Project description:Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world’s oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions.
Project description:Marine snow plays a central role in carbon cycling. It consists of organic particles and particle-associated (PA) microbMarine snow plays a central role in carbon cycling. It consists of organic particles and particle-associated (PA) microbial communities that are embedded in a sugary matrix. Metaproteomic analysis offers the unique opportunity to gain unprecedented insight into the microbial community composition and biomolecular activity of environmental samples. In order to realize this potential for marine PA microbial communities, new methods of protein extraction must be developed. In this study, we used 1D-SDS-PAGEs and LC-MS/MS to compare the efficiency of six established protein extraction protocols for their applicability of metaproteomic analyses of the PA microbial community in the North Sea. A combination of SDS-buffer extraction and bead beating resulted in the greatest number of identified protein groups. As expected, a metagenomic database of the same environmental sample increased the number of protein identification by approximately 50%. To demonstrate the application of our established protocol, particulate bacterioplankton samples collected during spring phytoplankton bloom in 2009 near the island Helgoland, were analysed by a GeLC-MS/MS-based metaproteomic approach. Our results indicated that there are only slight differences in the taxonomical distribution between free-living (FL) and PA bacteria but that the abundance of protein groups involved in polysaccharide degradation, motility and particle specific stress (oxygen limitation, nutrient limitation, heavy metal stress) is higher in the PA fractions. ial communities that are embedded in a sugary matrix. Metaproteomic analysis offers the unique opportunity to gain unprecedented insight into the microbial community composition and biomolecular activity of environmental samples. In order to realize this potential for marine PA microbial communities, new methods of protein extraction must be developed. In this study, we used 1D-SDS-PAGEs and LC-MS/MS to compare the efficiency of six established protein extraction protocols for the their applicability of metaproteomic analyses of the PA microbial community in the North Sea. A combination of SDS-buffer extraction and bead beating resulted in the greatest number of identified protein groups. As expected, a metagenomic database of the same environmental sample increased the number of protein identification by approximately 50%. To demonstrate the application of our established protocol, particulate bacterioplankton samples collected during spring phytoplankton bloom in 2009 near the island Helgoland, were analysed by a GeLC-MS/MS-based metaproteomic approach. Our results indicated that there are only slight differences in the taxonomical distribution between free-living (FL) and PA bacteria but that the abundance of protein groups involved in polysaccharide degradation, motility and particle specific stress (oxygen limitation, nutrient limitation, heavy metal stress) is higher in the PA fractions.
Project description:metagenomic and metaproteomic analyses were used to identify the changes in the dominant flora and related enzymes in amino acid synthesis and metabolism during Cantonese soy sauce fermentation.
Project description:Samples of oil and production water were collected from five wells of the Qinghai Oilfield, China, and subjected to GeoChip hybridization experiments for microbial functional diversity profiling. Unexpectedly, a remarkable microbial diversity in oil samples, which was higher than that in the corresponding water samples, was observed, thus challenging previously believed assumptions about the microbial diversity in this ecosystem. Hierarchical clustering separated oil and water samples, thereby indicating distinct functional structures in the samples. Genes involved in the degradation of hydrocarbons, organic remediation, stress response, and carbon cycling were significantly abundant in crude oil, which is consistent with their important roles in residing in oil. Association analysis with environmental variables suggested that oil components comprising aromatic hydrocarbons, aliphatic hydrocarbons, and a polar fraction with nitrogen-, sulfur-, and oxygen-containing compounds were mainly influential on the structure of the microbial community. Furthermore, a comparison of microbial communities in oil samples indicated that the structures were depth/temperature-dependent. To our knowledge, this is the first thorough study to profile microbial functional diversity in crude oil samples.
