Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics

Modern microbial mats are potential analogues of some of Earth''s earliest ecosystems. Excellent examples can be found in Shark Bay, Australia, with mats of various morphologies. To further our understanding of the functional genetic potential of these complex microbial ecosystems, we conducted for the first time shotgun metagenomic analyses. We assembled metagenomic next-generation sequencing data to classify the taxonomic and metabolic potential across diverse morphologies of marine mats in Shark Bay. The microbial community across taxonomic classifications using protein-coding and small subunit rRNA genes directly extracted from the metagenomes suggests that three phyla Proteobacteria, Cyanobacteria and Bacteriodetes dominate all marine mats. However, the microbial community structure between Shark Bay and Highbourne Cay (Bahamas) marine systems appears to be distinct from each other. The metabolic potential (based on SEED subsystem classifications) of the Shark Bay and Highbourne Cay microbial communities were also distinct. Shark Bay metagenomes have a metabolic pathway profile consisting of both heterotrophic and photosynthetic pathways, whereas Highbourne Cay appears to be dominated almost exclusively by photosynthetic pathways. Alternative non-rubisco-based carbon metabolism including reductive TCA cycle and 3-hydroxypropionate/4-hydroxybutyrate pathways is highly represented in Shark Bay metagenomes while not represented in Highbourne Cay microbial mats or any other mat forming ecosystems investigated to date. Potentially novel aspects of nitrogen cycling were also observed, as well as putative heavy metal cycling (arsenic, mercury, copper and cadmium). Finally, archaea are highly represented in Shark Bay and may have critical roles in overall ecosystem function in these modern microbial mats.     

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.     

Microbiome analysis of dairy cows fed pasture or total mixed ration diets

Understanding rumen microbial ecology is essential for the development of feed systems designed to improve livestock productivity, health and for methane mitigation strategies from cattle. Although rumen microbial communities have been studied previously, few studies have applied next-generation sequencing technologies to that ecosystem. The aim of this study was to characterize changes in microbial community structure arising from feeding dairy cows two widely used diets: pasture and total mixed ration (TMR). Bacterial, archaeal and protozoal communities were characterized by terminal restriction fragment length polymorphism of the amplified SSU rRNA gene and statistical analysis showed that bacterial and archaeal communities were significantly affected by diet, whereas no effect was observed for the protozoal community. Deep amplicon sequencing of the 16S rRNA gene revealed significant differences in the bacterial communities between the diets and between rumen solid and liquid content. At the family level, some important groups of rumen bacteria were clearly associated with specific diets, including the higher abundance of the Fibrobacteraceae in TMR solid samples and members of the propionate-producing Veillonelaceae in pasture samples. This study will be relevant to the study of rumen microbial ecology and livestock feed management.     

Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).     

Phylogeny and physiology of candidate phylum BRC1 inferred from the first complete metagenome-assembled genome obtained from deep subsurface aquifer

Candidate bacterial phylum BRC1 has been identified in a broad range of mostly organic-rich oxic and anoxic environments through molecular analysis of microbial communities. None of the members of BRC1 have been cultivated and only a few draft genome sequences have been obtained from metagenomes or as a result of single-cell sequencing. We have reconstructed complete genome of BRC1 bacterium, BY40, from metagenome of the microbial community of a deep subsurface thermal aquifer in the Tomsk Region of the Western Siberia, Russia, and used it for metabolic reconstruction and comparison with existing genomic data. Analysis of 3.3 Mb genome of BY40 bacterium revealed numerous glycoside hydrolases that could enable utilization of carbohydrates, including enzymes of chitin-degradation pathway. The bacterium lacks flagellar machinery but the twitching motility is encoded. The reconstructed central metabolism revealed pathways enabling the fermentation of organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Phylogenetic analysis using BY40 genome supported the phylum level classification of BRC1 lineage. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Sumerlaea chitinivorans, within a candidate phylum Sumerlaeota.     

Flexible Community Structure Correlates with Stable Community Function in Methanogenic Bioreactor Communities Perturbed by Glucose

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050–4057, 2000).     

