Comparison between DNA- and cDNA-based gut microbial community analyses using 16S rRNA gene sequences

조혜준,홍지완,운노타쯔야,Jo, Hyejun,Hong, Jiwan,Unno, Tatsuya The Microbiological Society of Korea 2019 미생물학회지 Vol.55 No.3

Studies based on microbial community analyses have increased in the recent decade since the development of next generation sequencing technology. Associations of gut microbiota with host's health are one of the major outcomes of microbial ecology filed. The major approach for microbial community analysis includes the sequencing of variable regions of 16S rRNA genes, which does not provide the information of bacterial activities. Here, we conducted RNA-based microbial community analysis and compared results obtained from DNA- and its cDNA-based microbial community analyses. Our results indicated that these two approaches differed in the ratio of Firmicutes and Bacteroidetes, known as an obesity indicator, as well as abundance of some key bacteria in gut metabolisms such as butyrate producers and probiotics strains. Therefore, cDNA-based microbial community may provide different insights regarding roles of gut microbiota compared to the previous studies where DNA-based microbial community analyses were performed. 최근 10년간 미생물생태분석 기반의 연구는 차세대염기서열분석법이 개발된 이래로 지속적으로 증가하고 있다. 장내미생물생태와 건강의 연관성은 미생물 생태학 분야에 있어서 중요한 결과로 여겨지고 있다. 미생물 군집 분석은 주로 16S rRNA 유전자 가변 영역의 염기서열을 통해 분석되지만 이는 미생물의 활성 정보를 제공하지 않는다. 본 연구에서는 cDNA 기반의 미생물 생태분석을 수행하고 DNA 및 cDNA기반의 미생물생태분석 결과를 비교하였다. 두 가지의 서로 다른 접근법이 Butyrate producer와 probiotics와 같이 장내 대사과정에서 중요한 미생물의 abundance 뿐만 아니라 비만 지표로 알려진 Firmicutes 와 Bacteroidetes의 비율에 있어서 차이가 있음을 나타내었다. 따라서, cDNA 기반 미생물 군집은 이전에 수행된 DNA 기반 미생물 군집 분석과 비교하여 장내미생물생태의 역할과 관련된 또 다른 분석 방향성을 제공한다.

Metabolic Modeling of Microbial Community Interactions for Health, Environmental and Biotechnological Applications

Ang, Kok Siong,Lakshmanan, Meiyappan,Lee, Na-Rae,Lee, Dong-Yup Bentham Science Publishers 2018 Current genomics Vol.19 No.8

<P>In nature, microbes do not exist in isolation but co-exist in a variety of ecological and biological environments and on various host organisms. Due to their close proximity, these microbes interact among themselves, and also with the hosts in both positive and negative manners. Moreover, these interactions may modulate dynamically upon external stimulus as well as internal community changes. This demands systematic techniques such as mathematical modeling to understand the intrinsic community behavior. Here, we reviewed various approaches for metabolic modeling of microbial communities. If detailed species-specific information is available, segregated models of individual organisms can be constructed and connected via metabolite exchanges; otherwise, the community may be represented as a lumped network of metabolic reactions. The constructed models can then be simulated to help fill knowledge gaps, and generate testable hypotheses for designing new experiments. More importantly, such community models have been developed to study microbial interactions in various niches such as host microbiome, biogeochemical and bioremediation, waste water treatment and synthetic consortia. As such, the metabolic modeling efforts have allowed us to gain new insights into the natural and synthetic microbial communities, and design interventions to achieve specific goals. Finally, potential directions for future development in metabolic modeling of microbial communities were also discussed.</P>

Microbial Community Analysis using RDP 2 (Ribosomal Database Project 2): Methods, Tools and New Advances

Erick Cardenas,James R. Cole,James M. Tiedje,Joon Hong Park 대한환경공학회 2009 Environmental Engineering Research Vol.14 No.1

Microorganisms play an important role in the geochemical cycles, industry, environmental cleanup, and biotechnology among other fields. Given the high microbial diversity, identification of the microorganism is essential in understanding and managing the processes. One of the most popular and powerful method for microbial identification is comparative 16S rRNA gene analysis. Due to the highly conserved nature of this essential gene, sequencing and later comparison of it against known rRNA databases can provide assignment of the bacteria into the taxonomy, and identity of its closest relatives. Isolation and sequencing of 16S rRNA genes directly from natural environments (either from DNA or RNA) can also be used to study the structure of the whole microbial community. Nowadays, novel sequencing technologies with massive outputs are giving researchers worldwide the chance to study the microbial world with a depth that was previously too expensive to achieve. In this article we describe commonly used research approaches for the study of individual microorganisms and microbial communities using the tools provided by Ribosomal Database Project website.

