Steven J. Hallam

Insights into the phylogeny and coding potential of microbial dark matter

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum

Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal–sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.

Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-Oxidizing Archaea

ABSTRACT Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.

Microbial ecology of expanding oxygen minimum zones

Dissolved oxygen concentration is a crucial organizing principle in marine ecosystems. As oxygen levels decline, energy is increasingly diverted away from higher trophic levels into microbial metabolism, leading to loss of fixed nitrogen and to production of greenhouse gases, including nitrous oxide and methane. In this Review, we describe current efforts to explore the fundamental factors that control the ecological and microbial biodiversity in oxygen-starved regions of the ocean, termed oxygen minimum zones. We also discuss how recent advances in microbial ecology have provided information about the potential interactions in distributed co-occurrence and metabolic networks in oxygen minimum zones, and we provide new insights into coupled biogeochemical processes in the ocean.

Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean

Planktonic bacteria dominate surface ocean biomass and influence global biogeochemical processes, but remain poorly characterized owing to difficulties in cultivation. Using large-scale single cell genomics, we obtained insight into the genome content and biogeography of many bacterial lineages inhabiting the surface ocean. We found that, compared with existing cultures, natural bacterioplankton have smaller genomes, fewer gene duplications, and are depleted in guanine and cytosine, noncoding nucleotides, and genes encoding transcription, signal transduction, and noncytoplasmic proteins. These findings provide strong evidence that genome streamlining and oligotrophy are prevalent features among diverse, free-living bacterioplankton, whereas existing laboratory cultures consist primarily of copiotrophs. The apparent ubiquity of metabolic specialization and mixotrophy, as predicted from single cell genomes, also may contribute to the difficulty in bacterioplankton cultivation. Using metagenome fragment recruitment against single cell genomes, we show that the global distribution of surface ocean bacterioplankton correlates with temperature and latitude and is not limited by dispersal at the time scales required for nucleotide substitution to exceed the current operational definition of bacterial species. Single cell genomes with highly similar small subunit rRNA gene sequences exhibited significant genomic and biogeographic variability, highlighting challenges in the interpretation of individual gene surveys and metagenome assemblies in environmental microbiology. Our study demonstrates the utility of single cell genomics for gaining an improved understanding of the composition and dynamics of natural microbial assemblages.

A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities.

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by “flow-controlled wetting,” a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

lin-14 regulates the timing of synaptic remodelling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans six GABAergic motor neurons, known as DDs,, remodel their patterns of synaptic connectivity during larval development. DD remodelling involves a complete reversal of the direction of information flow within nerve processes without marked changes in process morphology. We used a marker localized in vivo to DD presynaptic zones to analyse how the timing of DD remodelling is controlled. In wild-type animals, DDs remodel their synaptic outputs within a 3–5-hour period at the end of the first larval stage. We show that the heterochronic gene lin-14, which controls the timing of stage-specific cell lineages,, regulates the timing of DD synaptic output remodelling. In lin-14 loss-of-function mutants, DDs remodel precociously. The degree of precocious remodelling is correlated with the level of lin-14 activity. Expression of lin-14(+) in the DDs of lin-14-null mutants rescues the precocious remodelling, indicating that lin-14 can act cell-autonomously. Consistent with this hypothesis, LIN-14 protein levels decrease in the DDs before remodelling. Our observations reveal a role of heterochronic genes in non-dividing cells, and provide an example of cell-autonomous respecification of neuronal connectivity.

Microbial community dynamics in a seasonally anoxic fjord: Saanich Inlet, British Columbia.

Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the delta-proteobacteria, SAR406 and gamma-proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.

Significant and persistent impact of timber harvesting on soil microbial communities in Northern coniferous forests

Forest ecosystems have integral roles in climate stability, biodiversity and economic development. Soil stewardship is essential for sustainable forest management. Organic matter (OM) removal and soil compaction are key disturbances associated with forest harvesting, but their impacts on forest ecosystems are not well understood. Because microbiological processes regulate soil ecology and biogeochemistry, microbial community structure might serve as indicator of forest ecosystem status, revealing changes in nutrient and energy flow patterns before they have irreversible effects on long-term soil productivity. We applied massively parallel pyrosequencing of over 4.6 million ribosomal marker sequences to assess the impact of OM removal and soil compaction on bacterial and fungal communities in a field experiment replicated at six forest sites in British Columbia, Canada. More than a decade after harvesting, diversity and structure of soil bacterial and fungal communities remained significantly altered by harvesting disturbances, with individual taxonomic groups responding differentially to varied levels of the disturbances. Plant symbionts, like ectomycorrhizal fungi, and saprobic taxa, such as ascomycetes and actinomycetes, were among the most sensitive to harvesting disturbances. Given their significant ecological roles in forest development, the fate of these taxa might be critical for sustainability of forest ecosystems. Although abundant bacterial populations were ubiquitous, abundant fungal populations often revealed a patchy distribution, consistent with their higher sensitivity to the examined soil disturbances. These results establish a comprehensive inventory of bacterial and fungal community composition in northern coniferous forests and demonstrate the long-term response of their structure to key disturbances associated with forest harvesting.

Growth and methane oxidation rates of anaerobic methanotrophic archaea in a continuous-flow bioreactor

ABSTRACT Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.