Effects of Endogenous Substrates on Adaptation of Anaerobic Microbial Communities to 3-Chlorobenzoate

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.     

Subseafloor microbial communities associated with rapid turbidite deposition in the Gulf of Mexico continental slope (IODP Expedition 308)

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA , and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 × 10⁷ cells mL⁻¹ or copies g⁻¹ sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.     

Effects of endogenous substrates on adaptation of anaerobic microbial communities to 3-chlorobenzoate

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.     

Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf,Antarctica

Marine sediments of the Ross Sea, Antarctica, harbor microbial communities that play a significant role in the decomposition, mineralization, and recycling of organic carbon (OC). In this study, the cell densities within a 153‐cm sediment core from the Ross Sea were estimated based on microbial phospholipid fatty acid (PLFA) concentrations and acridine orange direct cell counts. The resulting densities were as high as 1.7 × 10⁷ cells mL^?1 in the top ten centimeters of sediments. These densities are lower than those calculated for most near‐shore sites but consistent with deep‐sea locations with comparable sedimentation rates. The δ¹³C measurements of PLFAs and sedimentary and dissolved carbon sources, in combination with ribosomal RNA (SSU rRNA) gene pyrosequencing, were used to infer microbial metabolic pathways. The δ¹³C values of dissolved inorganic carbon (DIC) in porewaters ranged downcore from ?2.5‰ to ?3.7‰, while δ¹³C values for the corresponding sedimentary particulate OC (POC) varied from ?26.2‰ to ?23.1‰. The δ¹³C values of PLFAs ranged between ?29‰ and ?35‰ throughout the sediment core, consistent with a microbial community dominated by heterotrophs. The SSU rRNA gene pyrosequencing revealed that members of this microbial community were dominated by β‐, δ‐, and γ‐Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Among the sequenced organisms, many appear to be related to known heterotrophs that utilize OC sources such as amino acids, oligosaccharides, and lactose, consistent with our interpretation from δ¹³C_PLFA analysis. Integrating phospholipids analyses with porewater chemistry, δ¹³C_DIC and δ¹³C_POC values and SSU rRNA gene sequences provides a more comprehensive understanding of microbial communities and carbon cycling in marine sediments, including those of this unique ice shelf environment.     

An experimental metagenome data management and analysis system

The application of shotgun sequencing to environmental samples has revealed a new universe of microbial community genomes (metagenomes) involving previously uncultured organisms. Metagenome analysis, which is expected to provide a comprehensive picture of the gene functions and metabolic capacity for microbial communities, needs to be conducted in the context of a comprehensive data management and analysis system. We present in this paper IMG/M, an experimental metagenome data management and analysis system that is based on the Integrated Microbial Genomes (IMG) system. IMG/M provides tools and viewers for analyzing both metagenomes and isolate genomes individually or in a comparative context. IMG/M is available at http://img.jgi.doe.gov/m.     

Metagenomic mining for microbiologists

Microbial ecologists can now start digging into the accumulating mountains of metagenomic data to uncover the occurrence of functional genes and their correlations to microbial community members. Limitations and biases in DNA extraction and sequencing technologies impact sequence distributions, and therefore, have to be considered. However, when comparing metagenomes from widely differing environments, these fluctuations have a relatively minor role in microbial community discrimination. As a consequence, any functional gene or species distribution pattern can be compared among metagenomes originating from various environments and projects. In particular, global comparisons would help to define ecosystem specificities, such as involvement and response to climate change (for example, carbon and nitrogen cycle), human health risks (eg, presence of pathogen species, toxin genes and viruses) and biodegradation capacities. Although not all scientists have easy access to high-throughput sequencing technologies, they do have access to the sequences that have been deposited in databases, and therefore, can begin to intensively mine these metagenomic data to generate hypotheses that can be validated experimentally. Information about metabolic functions and microbial species compositions can already be compared among metagenomes from different ecosystems. These comparisons add to our understanding about microbial adaptation and the role of specific microbes in different ecosystems. Concurrent with the rapid growth of sequencing technologies, we have entered a new age of microbial ecology, which will enable researchers to experimentally confirm putative relationships between microbial functions and community structures.     