Identification of microbial communities, with a focus on foodborne pathogens, during kimchi manufacturing process using culture-independent and -dependent analyses

Lee, H.W.,Yoon, S.R.,Kim, S.J.,Lee, H.M.,Lee, J.Y.,Lee, J.H.,Kim, S.H.,Ha, J.H. Academic Press, etc 2017 FOOD SCIENCE AND TECHNOLOGY -ZURICH- Vol.81 No.-

<P>Little is known about microbial communities associated with the kimchi manufacturing process. We investigated the structure of microbial communities, focusing on foodborne pathogens, such as Bacillus cereus, enterohemorrhagic Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Salmonella spp., Listeria monocytogenes, and Yersinia enterocolitica, during the kimchi manufacturing process, including the brining, washing, and condiment stuffing phases, using culture dependent and culture-independent analyses. Foodborne pathogens were not identified in any test sample by the culture-dependent analysis, whereas the culture-independent analysis targeting the hypervariable regions V3 and V4 of the 16S rRNA gene detected traces of general or potential foodborne pathogens, such as Campylobacter spp., Enterobacter spp., Escherichia or Shigella spp., Serratia spp., Vibrio spp., Aeromonas spp., Acinetobacter spp., Pseudomonas spp., and Enterococcus spp. However, as the kimchi manufacturing process proceeded, the structure of the microbial community changed, and potential foodborne pathogens were replaced by probiotics, such as lactic acid bacteria. Our results suggest that it may be worth utilizing the culture-independent analysis to complement the culture-dependent analysis for microbiological quality control of the kimchi manufacturing process. (C) 2017 Elsevier Ltd. All rights reserved.</P>

Investigation of microbial communities in water dispensers

박상정,Adeel Farooq,조혜준,김지혜,양미희,고영호,강성모,운노 타쯔야,정현미 한국응용생명화학회 2017 Applied Biological Chemistry (Appl Biol Chem) Vol.60 No.6

Water dispensers remove disinfectant residues from tap water and thus are commonly present in Korean households; however, microbial contamination in water dispensers has recently become a major issue. To understand the occurrence of microbial contamination in water dispensers, we investigated microbial contamination in different dispenser types through heterotrophic plate count (HPC) and MiSeq-based microbial community analyses. Two newly purchased water dispensers were placed in a basement room and left for approximately 2 months; the HPC analysis indicated microbial contamination in the drinking water collected from these dispensers (160,000 and 48,000 CFU/mL, respectively). Taxonomic classification indicated that the basement dispensers were likely contaminated by freshwater bacteria, such as Acidovorax and Methylotenera. However, two dispensers located at the half landing and the first floor showed lower microbial contamination (110 and 78 CFU/mL, respectively). Furthermore, frequently used dispenser on the first floor showed higher HPCs on the faucet surface, which were classified as general oral bacteria like Hyphobacterium. We also observed that a deserted dispenser (6-year-old with no maintenance) placed on the half landing showed the least HPCs, although it seemed to have lost its filtration ability. Our results suggested that removal of disinfectant residues leads to an increase in the freshwater bacterial population in water tanks within dispensers, which could be avoided by frequent water circulation.

메타프로테오믹스의 미생물생태학적 응용

김종식,우정희,김준태,박년호,김충곤,Kim, Jong-Shik,Woo, Jung-Hee,Kim, Jun-Tae,Park, Nyun-Ho,Kim, Choong-Gon 한국미생물학회 2010 미생물학회지 Vol.46 No.1

