B14A-01 INVITED
New Tools For Understanding Microbial Diversity Using High-throughput Sequence Data
High-throughput sequencing techniques such as 454 are straining the limits of tools traditionally used to build trees, choose OTUs, and perform other essential sequencing tasks. We have developed a workflow for phylogenetic analysis of large-scale sequence data sets that combines existing tools, such as the Arb phylogeny package and the NAST multiple sequence alignment tool, with new methods for choosing and clustering OTUs and for performing phylogenetic community analysis with UniFrac. This talk discusses the cyberinfrastructure we are developing to support the human microbiome project, and the application of these workflows to analyze very large data sets that contrast the gut microbiota with a range of physical environments. These tools will ultimately help to define core and peripheral microbiomes in a range of environments, and will allow us to understand the physical and biotic factors that contribute most to differences in microbial diversity.
B14A-02 INVITED
Natural Microbial Assemblages Reflect Distinct Organismal and Functional Partitioning
The ability to link microbial community structure to function has long been a primary focus of environmental microbiology. With the advent of community genomic and proteomic techniques, along with advances in microscopic imaging techniques, it is now possible to gain insights into the organismal and functional makeup of microbial communities. Biofilms growing within highly acidic solutions inside the Richmond Mine (Iron Mountain, Redding, California) exhibit distinct macro- and microscopic morphologies. They are composed of microorganisms belonging to the three domains of life, including archaea, bacteria and eukarya. The proportion of each organismal type depends on sampling location and developmental stage. For example, mature biofilms floating on top of acid mine drainage (AMD) pools exhibit layers consisting of a densely packed bottom layer of the chemoautolithotroph Leptospirillum group II, a less dense top layer composed mainly of archaea, and fungal filaments spanning across the entire biofilm. The expression of cytochrome 579 (the most highly abundant protein in the biofilm, believed to be central to iron oxidation and encoded by Leptospirillum group II) is localized at the interface of the biofilm with the AMD solution, highlighting that biofilm architecture is reflected at the functional gene expression level. Distinct functional partitioning is also apparent in a biological wastewater treatment system that selects for distinct polyphosphate accumulating organisms. Community genomic data from " Candidatus Accumulibacter phosphatis" dominated activated sludge has enabled high mass-accuracy shotgun proteomics for identification of key metabolic pathways. Comprehensive genome-wide alignment of orthologous proteins suggests distinct partitioning of protein variants involved in both core-metabolism and specific metabolic pathways among the dominant population and closely related species. In addition, strain- resolved proteogenomic analysis of the AMD biofilms also highlights the importance of strain heterogeneity for the maintenance of community structure and function. These findings explain the importance of genetic diversity in facilitating the stable performance of complex microbial processes. Furthermore, although very different in terms of habitat, both microbial communities exhibit distinct functional compartmentalization and demonstrate its role in sustaining microbial community structure.
B14A-03
Phototrophs vs. chemotrophs: surprising diversity at the intersection of hot springs communities
Access makes hot spring ecosystems ideal for combining geochemical data and thermodynamic models of energy supplies with cultivation-independent studies that shed light on biological diversity and function. We have recently undertaken combined 16S rRNA and metagenomic sampling of multiple communities found along a nearly 40 degree temperature gradient in a geochemically well-characterized alkaline hot spring in Yellowstone National Park (YNP). One unexpected result of this work comes at the so-called photosynthetic fringe, where "hot" chemotrophic metabolism gives way to "cool" phototrophy. This transition occurs between 55 and 73 degrees C in alkaline YNP springs for reasons that are poorly understood. While it is generally observed in other habitats that biodiversity increases as temperature decreases, 16S analysis reveals that diversity peaks at the fringe and tapers off at both higher and lower temperatures. Intriguingly, this increase is above and beyond what would be expected by merging the chemotrophic communities above the fringe with the photosynthetic communities below it, indicating that some facet of this unique intersection is supporting the potential for new niches to arise. Integrating metagenomic data with geochemical analyses offers molecular-level details for understanding how these niches develop and are sustained. The underlying interplay between geochemistry and genomics may well be driving the evolution and distribution of new metabolic capabilities including the presence of deeply branching RuBisCO and pyruvate:ferredoxin oxidoreductase homologs involved in autotrophic carbon fixation.
