scholarly journals Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities

2001 ◽  
Vol 67 (1) ◽  
pp. 190-197 ◽  
Author(s):  
John Dunbar ◽  
Lawrence O. Ticknor ◽  
Cheryl R. Kuske
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Restriction Fragment ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Soil Dna ◽  
Microbial Genomes ◽  
Analysis Technique ◽  
Phylogenetic Resolution

ABSTRACT Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from theProteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.

scholarly journals Respiration of 13C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13C-Labeled Soil DNA

2003 ◽  
Vol 69 (3) ◽  
pp. 1614-1622 ◽  
Author(s):  
P. Padmanabhan ◽  
S. Padmanabhan ◽  
C. DeRito ◽  
A. Gray ◽  
D. Gannon ◽  
...  
Keyword(s):  
Organic Matter ◽  
Soil Organic Matter ◽  
16S Rrna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gas Chromatography Mass Spectrometry ◽  
Rrna Gene ◽  
Labeled Compounds ◽  
Field Soil ◽  
Soil Dna

ABSTRACT Our goal was to develop a field soil biodegradation assay using 13C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13CO2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13CO2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF6, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13CO2. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13CO2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

scholarly journals Identification of a Stable Hydrogen-Driven Microbiome in a Highly Radioactive Storage Facility on the Sellafield Site

2020 ◽  
Vol 11 ◽  
Author(s):  
Sharon Ruiz-Lopez ◽  
Lynn Foster ◽  
Chris Boothman ◽  
Nick Cole ◽  
Katherine Morris ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Nuclear Fuel ◽  
Spent Nuclear Fuel ◽  
Quantitative Polymerase Chain Reaction ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Spent Fuel

The use of nuclear power has been a significant part of the United Kingdom’s energy portfolio with the Sellafield site being used for power production and more recently reprocessing and decommissioning of spent nuclear fuel activities. Before being reprocessed, spent nuclear fuel is stored in water ponds with significant levels of background radioactivity and in high alkalinity (to minimize fuel corrosion). Despite these challenging conditions, the presence of microbial communities has been detected. To gain further insight into the microbial communities present in extreme environments, an indoor, hyper-alkaline, oligotrophic, and radioactive spent fuel storage pond (INP) located on the Sellafield site was analyzed. Water samples were collected from sample points within the INP complex, and also the purge water feeding tank (FT) that supplies water to the pond, and were screened for the presence of the 16S and 18S rRNA genes to inform sequencing requirements over a period of 30 months. Only 16S rRNA genes were successfully amplified for sequencing, suggesting that the microbial communities in the INP were dominated by prokaryotes. Quantitative Polymerase Chain Reaction (qPCR) analysis targeting 16S rRNA genes suggested that bacterial cells in the order of 104–106 mL–1 were present in the samples, with loadings rising with time. Next generation Illumina MiSeq sequencing was performed to identify the dominant microorganisms at eight sampling times. The 16S rRNA gene sequence analysis suggested that 70% and 91% from of the OTUs samples, from the FT and INP respectively, belonged to the phylum Proteobacteria, mainly from the alpha and beta subclasses. The remaining OTUs were assigned primarily to the phyla Acidobacteria, Bacteroidetes, and, Cyanobacteria. Overall the most abundant genera identified were Hydrogenophaga, Curvibacter, Porphyrobacter, Rhodoferax, Polaromonas, Sediminibacterium, Roseococcus, and Sphingomonas. The presence of organisms most closely related to Hydrogenophaga species in the INP areas, suggests the metabolism of hydrogen as an energy source, most likely linked to hydrolysis of water caused by the stored fuel. Isolation of axenic cultures using a range of minimal and rich media was also attempted, but only relatively minor components (from the phylum Bacteroidetes) of the pond water communities were obtained, emphasizing the importance of DNA-based, not culture-dependent techniques, for assessing the microbiome of nuclear facilities.

scholarly journals High Diversity in Iron Cycling Microbial Communities in Acidic, Iron-Rich Water of the Pyhäsalmi Mine, Finland

Geofluids ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Malin Bomberg ◽  
Jarno Mäkinen ◽  
Marja Salo ◽  
Päivi Kinnunen
Keyword(s):  
16S Rrna ◽  
Microbial Diversity ◽  
Microbial Communities ◽  
Mine Water ◽  
Ferrous Iron ◽  
Great Part ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Iron Oxidizers

