Genomic methods in analyzing the communities of soil bacteria

Culture-independent examination of complex microbial communities has been made possible by recent advances in molecular biology. Terminal restriction fragment length polymorphism is one of such techniques that allow rapid assessment of the diversity of microbial community

Application of terminal restriction fragment length polymorphism to environmental biotechnology

制度:新 ; 文部省報告番号:甲2608号 ; 学位の種類:博士(工学) ; 授与年月日:2008/3/15 ; 早大学位記番号:新476

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common technique used to characterize soil microbial diversity. The fidelity of this technique in accurately reporting diversity has not been thoroughly evaluated. Here we determine if rare fungal species can be reliably detected by T-RFLP analysis. Spores from three arbuscular mycorrhizal fungal species were each mixed at a range of concentrations (1%, 10%, 50%, and 100%) with Glomus irregulare to establish a minimum detection threshold. T-RFLP analysis was capable of detecting diagnostic peaks of rare taxa at concentrations as low as 1%. The relative proportion of the target taxa in the sample and DNA concentration influenced peak detection reliability. However, low concentrations produced small, inconsistent electropherogram peaks contributing to difficulty in differentiating true peaks from signal noise. The results of this experiment suggest T-RFLP is a reproducible and high fidelity procedure, which requires careful data interpretation in order to accurately characterize sample diversity

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Includes bibliographical references (page 932).Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units

Terminal restriction fragment length polymorphism is an “old school” reliable technique for swift microbial community screening in anaerobic digestion

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants

Profile of Gut Bacteria in Hypertensive Patients Based on Terminal Restriction Fragment Length Polymorphism Analysis

Hypertension is a severe public health problem due to its high prevalence worldwide. About 7.5 million deaths or 12.8% of all annual deaths worldwide occur due to high blood pressure. The hypertensive disease is also associated with the intestinal bacteria. To our knowledge, the diversity of the gut bacteria in hypertensive patients has not been reported yet in Indonesia. Thus, the aims of this study were to analyze profile of gut bacteria in hypertensive patients compared to non-hypertensive based on metagenomic analysis, Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results of the affiliation analysis of the entire Terminal Restriction Fragments (TRF) contained 6 groups of bacteria from 26 TRFs in hypertensive and non-hypertensive respondents. Cutting the 16S rRNA gene with the BslI restriction enzyme successfully detected intestinal bacterial groups, namely Clostridium subcluster XIVa, Prevotella, Clostridium cluster IV, Clostridium cluster XI, Bacteroides and others. In hypertensive patients, a higher relative abundance of bacterial groups showed in Clostridium cluster XI, Clostridium cluster IV and Clostridium subcluster XIVa. The abundance of  Bacteroides and Prevotella groups in hypertensive patients were lower than non-hypertensive. The abundance of enterotype I and enterotype II was lower in hypertensive patients than non-hypertensive. Contrarily to that enterotype III cluster. It is worth noting that the F/B ratio was higher in hypertensive patients than that in non-hypertensive. Our data suggest that the gut bacteria profile of hypertensive patients differs to that non-hypertensive