미생물 군집과 기능연구를 위한 최근의 새로운 분석기술의 발전은 다양한 유전 관련 정보를 제공해왔다. mRNA를 포함하는 핵산을 기초로 한 연구를 뛰어넘어서 메타프로테오믹스는 미생물 군집의 유전형 및 표현형의 특징적인 정보를 보다 정교하게 제공할 수 있다. 이미 서로 다른 미생물 생태계인 해수, 인간의 배설물, 활성 슬러지, 산성 광산 폐수 생물막, 토양 등에 메타프로테오믹스 기술이 유용하게 사용되었다. 이들 연구는 여러 측면에서 상당히 다르지만 미생물 군집의 구조, 기능, 생리, 상호관계, 생태, 진화적 측면을 결정적으로 상호 연결한다는 것을 밝혀냈다. 본 총설은 메타프로테오믹스에 대한 현재까지의 가장 최신의 정보를 요약하여 제공함으로써 메타프로테오믹스에 대한 정확한 이해와 활용을 통해 다방면의 메타프로테오믹스가 가능하도록 하고자 하였다. New technologies are providing unprecedented knowledge into microbial community structure and functions. Even though nucleic acid based approaches provide a lot of information, metaproteomics could provide a high-resolution representation of genotypic and phenotypic traits of distinct microbial communities. Analyzing the metagenome from different microbial ecosystems, metaproteomics has been applied to seawater, human guts, activated sludge, acid mine drainage biofilm, and soil. Although these studies employed different approaches, they elucidated that metaproteomics could provide a link among microbial community structure, function, physiology, interaction, ecology, and evolution. These approaches are reviewed here to help gain insights into the function of microbial community in ecosystems.

유류오염대수층 고온공기분사공정시 제한효소다형성 미생물 군집

이준호 ( Jun Ho Lee ),박갑성 ( Kap Song Park ) 한국물환경학회 2008 한국물환경학회지 Vol.24 No.1

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air (34.9±2.7°C) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, γ-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.

절식이 마우스 장내미생물에 미치는 영향

홍지완 ( Jiwan Hong ),조혜준 ( Hyejun Jo ),운노타쯔야 ( Tatsuya Unno ) 한국응용생명화학회(구 한국농화학회) 2019 Journal of Applied Biological Chemistry (J. Appl. Vol.62 No.4

본 연구에서는 절식이 일반사료 및 고지방사료를 섭취한 마우스의 장내미생물생태에 미치는 영향에 관하여 조사하였다. 일반사료 및 고지방사료를 마우스에 16주 동안 무한급이 형태로 섭취하게 하여 실험종료 직전에 1일(24시간) 간 절식시켰으며, 절식 전 후의 분변 샘플을 채집하고 MiSeq을 이용하여 장내미생 물생태 분석을 진행하였다. 본 연구의 결과는 고지방사료를 섭취한 마우스에서는 절식 전후 장내미생물생태 내의 종 풍부성과 균등성이 감소한 반면, 일반사료를 섭취한 마우스에서는 차이를 나타내지 않았다. 또한, 고지방사료를 섭취한 마우스의 장내미생물생태에서는 절식 후 변화하는 것을 확인하였으며, 일반사료를 섭취한 마우스의 절식 전후 변화가 없었다. Difference abundance analysis는 일반사료를 섭취한 마우스에서 절식 이후S24-7로 분류되는 OTU는 증가하고, Ruminococcaceae로 분류되는 OTU가 감소하는 것으로 확인되었다. 반면, 고지방사료를 섭취한 마우스에서는 절식 이후 10OTUs가 감소하고, 3OTUs가 증가하였는데, 대부분 Ruminococcaceae로 분류되는 것으로 확인되었다. 본 연구 결과는 마우스에서의 절식이 장내미생물생태 중 Ruminococcaceae의 abundance에 영향을 미치며, 일반사료를 섭취한 마우스에 비해 고지방사료를 섭취한 마우스에서 절식에 대한 효과가 더 명확하게 나타난다는 것을 시사한다. In this study, we investigated the effects of fasting on gut microbiota of mice fed normal (CTL) or high-fat diets (HF). Mice were raised for 16 weeks and fasted for a day at the end of the experiment. Fecal samples were collected one day before and after fasting, which were analyzed using MiSeq. Our results showed that the species richness and evenness were decreased in fasted HF group, whereas no difference was observed for CTL groups. Moreover, HF fed mice gut microbiota showed different microbial communities after fasting, while CTL groups did not show microbiota shifts. Differential abundance analysis showed that fasting CTL group mice increased and decreased one operational taxonomic unit (OTU) in S24_7 and one OTU in Ruminococcaceae, respectively. On the other hand, fasting HF group mice decreased 10 OTUs and increased 3 OTUs most of which were classified to Ruminococcaceae. Our results suggest that fasting mice may affect the abundance of Ruminococcaceae species and effects of fasting seem to be more obvious for HF-fed mice compared to those of mice fed CTL-diet.