Project description:Increasing atmospheric CO2 concentrations are causing decreased pH over vast expanses of the ocean. This decreasing pH may alter biogeochemical cycling of carbon and nitrogen via the microbial process of nitrification, a key process that couples these cycles in the ocean, but which is often sensitive to acidic conditions. Recent reports indicate a decrease in oceanic nitrification rates under experimentally lowered pH. How composition and abundance of ammonia oxidizing bacteria (AOB) and archaea (AOA) assemblages respond to decreasing oceanic pH, however, is unknown. We sampled microbes from two different acidification experiments and used a combination of qPCR and functional gene microarrays for the ammonia monooxygenase gene (amoA) to assess how acidification alters the structure of ammonia oxidizer assemblages. We show that despite widely different experimental conditions, acidification consistently altered the community composition of AOB by increasing the relative abundance of taxa related to the Nitrosomonas ureae clade. In one experiment this increase was sufficient to cause an increase in the overall abundance of AOB. There were no systematic shifts in the community structure or abundance of AOA in either experiment. These different responses to acidification underscore the important role of microbial community structure in the resiliency of marine ecosystems. SUBMITTER_CITATION: Title: Acidification alters the composition of ammonia oxidizing microbial assemblages in marine mesocosms Journal: Marine Ecology Progress Series Issue: 492 Pages: 1-8 DOI: 10.3354/meps 10526 Authors: Jennifer L Bowen Patrick J Kearns Michael Holcomb Bess B Ward
Project description:Samples of oil and production water were collected from five wells of the Qinghai Oilfield, China, and subjected to GeoChip hybridization experiments for microbial functional diversity profiling. Unexpectedly, a remarkable microbial diversity in oil samples, which was higher than that in the corresponding water samples, was observed, thus challenging previously believed assumptions about the microbial diversity in this ecosystem. Hierarchical clustering separated oil and water samples, thereby indicating distinct functional structures in the samples. Genes involved in the degradation of hydrocarbons, organic remediation, stress response, and carbon cycling were significantly abundant in crude oil, which is consistent with their important roles in residing in oil. Association analysis with environmental variables suggested that oil components comprising aromatic hydrocarbons, aliphatic hydrocarbons, and a polar fraction with nitrogen-, sulfur-, and oxygen-containing compounds were mainly influential on the structure of the microbial community. Furthermore, a comparison of microbial communities in oil samples indicated that the structures were depth/temperature-dependent. To our knowledge, this is the first thorough study to profile microbial functional diversity in crude oil samples. From the Qinghai Oilfield located in the Tibetan Plateau, northwest China, oil production mixtures were taken from four oil production wells (No. 813, 516, 48 and 27) and one injection well (No. 517) in the Yue-II block. The floating oil and water phases of the production mixtures were separated overnight by gravitational separation. Subsequently, the microbial community and the characteristics of the water solution (W813, W516, W48, and W27) and floating crude oil (O813, O516, O48, and O27) samples were analyzed. A similar analysis was performed with the injection water solution (W517).
Project description:To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild. To gain improved temporal, spatial and phylogenetic resolution of marine microbial communities, in this study we expanded the original prototype genome proxy array (an oligonucleotide microarray targeting marine microbial genome fragments and genomes), evaluated it against metagenomic sequencing, and applied it to time series samples from the Monterey Bay long term ecological research site. The expanded array targeted 268 microbial genotypes (vs. 14 in the original prototype) across much of the known diversity of cultured and uncultured marine microbes. The target abundances measured by the genome proxy array were highly correlated to pyrosequence-based abundances (linear regression R2 = 0.85-0.91, p<0.0001). Fifty-seven samples from ~4-years in Monterey Bay were examined with the array, spanning the photic zone (0m), the base of the surface mixed layer (30m), and the subphotic zone (200m). A significant portion of the expanded genome proxy array’s targets showed signal (95 out of 268 targets present in ≥ 1 sample). The multi-year community survey showed the consistent presence of a core group of common and abundant targeted taxa at each depth in Monterey Bay, higher variability among shallow than deep samples, and episodic occurrences of more transient marine genotypes. The abundance of the most dominant genotypes peaked after strong episodic upwelling events. The genome-proxy array’s ability to track populations of closely-related genotypes indicated population shifts within several abundant target taxa, with specific populations in some cases clustering by depth or oceanographic season. Although 51 cultivated organisms were targeted (representing 19% of the array) the majority of targets detected and of total target signal (85% and ~92%, respectively) were from uncultivated lineages, often those derived from Monterey Bay. The array provided cost-effective (~$15 per array, for construction and use) insights into the natural history of uncultivated lineages in the wild.