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Project description:Meta-barcoded evaluation of the ISO Standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition
| PRJEB4825 | ENA
Project description:D. vulgaris is a model sulfate reducing bacteria whose genome encodes a high number of two component systems, including 29 DNA-binding response regulators (RRs) whose functions are unknown, but are very likley to be important to the environmental lifestyle of the organism. We determined the gene targets for 24 of these RRs using purified His-tagged RR and sheared genomic DNA in an in vitro DAP-chip (DNA-affinity-purified - chip) assay. For each RR, one target was identified first using gel shift assays, and qPCR was used to ensure that the target was enriched in the RR-bound DNA before the samples were hybridized to a tiling array. Based on the peaks generated by the array analysis, we determined that at least 200 genes are regulated by two component systems in this organism. We also predicted binding site motifs and validated them for 15 RRs using gel shift assays.
2011-05-31 | GSE25163 | GEO
Project description:Because of severe abiotic limitations, Antarctic soils represent simplified ecosystems, where microorganisms are the principle drivers of nutrient cycling. This relative simplicity makes these ecosystems particularly vulnerable to perturbations, like global warming, and the Antarctic Peninsula is among the most rapidly warming regions on the planet. However, the consequences of the ongoing warming of Antarctica on microorganisms and the processes they mediate are unknown. Here, using 16S rRNA gene pyrosequencing and qPCR, we report a number of highly consistent changes in microbial community structure and abundance across very disparate sub-Antarctic and Antarctic environments following three years of experimental field warming (+ 0.5-2°C). Specifically, we found significant increases in the abundance of fungi and bacteria and in the Alphaproteobacteria-to-Acidobacteria ratio. These alterations were linked to a significant increase in soil respiration. Furthermore, the shifts toward generalist or opportunistic bacterial communities following warming weakened the linkage between bacterial diversity and functional diversity. Warming also increased the abundance of some organisms related to the N-cycle, detected as an increase in the relative abundance of nitrogenase genes via GeoChip microarray analyses. Our results demonstrate that soil microorganisms across a range of sub-Antarctic and Antarctic environments can respond consistently and rapidly to increasing temperatures, thereby potentially disrupting soil functioning. We conducted in situ warming experiments for three years using open-top chambers (OTCs) at one sub-Antarctic (Falkland Islands, 52ºS) and two Antarctic locations (Signy and Anchorage Islands, 60ºS and 67ºS respectively) (see Supplementary Fig. 1 for a map). OTCs increased annual soil temperature by an average of 0.8°C (at a depth of 5 cm), resulting in 8-43% increase in positive-degree days annually and a decrease in freeze-thaw cycle frequency by an average of 15 cycles per year (8). At each location, we included densely vegetated and bare fell-field soils in the experimental design for a total of six environments. Densely vegetated and bare environments represent two contrasting environments for Antarctic soil microorganisms, with large differences in terms of C and N inputs to soils. Massively parallel pyrosequencing (Roche 454 GS FLX Titanium) of 16S rRNA gene amplicons was used to follow bacterial diversity and community composition [GenBank Accession Numbers: HM641909-HM744649], and functional gene microarrays (GeoChip 2.0)(11) were used to assess changes in functional gene distribution. Bacterial and fungal communities were also quantified using real-time PCR.
2010-12-30 | E-GEOD-22825 | biostudies-arrayexpress
Project description:D. vulgaris is a model sulfate reducing bacteria whose genome encodes a high number of two component systems, including 29 DNA-binding response regulators (RRs) whose functions are unknown, but are very likley to be important to the environmental lifestyle of the organism. We determined the gene targets for 24 of these RRs using purified His-tagged RR and sheared genomic DNA in an in vitro DAP-chip (DNA-affinity-purified - chip) assay. For each RR, one target was identified first using gel shift assays, and qPCR was used to ensure that the target was enriched in the RR-bound DNA before the samples were hybridized to a tiling array. Based on the peaks generated by the array analysis, we determined that at least 200 genes are regulated by two component systems in this organism. We also predicted binding site motifs and validated them for 15 RRs using gel shift assays. In vitro DAP-chip for 28 response regulators where RR-bound enriched DNA is compared to input total genomic DNA
2011-05-31 | E-GEOD-25163 | biostudies-arrayexpress
Project description:Identification of the direct target genes of a response regulators (RRs) of a bacterial two-component system (TCS) is critical to understand the roles of the TCS in bacterial environmental adaption and pathogenesis. A. pleuropneumoniae is an important respiratory bacterial pathogen causing great economic losses to swine industry worldwide. The triggering signal(s) and targets of the RR NarP belonging to the TCS NarQ/NarP of A. pleuropneumoniae are still unknown. In the present study, a DNA-affinity-purified sequencing (DAP-Seq) approach was established. The upstream regions of a total of 131 candidate genes from the genome of A. pleuropneumoniae were shown to be co-purified with the activated NarP protein. Electrophoretic mobility shift assay results confirmed the interactions of NarP with the promoter regions of five selected target genes, including dmsA, pgaA, ftpA, cstA and ushA. Then, the EMSA-confirmed target genes were shown to be significantly up-regulated in the narP deleted mutant in the presence of additional nitrate, while the transcriptional changes were restored in the complemented strain. The NarP binding motif in the upstream regions of the target gene dmsA and ftpA were further identified and confirmed by EMSA using the truncated binding motif. The NarP binding sites were present in a total of 25.2% of the DNA fragments captured by DAP-Seq. These results demonstrated that the DAP-Seq method established in this study was effective to explore the direct targets of RRs of bacterial TCSs, and indicated that the A. pleuropneumoniae NarP could be a repressor in response to nitrate.
2019-11-30 | GSE107526 | GEO
Project description:Methods for the extraction of bacterial DNA
| PRJEB25932 | ENA
Project description:Because of severe abiotic limitations, Antarctic soils represent simplified ecosystems, where microorganisms are the principle drivers of nutrient cycling. This relative simplicity makes these ecosystems particularly vulnerable to perturbations, like global warming, and the Antarctic Peninsula is among the most rapidly warming regions on the planet. However, the consequences of the ongoing warming of Antarctica on microorganisms and the processes they mediate are unknown. Here, using 16S rRNA gene pyrosequencing and qPCR, we report a number of highly consistent changes in microbial community structure and abundance across very disparate sub-Antarctic and Antarctic environments following three years of experimental field warming (+ 0.5-2°C). Specifically, we found significant increases in the abundance of fungi and bacteria and in the Alphaproteobacteria-to-Acidobacteria ratio. These alterations were linked to a significant increase in soil respiration. Furthermore, the shifts toward generalist or opportunistic bacterial communities following warming weakened the linkage between bacterial diversity and functional diversity. Warming also increased the abundance of some organisms related to the N-cycle, detected as an increase in the relative abundance of nitrogenase genes via GeoChip microarray analyses. Our results demonstrate that soil microorganisms across a range of sub-Antarctic and Antarctic environments can respond consistently and rapidly to increasing temperatures, thereby potentially disrupting soil functioning.
2010-12-30 | GSE22825 | GEO
Project description:Soil is an inherently complex matrix and as such, we believe when performing culture-independent microbial community analyses using the 'omics' suite of tools, all biomolecules investigated should be co-extracted from the same biological sample. To this end, we developed a robust, cost-effective DNA, RNA and protein co-extraction method for soil. The samples deposited here represent 3 biological replicates from one of eight soil types tested in this work.
2019-07-15 | PXD007077 | Pride