B14A-04
Using Small Subunit Ribosomal RNA to Follow Dark Incorporation of 14C-bicarbonate by Bacteria and Archaea in Sandy Sediment
Small subunit ribosomal RNA (SSU rRNA) and the genes encoding it have become the basis of modern microbial phylogeny, and of numerous methods for characterizing the composition of bacterial, archaeal, and even eukaryotic communities as they occur in nature. A limitation of this approach has been that phylogeny alone is not a reliable guide to physiology, particularly for groups with no close relatives in culture. We have been developing ways of using the SSU rRNA molecule itself to identify and (eventually) quantify the carbon sources incorporated by particular phylogenetic groups. This can be done by taking advantage of natural variations in carbon isotopic composition among growth substrates, or by following incorporation of 13C- or 14C-labeled compounds. 14C has the advantage that natural background levels are negligible. In the present study, our goal is to identify species responsible for non-photosynthetic CO2 incorporation in sandy sediments of the German Wadden Sea. Sediment cores collected from the Janssand sand flats were percolated with 14C-bicarbonate at in situ temperature for 36-38h in the dark, total RNA isolated, and domain-specific oligonucleotide probes used to capture bacterial and archaeal SSU rRNA. Total and/or captured RNA was separated by denaturing polyacrylamide gel electrophoresis, and 14C detected by phosphor imager, autoradiography, or beta imager. Detection was fastest and most sensitive with the beta imager. Both Bacteria and Archaea had incorporated label, suggesting both groups may harbor non-photosynthetic autotrophs. The next step will be to use more specific capture probes. We are currently working to separate the captured domain-specific SSU rRNA on non-denaturing gels, with detection by the high-resolution mode of the beta imager, so that individual species incorporating label can be identified by RT-PCR and sequencing of labeled bands.
B14A-05 INVITED
A Thermodynamically-Based Model For Predicting Microbial Growth And Community Composition Coupled To System Geochemistry
We present an approach that couples thermodynamic descriptions for microbial growth and geochemical reactions to provide quantitative predictions for the effects of substrate addition or other enviornmental perturbations on microbial community composition. A synthetic microbial community is defined as a collection of defined microbial groups; each with a growth equation derived from bioenergetic principles. The growth equations and standard-state free energy yields are appended to a thermodynamic database for geochemical reactions and the combined equations are solved simultaneously to predict coupled changes in microbial biomass, community composition, and system geochemistry. This approach, with a single set of thermodynamic parameters (one for each growth equation), was used to predict the results of laboratory and field experiments at three geochemically diverse research sites. Predicted effects of ethanol or acetate addition on radionuclide and heavy metal solubility, major ion geochemistry, mineralogy, microbial biomass and community composition were in general agreement with experimental observations although the available experimental data precluded rigorous model testing. Model simulations provide insight into the long-standing difficulty in transferring experimental results from the laboratory to the field and from one site to the next, especially if the form, concentration, or delivery of growth substrate is varied from one experiment to the next. Although originally developed for use in better understanding bioimmobilization of radionuclides and heavy metals via reductive precipitation, the modeling approach is potentially useful for exploring the coupling of microbial growth and geochemical reactions in a variety of basic and applied biotechnology research settings.
