A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS-). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS2). Recently, paradigm-shifting studies indicate that Fe and S in FeS2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS2, as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS2, Fe(II)/HS-, or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS-, either as a component of the growth medium [Fe(II)/HS-] or generated through reduction of FeS2, were likely a major factor underpinning these phenotypic differences.IMPORTANCEThe methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life's origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

Methanogens acquire and bioaccumulate nickel during reductive dissolution of nickelian pyrite.

Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni,Fe)S2], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens (Methanosarcina barkeri, Methanococcus voltae, Methanococcus maripaludis) were shown to reductively dissolve pyrite (FeS2) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using M. barkeri Fusaro, and laboratory-synthesized (Ni,Fe)S2 that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni,Fe)S2, M. barkeri promoted the reductive dissolution of (Ni,Fe)S2 and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni,Fe)S2 bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni,Fe)S2 is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth. IMPORTANCE Nickel is an essential metal, and its availability has changed dramatically over Earth history due to shifts in the predominant type of volcanism in the late Archean that limited its availability and an increase in euxinic conditions in the early Proterozoic that favored its precipitation as nickel sulfide minerals. Observations presented herein indicate that the methanogen, Methanosarcina barkeri, can acquire nickel at low concentration (<20 nM) from soluble and mineral sources. Furthermore, M. barkeri was shown to actively reduce nickelian pyrite; use dissolution products to meet their iron, sulfur, and nickel demands; and bioaccumulate nickel. These data help to explain how M. barkeri (and possibly other methanogens and anaerobes) can acquire nickel in contemporary and past anoxic or euxinic environments.

Natural and anthropogenic carbon input affect microbial activity in salt marsh sediment.

Salt marshes are dynamic, highly productive ecosystems positioned at the interface between terrestrial and marine systems. They are exposed to large quantities of both natural and anthropogenic carbon input, and their diverse sediment-hosted microbial communities play key roles in carbon cycling and remineralization. To better understand the effects of natural and anthropogenic carbon on sediment microbial ecology, several sediment cores were collected from Little Sippewissett Salt Marsh (LSSM) on Cape Cod, MA, USA and incubated with either Spartina alterniflora cordgrass or diesel fuel. Resulting shifts in microbial diversity and activity were assessed via bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Both Spartina and diesel amendments resulted in initial decreases of microbial diversity as well as clear, community-wide shifts in metabolic activity. Multi-stage degradative frameworks shaped by fermentation were inferred based on anabolically active lineages. In particular, the metabolically versatile Marinifilaceae were prominent under both treatments, as were the sulfate-reducing Desulfovibrionaceae, which may be attributable to their ability to utilize diverse forms of carbon under nutrient limited conditions. By identifying lineages most directly involved in the early stages of carbon processing, we offer potential targets for indicator species to assess ecosystem health and highlight key players for selective promotion of bioremediation or carbon sequestration pathways.

Influence of sulfide on diazotrophic growth of the methanogen Methanococcus maripaludis and its implications for the origin of nitrogenase.

Methanogens inhabit euxinic (sulfide-rich) or ferruginous (iron-rich) environments that promote the precipitation of transition metals as metal sulfides, such as pyrite, reducing metal or sulfur availability. Such environments have been common throughout Earth's history raising the question as to how anaerobes obtain(ed) these elements for the synthesis of enzyme cofactors. Here, we show a methanogen can synthesize molybdenum nitrogenase metallocofactors from pyrite as the source of iron and sulfur, enabling nitrogen fixation. Pyrite-grown, nitrogen-fixing cells grow faster and require 25-fold less molybdenum than cells grown under euxinic conditions. Growth yields are 3 to 8 times higher in cultures grown under ferruginous relative to euxinic conditions. Physiological, transcriptomic, and geochemical data indicate these observations are due to sulfide-promoted metal limitation, in particular molybdenum. These findings suggest that molybdenum nitrogenase may have originated in a ferruginous environment that titrated sulfide to form pyrite, facilitating the availability of sufficient iron, sulfur, and molybdenum for cofactor biosynthesis.

Diversity and function of methyl-coenzyme M reductase-encoding archaea in Yellowstone hot springs revealed by metagenomics and mesocosm experiments.

