전자 분석방법을 이용한 동해 울릉분지 심해 퇴적물 내 고세균 군집 구조 및 다양성의 수직분포 특성연구)

  • Kim, Bo-Bae;Cho, Hye-Youn;Hyun, Jung-Ho
    • Ocean and Polar Research
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    • v.32 no.3
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    • pp.309-319
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    • 2010
  • To assess community structure and diversity of archaea, a clone sequencing analysis based on an archaeal 16S rRNA gene was conducted at three sediment depths of the continental slope and Ulleung Basin in the East Sea. A total of 311 and 342 clones were sequenced at the slope and basin sites, respectively. Marine Group I, which is known as the ammonia oxidizers, appeared to predominate in the surface sediment of both sites (97.3% at slope, 88.5% at basin). In the anoxic subsurface sediment of the slope and basin, the predominant archaeal group differed noticeably. Marine Benthic Group B dominated in the subsurface sediment of the slope. Marine Benthic Group D and Miscellaneous Crenarchaeotal Group were the second largest archaeal group at 8-9 cm and 18-19 cm depth, respectively. Marine Benthic Group C of Crenarchaeota occupied the highest proportion by accounting for more than 60% of total clones in the subsurface sediments of the basin site. While archaeal groups that use metal oxide as an electron acceptor were relatively more abundant at the basin sites with manganese (Mn) oxide-enriched surface sediment, archaeal groups related to the sulfur cycle were more abundant in the sulfidogenic sediments of the slope. Overall results indicate that archaeal communities in the Ulleung Basin show clear spatial variation with depth and sites according to geochemical properties the sediment. Archaeal communities also seem to play a significant role in the biogeochemical carbon (C), nitrogen (N), sulfur (S), and metal cycles at each site.

Analysis of Microbial Communities Using Culture-dependent and Culture-independent Approaches in an Anaerobic/Aerobic SBR Reactor

  • Lu Shipeng;Park Min-Jeong;Ro Hyeon-Su;Lee Dae-Sung;Park Woo-Jun;Jeon Che-Ok
    • Journal of Microbiology
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    • v.44 no.2
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    • pp.155-161
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    • 2006
  • Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

Microbiological Characteristics of Nocardia takedensis Isolated from Skin Lesion, in Korea

  • Kang, Hye-Sook;Park, Gyu-Nam;Kim, Hye-Ran;Chang, Kyung-Soo
    • Biomedical Science Letters
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    • v.23 no.2
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    • pp.96-103
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    • 2017
  • Nocardia species (spp.) are opportunistic pathogen in immunocompromised hosts. The genus Nocardia contains more than 70 species. Nocardia takedensis has been recently reported as a new species of the genus Nocardia. In this study, we describes the first clinical isolate of N. takedensis from the skin lesion in Busan, Korea. For the identification of clinical isolate to the species level as N. takedensis, classical methods (colony morphology, biochemical characteristics, and antimicrobial susceptibility), molecular method (16S rRNA gene sequencing), and MS (mass spectrometry) analysis were conducted. Clinical isolates grew slowly on the culture media (5% sheep blood agar and chocolate agar) under 5% $CO_2$ condition. Especially, carotene pigmentation was detected well on the media. Using mass spectrometry, Nocardia isolate was not identified to the species level. However, molecular method based on 16S rRNA sequencing confirmed the isolate as N. takedensis correctly. N. takedensis isolate was partial positive for acid-fast bacilli on the Ziehl-Neelsen method. And it was observed to be resistance to amoxicillin/clavulanic acid and ciprofloxacin. Our results provide useful information to develop optimal identification protocol of N. takedensis in clinical diagnostic laboratories.

Seasonal Changes in the Microbial Communities on Lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea

  • Woojung Lee;Min-Hee Kim;Juyeon Park;You Jin Kim;Eiseul Kim;Eun Jeong Heo;Seung Hwan Kim;Gyungcheon Kim;Hakdong Shin;Soon Han Kim;Hae-Yeong Kim
    • Journal of Microbiology and Biotechnology
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    • v.33 no.2
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    • pp.219-227
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    • 2023
  • Lettuce is one of the most consumed vegetables worldwide. However, it has potential risks associated with pathogenic bacterial contamination because it is usually consumed raw. In this study, we investigated the changes in the bacterial community on lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea, and the prevalence of foodborne pathogens on lettuce in different seasons using 16S rRNA gene-based sequencing. Our data revealed that the Shannon diversity index showed the same tendency in term of the number of OTUs, with the index being greatest for summer samples in comparison to other seasons. Moreover, the microbial communities were significantly different between the four seasons. The relative abundance of Actinobacteriota varied according to the season. Family Micrococcaceae was most dominant in all samples except summer, and Rhizobiaceae was predominant in the microbiome of the summer sample. At the genus level, the relative abundance of Bacillus was greatest in spring samples, whereas Pseudomonas was greatest in winter samples. Potential pathogens, such as Staphylococcus and Clostridium, were detected with low relative abundance in all lettuce samples. We also performed metagenome shotgun sequencing analysis on the selected summer and winter samples, which were expected to be contaminated with foodborne pathogens, to support 16S rRNA gene-based sequencing dataset. Moreover, we could detect seasonal biomarkers and microbial association networks of microbiota on lettuce samples. Our results suggest that seasonal characteristics of lettuce microbial communities, which include diverse potential pathogens, can be used as basic data for food safety management to predict and prevent future outbreaks.