B14A-06
Targeted Proteomics Approaches To Monitor Microbial Activity In Basalt Aquifer
Microorganisms play a major role in biogeochemical cycles of the Earth. Information regarding microbial community composition can be very useful for environmental monitoring since the short generation times of microorganisms allows them to respond rapidly to changing environmental conditions. Microbial mediated attenuation of toxic chemicals offers great potential for the restoration of contaminated environments in an ecologically acceptable manner. Current knowledge regarding the structure and functional activities of microbial communities is limited, but more information is being acquired every day through many genomic- and proteomic- based methods. As of today, only a small fraction of the Earth's microorganisms has been cultured, and so most of the information regarding the biodegradation and therapeutic potentials of these uncultured microorganisms remains unknown. Sequence analysis of DNA and/or RNA has been used for identifying specific microorganisms, to study the community composition, and to monitor gene expression providing limited information about metabolic state of given microbial system. Proteomic studies can reveal information regarding the real-time metabolic state of the microbial communities thereby aiding in understanding their interaction with the environment. In research described here the involvement of microbial communities in the degradation of anthropogenic contaminants such as trichloroethylene (TCE) was studied using mass spectrometry-based proteomics. The co- metabolic degradation of TCE in the groundwater of the Snake River Plain Aquifer at the Test Area North (TAN) site of Idaho National Laboratory (INL) was monitored by the characterization of peptide sequences of enzymes such as methane monooxygenases (MMOs). MMOs, expressed by methanotrophic bacteria are involved in the oxidation of methane and non-specific co-metabolic oxidation of TCE. We developed a time- course cell lysis method to release proteins from complex microbial communities to allow for better identification of specific proteins. This method not only identified the proteins of interest but also increased the peptide coverage and increased the number of proteins identified. We were able to identify methane monooxygenase proteins within TAN site microbial communities supporting the occurrence of co-metabolic oxidation of TCE in this aquifer. We correlated methane monooxygenase presence with the number of methanotrophs in the samples obtained through quantitative PCR and quantitative proteomic methods. Utilization of this extraction method in combination with UPLC/MS/MS resulted in successful extraction, identification, and quantification of MMO-derived biomarker peptides from both pure cultures and environmental samples. Along with MMO proteins, we identified proteins from non-methanotrophic organisms that may play major roles in macronutrients turnover and the attenuation of TCE in the TAN aquifer. http://www.ebi.uidaho.edu/default.aspx?pid=99130
B14A-07
Links between evolutionary history and soil microbial community structure revealed by DNA microarrays
Soils harbor a vast diversity of uncultured microorganisms whose occurrence and habitat preferences across the Earth's surface are largely uncharted. We used phylogenetic DNA microarrays to make high-resolution assessments of microbial community structure and its relationship to environmental parameters in soils from different temperate and tropical ecosystems. Diverse communities (>2000 detected taxa) of soil bacteria were strongly structured in relation to single environmental predictors, particularly variables related to soil moisture. Bacteria within high-ranking taxonomic groups (up to the Phylum level) generally displayed coherent responses to changes in moisture, indicating that these responses have remained stable through evolutionary time. Drought-tolerant bacteria have evolved in remarkably few bacterial lineages, and consequently the variety of soil bacteria present in drier ecosystems is reduced compared to wet ecosystems like rainforests. From these results we predict that genomic diversity in soil is likely to decline where climate or land-use change increases drought conditions.
B14A-08
Global Patterns in Bacterial Diversity
Microbes are difficult to culture. Consequently, the primary source of information about a fundamental evolutionary topic, life's diversity, is the environmental distribution of gene sequences. We report the most comprehensive analysis of the environmental distribution of bacteria to date, based on 21,752 16S rRNA sequences compiled from 111 studies of diverse physical environments. We clustered the samples based on similarities in the phylogenetic lineages that they contain and found that, surprisingly, the major environmental determinant of microbial community composition is salinity rather than extremes of temperature, pH, or other physical and chemical factors represented in our samples. We find that sediments are more phylogenetically diverse than any other environment type. Surprisingly, soil, which has high species-level diversity, has below-average phylogenetic diversity. This work provides a framework for understanding the impact of environmental factors on bacterial evolution and for the direction of future sequencing efforts to discover new lineages.