Novel bacterial lineages associated with boreal moss species

Mosses are critical components of boreal ecosystems where they typically account for a large proportion of net primary productivity and harbour diverse bacterial communities that can be the major source of biologically‐fixed nitrogen in these ecosystems. Despite their ecological importance, we have limited understanding of how microbial communities vary across boreal moss species and the extent to which local site conditions may influence the composition of these bacterial communities. We used marker gene sequencing to analyze bacterial communities associated with seven boreal moss species collected near Fairbanks, AK, USA. We found that host identity was more important than site in determining bacterial community composition and that mosses harbour diverse lineages of potential N₂‐fixers as well as an abundance of novel taxa assigned to understudied bacterial phyla (including candidate phylum WPS‐2). We performed shotgun metagenomic sequencing to assemble genomes from the WPS‐2 candidate phylum and found that these moss‐associated bacteria are likely anoxygenic phototrophs capable of carbon fixation via RuBisCo with an ability to utilize byproducts of photorespiration from hosts via a glyoxylate shunt. These results give new insights into the metabolic capabilities of understudied bacterial lineages that associate with mosses and the importance of plant hosts in shaping their microbiomes.     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

Shedding light on biogas: Phototrophic biofilms in anaerobic digesters hold potential for improved biogas production

Conventional anaerobic digesters intended for the production of biogas usually operate in complete darkness. Therefore, little is known about the effect of light on their microbial communities. In the present work, 16S rRNA gene amplicon Nanopore sequencing and shotgun metagenomic sequencing were used to study the taxonomic and functional structure of the microbial community forming a biofilm on the inner wall of a laboratory-scale transparent anaerobic biodigester illuminated with natural sunlight. The biofilm was composed of microorganisms involved in the four metabolic processes needed for biogas production, and it was surprisingly rich in Rhodopseudomonas faecalis, a versatile bacterium able to carry out photoautotrophic metabolism when grown under anaerobic conditions. The results suggested that this bacterium, which is able to fix carbon dioxide, could be considered for use in transparent biogas fermenters in order to contribute to the production of optimized biogas with a higher CH₄:CO₂ ratio than the biogas produced in regular, opaque digesters. To the best of our knowledge, this is the first study characterising the phototrophic biofilm associated with illuminated bioreactors.     

Analysis of the global ocean sampling (GOS) project for trends in iron uptake by surface ocean microbes

Microbial metagenomes are DNA samples of the most abundant, and therefore most successful organisms at the sampling time and location for a given cell size range. The study of microbial communities via their DNA content has revolutionized our understanding of microbial ecology and evolution. Iron availability is a critical resource that limits microbial communities' growth in many oceanic areas. Here, we built a database of 2319 sequences, corresponding to 140 gene families of iron metabolism with a large phylogenetic spread, to explore the microbial strategies of iron acquisition in the ocean's bacterial community. We estimate iron metabolism strategies from metagenome gene content and investigate whether their prevalence varies with dissolved iron concentrations obtained from a biogeochemical model. We show significant quantitative and qualitative variations in iron metabolism pathways, with a higher proportion of iron metabolism genes in low iron environments. We found a striking difference between coastal and open ocean sites regarding Fe(2+) versus Fe(3+) uptake gene prevalence. We also show that non-specific siderophore uptake increases in low iron open ocean environments, suggesting bacteria may acquire iron from natural siderophore-like organic complexes. Despite the lack of knowledge of iron uptake mechanisms in most marine microorganisms, our approach provides insights into how the iron metabolic pathways of microbial communities may vary with seawater iron concentrations.     