Microbial Diversity in Raw and Pasteurized Milk with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Abstract In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Overall, the results demonstrated that T-RFLP is a powerful tool for analyzing microbiota in foods. In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The initial hypothesis, that the two pasteurization techniques affect different parts of the microbiota of the raw milk, was confirmed. As expected, the molecular approach of DNA extraction direct from the milk detected a more differentiated microflora, compared to DNA extracted from cultivated bacteria from the milk. The results also indicated that the molecular approach was more reproducible between the sampling occasions. The cultivation and sequencing showed that the microbiota mainly consisted of Firmicutes and Actinobacteria, as well as Bacteriodetes. Popular science summary: Microbial Diversity in Raw and Pasteurized milk In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Much of the flavor and aroma of milk and cheese originates in its microbial content. Pasteurization is used to minimize the pathogens and extend the shelf-life for food. It decreases the number of microorganisms in the product by heating, followed by an immediate cooling. The treatment does not sterilize the product, but reduces the number of viable microorganisms. The awareness and control of the bacterial diversity in the milk is therefore a very important aspect in production of cheese, dairy product and other food products. Traditional methods to assess community diversity often include culturing on plates. In addition to being labor-intensive, it has proven to give a deceptive reflection of the original community structure. Molecular biology methods are based on variations in the DNA sequences between bacteria species. T-RFLP visualizes differences in the prokaryotic 16S rRNA. The analysis typically involve four steps: DNA isolation and purification, PCR amplification and restriction enzyme digestion, separation of digested products via capillary gel electrophoresis and finally analysis and clustering of data to generate a fragment profile for each sample. Identity of the terminal restriction fragments can be obtained by creation of a clone library. Effect of pasteurization The two pasteurization techniques affect different parts of the microbiota of the raw milk. Traditional pasteurization showed the greatest decrease in number of bacterial species. The cheese microbiota was considerably less diverse. As expected, the number of bacterial species found from the non-cultured population was greater than from the cultivatable population. The results also indicated that the molecular approach was more reproducible between the sampling occasions. A majority of the identified bacteria belonged to the Firmicutes phylum and were lactic acid bacteria (LAB). In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The results confirmed the advantages of using molecular opposed to culture-based approaches to characterize microbiota. The study also verified the applicability of T-RFLP for analyzing microbiota in foods. Advisor: Klara Båth Degree Project 30 credits in Cell and Molecular Biology 2012 Department of Biology, Lund Universit

Stable isotope probing: Technical considerations when resolving ¹⁵N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. ¹⁵N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using ¹⁵N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used ¹⁵N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with ¹⁵N-ammonium chloride or ¹⁵N₂ we assessed the fractionation resolution of ¹⁵N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between ¹⁵N- and ¹⁴N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments

It Takes an Individual Plant to Raise a Community: TRFLP Analysis of the Rhizosphere Microbial Community of Two Pairs of High- and Low-Metal-Accumulating Plants

We used terminal restriction fragment length polymorphism (TRFLP) analysis to look at the microbial community profiles of the rhizosphere surrounding two pairs of high- and low-metal (Cd)-accumulating plants (Brassica and Triticum). Unexpectedly, the microbial community did not vary with soil type, time, plant type, or metal-accumulating ability of the plant. Instead, when a plant\u27s metal-accumulating ability was well matched to the level of metal contamination in the soil, the microbial populations in the rhizosphere were different than those of the seed endophytes and bulk soil. Unmatched plants had the same microbial community as bulk soil. The plant interaction with the soil, therefore, is essential to forming the bacterial community in the rhizosphere

Comparison of bacterioneuston and bacterioplankton dynamics during a phytoplankton bloom in a fjord mesocosm

The bacterioneuston is the community of Bacteria present in surface microlayers, the thin surface film that forms the interface between aquatic environments and the atmosphere. In this study we compared bacterial cell abundance and bacterial community structure of the bacterioneuston and the bacterioplankton (from the subsurface water column) during a phytoplankton bloom mesocosm experiment. Bacterial cell abundance, determined by flow cytometry, followed a typical bacterioplankton response to a phytoplankton bloom, with Synechococcus and high nucleic acid (HNA) bacterial cell numbers initially falling, probably due to selective protist grazing. Subsequently HNA and low nucleic acid (LNA) bacterial cells increased in abundance but Synechococcus did not. There was no significant difference between bacterioneuston and bacterioplankton cell abundances during the experiment. Conversely, distinct and consistent differences between the bacterioneuston and the bacterioplankton community structure were observed. This was monitored simultaneously by Bacteria 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). The conserved patterns of community structure observed in all of the mesocosms indicate that the bacterioneuston is distinctive and non-random