Project description:This experiment aims on the identification of serine hydrolases from a complex thermophile community that live in a hot vent in Kamchatka Peninsula based on in vivo labelling with FP-alkyne directly in the hot spring and subsequent analysis using metagenomics/metaproteomics. To this end, sediment samples were collected and treated using the following three conditions. DMSO- treated control FP-alkyne labelled Samples for each condition were prepared in triplicate, resulting a total number of 6 samples per spring. Labelling was performed using 4 µM of the probe FP-alkyne and incubation for 2 h in the hot spring.
Project description:To determine whether and how warming affects the functional capacities of the active microbial communities, GeoChip 5.0 microarray was used. Briefly, four fractions of each 13C-straw sample were selected and regarded as representative for the active bacterial community if 16S rRNA genes of the corresponding 12C-straw samples at the same density fraction were close to zero.
Project description:The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the application of amplicon pyrosequencing. The possibility of barcode-tagged sequencing of templates gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing and, as in the early days of genetic community fingerprinting, pros and cons are continuously provided. In this study we investigate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of natural microbiota. Moreover, via quantitatively defined template spiking to the natural community, we explore the potential for recovering specific template ratios within complex microbial communities. For this reason, we pyrotag sequenced three biological replicates of three samples, each belonging from yearly sampling campaigns of sediment from a tar oil contaminated aquifer in Düsseldorf, Germany. Furthermore, we subjected one DNA extract to replicate technical analyses as well as to increasing ratios (0, 0.2, 2 and 20%) of 16S rRNA genes from a pure culture (Aliivibrio fisheri) originally not present in the sample. Unexpectedly, taxa abundances were highly reproducible in our hands, with max standard deviation of ~3% abundance across biological and ~2% for technical replicates. Furthermore, our workflow was also capable of recovering A. fisheri amendmend ratios in reliable amounts (0, 0.29, 3.9 and 23.8%). These results highlight that pyrotag sequencing, if done and evaluated with due caution, has the potential to robustly recapture taxa template abundances within environmental microbial communities. 9 Biological and 3 technical replicates were evaluated, as well as potential to recover qPCR-defined ratios of DNA, in 454 pyrotag sequencing
Project description:Anaerobic digestion is a popular and effective microbial process for waste treatment. The performance of anaerobic digestion processes is contingent on the balance of the microbial food web in utilizing various substrates. Recently, co-digestion, i.e., supplementing the primary substrate with an organic-rich co-substrate has been exploited to improve waste treatment efficiency. Yet the potential effects of elevated organic loading on microbial functional gene community remains elusive. In this study, functional gene array (GeoChip 5.0) was used to assess the response of microbial community to the addition of poultry waste in anaerobic digesters treating dairy manure. Consistent with 16S rRNA gene sequences data, GeoChip data showed that microbial community compositions were significantly shifted in favor of copiotrophic populations by co-digestion, as taxa with higher rRNA gene copy number such as Bacilli were enriched. The acetoclastic methanogen Methanosarcina was also enriched, while Methanosaeta was unaltered but more abundant than Methanosarcina throughout the study period. The microbial functional diversity involved in anaerobic digestion were also increased under co-digestion.
Project description:The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the application of amplicon pyrosequencing. The possibility of barcode-tagged sequencing of templates gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing and, as in the early days of genetic community fingerprinting, pros and cons are continuously provided. In this study we investigate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of natural microbiota. Moreover, via quantitatively defined template spiking to the natural community, we explore the potential for recovering specific template ratios within complex microbial communities. For this reason, we pyrotag sequenced three biological replicates of three samples, each belonging from yearly sampling campaigns of sediment from a tar oil contaminated aquifer in Düsseldorf, Germany. Furthermore, we subjected one DNA extract to replicate technical analyses as well as to increasing ratios (0, 0.2, 2 and 20%) of 16S rRNA genes from a pure culture (Aliivibrio fisheri) originally not present in the sample. Unexpectedly, taxa abundances were highly reproducible in our hands, with max standard deviation of ~3% abundance across biological and ~2% for technical replicates. Furthermore, our workflow was also capable of recovering A. fisheri amendmend ratios in reliable amounts (0, 0.29, 3.9 and 23.8%). These results highlight that pyrotag sequencing, if done and evaluated with due caution, has the potential to robustly recapture taxa template abundances within environmental microbial communities.
Project description:Sequencing of 16S ribosomal RNA (rRNA) gene, which has improved the characterization of microbial community, has made it possible to detect a low level Helicobacter pylori (HP) sequences even in HP-negative subjects which were determined by a combination of conventional methods. This study was conducted to obtain a cutoff value for HP colonization in gastric mucosa biopsies and gastric juices by the pyrosequencing method. Corresponding author: Department of Internal Medicine, Seoul National University Bundang Hospital, Seoungnam, Gyeonggi-do, Korea; Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea (Tel., +82-31-787-7008; e-mail, nayoungkim49@empas.com). Microbial DNA from gastric mucosal samples [gastric antrum (n=63, mucosal biopsy), follow-up sample on gastric antrum (n=16, mucosal biopsy), and gastric body (n=18, mucosal biopsy)] and gastric juices (n=4, not mucosal biopsy) was amplified by nested PCR using universal bacterial primers, and the 16S rRNA genes were pyrosequenced.
Project description:Investigation of microbial community composition in mouse models using an intestinal epithial-specific and inducible VilCreERT2-mediated conditional knockout of Jup under basal conditions and in acute dextran-sodium sulfate (DSS)-induced colitis We investigated the gut microbiome composition in a dextran sulfate sodium (DSS) colitis model using Jupfl/fl and iVilCreERT2Jupfl/fl mice. Fecal samples were collected after DSS treatment, and 16S rRNA sequencing was employed to analyze microbial communities. Our findings revealed no significant differences in microbial profiles between Jupfl/fl and iVilCreERT2Jupfl/fl under DSS treatment.
Project description:Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply, despite demonstrable and diverse nutrient–induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.
Project description:Despite the global importance of forests, it is virtually unknown how their soil microbial communities adapt at the phylogenetic and functional level to long term metal pollution. Studying twelve sites located along two distinct gradients of metal pollution in Southern Poland revealed that both community composition (via MiSeq Illumina sequencing of 16S rRNA genes) and functional gene potential (using GeoChip 4.2) were highly similar across the gradients despite drastically diverging metal contamination levels. Metal pollution level significantly impacted microbial community structure (p = 0.037), but not bacterial taxon richness. Metal pollution altered the relative abundance of specific bacterial taxa, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes and Proteobacteria. Also, a group of metal resistance genes showed significant correlations with metal concentrations in soil, although no clear impact of metal pollution levels on overall functional diversity and structure of microbial communities was observed. While screens of phylogenetic marker genes, such as 16S rRNA, provided only limited insight into resilience mechanisms, analysis of specific functional genes, e.g. involved in metal resistance, appeared to be a more promising strategy. This study showed that the effect of metal pollution on soil microbial communities was not straightforward, but could be filtered out from natural variation and habitat factors by multivariate statistical analysis and spatial sampling involving separate pollution gradients.
Project description:We aimed to investigate the microbial community composition in patients with intracerebral hemorrhage (ICH) and its effect on prognosis. The relationship between changes in bacterial flora and the prognosis of spontaneous cerebral hemorrhage was studied in two cohort studies. Fecal samples from healthy volunteers and patients with intracerebral hemorrhage were subjected to 16S rRNA sequencing at three time points: T1 (within 24 hours of admission), T2 (3 days post-surgery), and T3 (7 days post-surgery) using Illumina high-throughput sequencing technology.