Optimizing the analysis of human intestinal microbiota with phylogenetic microarray

Phylogenetic microarrays present an attractive strategy to high-throughput interrogation of complex microbial communities. In this work, we present several approaches to optimize the analysis of intestinal microbiota with the recently developed Microbiota Array. First, we determined how 16S rDNA-specific PCR amplification influenced bacterial detection and the consistency of measured abundance values. Bacterial detection improved with an increase in the number of PCR amplification cycles, but 25 cycles were sufficient to achieve the maximum possible detection. A PCR-caused deviation in the measured abundance values was also observed. We also developed two mathematical algorithms that aimed to account for a predicted cross-hybridization of 16S rDNA fragments among different species, and to adjust the measured hybridization signal based on the number of 16S rRNA gene copies per species genome. The 16S rRNA gene copy adjustment indicated that the presence of members of the class Clostridia might be overestimated in some 16S rDNA-based studies. Finally, we show that the examination of total community RNA with phylogenetic microarray can provide estimates of the relative metabolic activity of individual community members. Complementary profiling of genomic DNA and total RNA isolated from the same sample presents an opportunity to assess population structure and activity in the same microbial community.     

Dryland biological soil crust cyanobacteria show unexpected decreases in abundance under long‐term elevated CO2

Biological soil crusts (biocrusts) cover soil surfaces in many drylands globally. The impacts of 10 years of elevated atmospheric CO₂ on the cyanobacteria in biocrusts of an arid shrubland were examined at a large manipulated experiment in Nevada, USA. Cyanobacteria‐specific quantitative PCR surveys of cyanobacteria small‐subunit (SSU) rRNA genes suggested a reduction in biocrust cyanobacterial biomass in the elevated CO₂ treatment relative to the ambient controls. Additionally, SSU rRNA gene libraries and shotgun metagenomes showed reduced representation of cyanobacteria in the total microbial community. Taxonomic composition of the cyanobacteria was similar under ambient and elevated CO₂ conditions, indicating the decline was manifest across multiple cyanobacterial lineages. Recruitment of cyanobacteria sequences from replicate shotgun metagenomes to cyanobacterial genomes representing major biocrust orders also suggested decreased abundance of cyanobacteria sequences across the majority of genomes tested. Functional assignment of cyanobacteria‐related shotgun metagenome sequences indicated that four subsystem categories, three related to oxidative stress, were differentially abundant in relation to the elevated CO₂ treatment. Taken together, these results suggest that elevated CO₂ affected a generalized decrease in cyanobacteria in the biocrusts and may have favoured cyanobacteria with altered gene inventories for coping with oxidative stress.     

Metabolic reconstruction for metagenomic data and its application to the human microbiome

Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes including metabolism and immune homeostasis in the human microbiome. Shotgun sequencing of such communities' metagenomes provides information complementary to organismal abundances from taxonomic markers, but the resulting data typically comprise short reads from hundreds of different organisms and are at best challenging to assemble comparably to single-organism genomes. Here, we describe an alternative approach to infer the functional and metabolic potential of a microbial community metagenome. We determined the gene families and pathways present or absent within a community, as well as their relative abundances, directly from short sequence reads. We validated this methodology using a collection of synthetic metagenomes, recovering the presence and abundance both of large pathways and of small functional modules with high accuracy. We subsequently applied this method, HUMAnN, to the microbial communities of 649 metagenomes drawn from seven primary body sites on 102 individuals as part of the Human Microbiome Project (HMP). This provided a means to compare functional diversity and organismal ecology in the human microbiome, and we determined a core of 24 ubiquitously present modules. Core pathways were often implemented by different enzyme families within different body sites, and 168 functional modules and 196 metabolic pathways varied in metagenomic abundance specifically to one or more niches within the microbiome. These included glycosaminoglycan degradation in the gut, as well as phosphate and amino acid transport linked to host phenotype (vaginal pH) in the posterior fornix. An implementation of our methodology is available at http://huttenhower.sph.harvard.edu/humann. This provides a means to accurately and efficiently characterize microbial metabolic pathways and functional modules directly from high-throughput sequencing reads, enabling the determination of community roles in the HMP cohort and in future metagenomic studies.