Denitrifying Phosphate Accumulating Organisms (dPAOs)을 이용한 영양소제거 및 반응조내 미생물 분포 조사

박용근,이진우,이한웅,이수연,최의소 한국미생물학회 2002 미생물학회지 Vol.38 No.2

혐기-무산소조건으로 구성된 회분식 반응조에서 질산염을 이용하여 인(P)도 동시에 제거될 수 있는 가능성을 알아보기 위해서 인의 제거 양상을 혐기-호기조건의 반응조와 비교하여 조사하였고, 질산염과 인을 동시에 제거하는 미생물분포를 분석하였다. 그 결과 비교적 낮은 농도의 유기물이 적용되었을 때(평균 CODcr=130mg/ι)두 반응조 모두 인이 효과적으로 제거되었으며 반응조내의 최종 인의 농도를 1 mg P/L. 이하로 유지하였다. 특히, 질산염을 전자수용체로 이용한 혐기-무산소조건의 반응조는 기존의 영양소제거 시스템과 비교하여 5-7 mg (P+N)/ι의 영양소를 추가적으로 제거하여 유기물의 효과적 인 이용이 가능한 것으로 판명되었다. 혐기-무산조 조건의 방응조내 미생물 분포를 조사 한 결과 질소원을 제거하는 미생물군(denitrifying bacteria)과 인을 제거하는 미생물군(polyphosphate accumulating bacteria)이 함께 존재하고 있음이 밝혀졌고, 이들 중 $\beta$-proteobacteria에 속하는 Zoogloea ramigera와 Rhodocyclus에 포함되는Alcaligenes defragrans 등은 탈질능력 이 있으면서 anoxic상태에서 인을 동시에 축적할 수 있는 탈질-탈인균주(denitrifying phosphate accumulating organisms; dPAO)로 조사되었다. Laboratory experiments were aimed to evaluate the effect of nitrate as a electron acceptor during the biological phosphorus uptake and to investigate the microbial community. Anaerobic-anoxic sequencing batch reactor (SBR) compared the removal behaviour to anaerobic-oxic SBR, both SBRs maintained lower effluent quality with 1.0 mgp/1. Anaerobic-anoxic SBR was able to remove additional 5.0 to 7.0 mg (P+N)/ι than other biological nutrient removal (BM) system. Therefore, it was proposed that the anaerobic-anoxic SBR was more effective at weak sewage. From the results of the maicrobial community analysis, it can be inferred that denitrifying bacteria and polyphosphate accumulating bacteria coexist in anaerobic-anoxic SBR during stable condition for removing the nitrogen and phosphorus. Particularly, it was suggested that the Zoogloea ramigera in the $\beta$-subclass of proteobacteria and the Alcaligenes defragrans of the Rhodocyclus group in the $\beta$-subclass of proteobacteria played a major role for removing the nitrogen and phosphorus as dPAOs (denitrifying phosphate accumulating organisms).

Using T-RFLP to Assess the Impact on Soil Microbial Communities by Transgenic Lines of Watermelon Rootstock Resistant to Cucumber Green Mottle Mosaic Virus (CGMMV)

Hoonbok Yi,김효정,김창기,한지학,Hwan Mook Kim,Sangkyu Park 한국식물학회 2009 Journal of Plant Biology Vol.52 No.6

To establish quantitative methods for ecological risk assessment, we assessed the impacts of transgenic watermelon rootstock (Citrullus lanatus (Twinser) cv. Gongdae) that was resistant to cucumber green mottle mosaic virus. The diversity of soil bacteria and fungi was monitored from May to July of 2005. Terminal restriction fragment length polymorphism (T-RFLP) was used with 16S ribosomal RNA (rRNA) coding genes for bacterial communities and with internal transcribed spacer (ITS) regions of rRNA coding genes for fungal communities. Multivariate analysis of variance (MANOVA) on the principal component analysis (PCA) scores of T-RF profiles detected no significant difference between microbial communities with transgenic or non-transgenic watermelon. Likewise, the results of our multi-response permutation procedure (MRPP) tests on non-metric multidimensional scaling (NMS) showed no significant difference between plant types. However, both MANOVA on PCA and MRPP on NMS revealed significant changes in the microbial community during the growing season. We used loading values of PCA to rank the abundances of bacterial species and found increases of some species in June and July.