Microbial communities of iron-rich water in the Pyhäsalmi mine, Finland, were investigated with high-throughput amplicon sequencing and qPCR targeting bacteria, archaea, and fungi. In addition, the abundance ofLeptospirillumandAcidithiobacilluswas assessed with genus-specific qPCR assays, and enrichment cultures targeting aerobic ferrous iron oxidizers and ferric iron reducers were established. The acidic (pH 1.4–2.3) mine water collected from 240 m, 500 m, and 600 m depth from within the mine had a high microbial diversity consisting of 63-114 bacterial, 10-13 archaeal, and 104-117 fungal genera. The most abundant microorganisms in the mine water were typical acid mine drainage (AMD) taxa, such as acidophilic, iron-oxidizingLeptospirillum,Acidiphilum,Acidithiobacillus,Ferrovum, andThermoplasma. The fungi belonged mostly to the phylum Ascomycetes, although a great part of the fungal sequences remained unclassified. The number of archaeal 16S rRNA genes in the mine water was between 0.3 and 1.2 × 107copies mL−1in the samples from 500 m and 600 m, but only 3.9 × 103at 240 m and archaea were in general not enriched in cultures. The number of fungal 5.8S rRNA genes was high only in the mine water from 500 m and 600 m, where 0.2–3.4 × 104spore equivalents mL−1were detected. A high number ofLeptospirillum16S rRNA genes, 0.6–1.6 × 1010copies mL−1, were detected at 500 m and 600 m depth and in cultures containing ferrous iron, showing the importance of iron oxidizers in this environment. The abundance of bacteria in general was between 103and 10616S rRNA gene copies mL−1. Our results showed a high microbial diversity in the acid- and iron-impacted waters of the Pyhäsalmi mine, whereLeptospirillumbacteria were especially prominent. These iron oxidizers are also the main nitrogen-fixing microorganisms in this ecosystem.

scholarly journals Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products

2004 ◽  
Vol 70 (5) ◽  
pp. 2577-2587 ◽  
Author(s):  
Manuel Pesaro ◽  
Gilles Nicollier ◽  
Josef Zeyer ◽  
Franco Widmer
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Stress Responses ◽  
Crop Protection ◽  
16S Rrna Genes ◽  
Cell Counting ◽  
Rrna Genes ◽  
Soil Dna ◽  
Degradation Rates ◽  
The Impact

ABSTRACT Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions. For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests. During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates. We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days. The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference. The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively. The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration. The effects on Bacteria, Archaea, and Pseudomonas were investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting. Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress. In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities. We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated.

scholarly journals Comparative genomics of the genus Bifidobacterium

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3243-3254 ◽  
Author(s):  
Francesca Bottacini ◽  
Duccio Medini ◽  
Angelo Pavesi ◽  
Francesca Turroni ◽  
Elena Foroni ◽  
...  
Keyword(s):  
16S Rrna ◽  
Genome Structure ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pan Genome ◽  
Single Genome ◽  
Phylogenetic Resolution ◽  
Functional Analyses ◽  
The 16S Rrna Gene

Whole-genome sequencing efforts have revolutionized the study of bifidobacterial genetics and physiology. Unfortunately, the sequence of a single genome does not provide information on bifidobacterial genetic diversity and on how genetic variability supports improved adaptation of these bacteria to the environment of the human gastrointestinal tract (GIT). Analysis of nine genomes from bifidobacterial species showed that such genomes display an open pan-genome structure. Mathematical extrapolation of the data indicates that the genome reservoir available to the bifidobacterial pan-genome consists of more than 5000 genes, many of which are uncharacterized, but which are probably important to provide adaptive abilities pertinent to the human GIT. We also define a core bifidobacterial gene set which will undoubtedly provide a new baseline from which one can examine the evolution of bifidobacteria. Phylogenetic investigation performed on a total of 506 orthologues that are common to nine complete bifidobacterial genomes allowed the construction of a Bifidobacterium supertree which is largely concordant with the phylogenetic tree obtained using 16S rRNA genes. Moreover, this supertree provided a more robust phylogenetic resolution than the 16S rRNA gene-based analysis. This comparative study of the genus Bifidobacterium thus presents a foundation for future functional analyses of this important group of GIT bacteria.

scholarly journals Application of the stochastic labeling methods with random-sequence barcodes for simultaneous quantification and sequencing of environmental 16S rRNA genes

2016 ◽  
Author(s):  
Tatsuhiko Hoshino ◽  
Fumio Inagaki
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Copy Number ◽  
Random Sequence ◽  
Digital Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
The 16S Rrna Gene

AbstractNext-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5’ end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103to 5.0 × 104copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

scholarly journals Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

1999 ◽  
Vol 65 (9) ◽  
pp. 4280-4284 ◽  
Author(s):  
Katrina A. O’Farrell ◽  
Peter H. Janssen
Keyword(s):  
16S Rrna ◽  
Restriction Analysis ◽  
Comparative Sequence Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Soil Dna ◽  
Probable Number ◽  
Pasture Soil

ABSTRACT Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial divisionVerrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.

scholarly journals Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles

2014 ◽  
Vol 80 (18) ◽  
pp. 5717-5722 ◽  
Author(s):  
Katherine Kennedy ◽  
Michael W. Hall ◽  
Michael D. J. Lynch ◽  
Gabriel Moreno-Hagelsieb ◽  
Josh D. Neufeld
Keyword(s):  
16S Rrna ◽  
Sample Preparation ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Massively Parallel Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Sample Sequence ◽  
Multiple Pcr

ABSTRACTMassively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

scholarly journals Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis

2000 ◽  
Vol 66 (7) ◽  
pp. 2943-2950 ◽  
Author(s):  
John Dunbar ◽  
Lawrence O. Ticknor ◽  
Cheryl R. Kuske
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Community Diversity ◽  
Detection Sensitivity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Clone Libraries

ABSTRACT The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 16S rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662–1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, HhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

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