Metagenomic studies on geothermal environments have been central in recent discoveries on the diversity of archaeal methane and alkane metabolism. Here, we investigated methanogenic populations inhabiting terrestrial geothermal features in Yellowstone National Park (YNP) by combining amplicon sequencing with metagenomics and mesocosm experiments. Detection of methyl-coenzyme M reductase subunit A (mcrA) gene amplicons demonstrated a wide diversity of Mcr-encoding archaea inhabit geothermal features with differing physicochemical regimes across YNP. From three selected hot springs we recovered twelve Mcr-encoding metagenome assembled genomes (MAGs) affiliated with lineages of cultured methanogens as well as Candidatus (Ca.) Methanomethylicia, Ca. Hadesarchaeia, and Archaeoglobi. These MAGs encoded the potential for hydrogenotrophic, aceticlastic, hydrogen-dependent methylotrophic methanogenesis, or anaerobic short-chain alkane oxidation. While Mcr-encoding archaea represent minor fractions of the microbial community of hot springs, mesocosm experiments with methanogenic precursors resulted in the stimulation of methanogenic activity and the enrichment of lineages affiliated with Methanosaeta and Methanothermobacter as well as with uncultured Mcr-encoding archaea including Ca. Korarchaeia, Ca. Nezhaarchaeia, and Archaeoglobi. We revealed that diverse Mcr-encoding archaea with the metabolic potential to produce methane from different precursors persist in the geothermal environments of YNP and can be enriched under methanogenic conditions. This study highlights the importance of combining environmental metagenomics with laboratory-based experiments to expand our understanding of uncultured Mcr-encoding archaea and their potential impact on microbial carbon transformations in geothermal environments and beyond.

A naturalist perspective of microbiology: Examples from methanogenic archaea.

Storytelling has been the primary means of knowledge transfer over human history. The effectiveness and reach of stories are improved when the message is appropriate for the target audience. Oftentimes, the stories that are most well received and recounted are those that have a clear purpose and that are told from a variety of perspectives that touch on the varied interests of the target audience. Whether scientists realize or not, they are accustomed to telling stories of their own scientific discoveries through the preparation of manuscripts, presentations, and lectures. Perhaps less frequently, scientists prepare review articles or book chapters that summarize a body of knowledge on a given subject matter, meant to be more holistic recounts of a body of literature. Yet, by necessity, such summaries are often still narrow in their scope and are told from the perspective of a particular discipline. In other words, interdisciplinary reviews or book chapters tend to be the rarity rather than the norm. Here, we advocate for and highlight the benefits of interdisciplinary perspectives on microbiological subjects.

Reductive biomining of pyrite by methanogens.

Pyrite (FeS2) is the most abundant iron sulfide mineral in Earth's crust. Until recently, FeS2 has been considered a sink for iron (Fe) and sulfur (S) at low temperature in the absence of oxygen or oxidative weathering, making these elements unavailable to biology. However, anaerobic methanogens can transfer electrons extracellularly to reduce FeS2 via direct contact with the mineral. Reduction of FeS2 occurs through a multistep process that generates aqueous sulfide (HS-) and FeS2-associated pyrrhotite (Fe1-xS). Subsequent dissolution of Fe1-xS provides Fe(II)(aq), but not HS-, that rapidly complexes with HS-(aq) generated from FeS2 reduction to form soluble iron sulfur clusters [nFeS(aq)]. Cells assimilate nFeS(aq) to meet Fe/S nutritional demands by mobilizing and hyperaccumulating Fe and S from FeS2. As such, reductive dissolution of FeS2 by methanogens has important implications for element cycling in anoxic habitats, both today and in the geologic past.

Investigating Abiotic and Biotic Mechanisms of Pyrite Reduction.

Pyrite (FeS2) has a very low solubility and therefore has historically been considered a sink for iron (Fe) and sulfur (S) and unavailable to biology in the absence of oxygen and oxidative weathering. Anaerobic methanogens were recently shown to reduce FeS2 and assimilate Fe and S reduction products to meet nutrient demands. However, the mechanism of FeS2 mineral reduction and the forms of Fe and S assimilated by methanogens remained unclear. Thermodynamic calculations described herein indicate that H2 at aqueous concentrations as low as 10-10 M favors the reduction of FeS2, with sulfide (HS-) and pyrrhotite (Fe1- x S) as products; abiotic laboratory experiments confirmed the reduction of FeS2 with dissolved H2 concentrations greater than 1.98 × 10-4 M H2. Growth studies of Methanosarcina barkeri provided with FeS2 as the sole source of Fe and S resulted in H2 production but at concentrations too low to drive abiotic FeS2 reduction, based on abiotic laboratory experimental data. A strain of M. barkeri with deletions in all [NiFe]-hydrogenases maintained the ability to reduce FeS2 during growth, providing further evidence that extracellular electron transport (EET) to FeS2 does not involve H2 or [NiFe]-hydrogenases. Physical contact between cells and FeS2 was required for mineral reduction but was not required to obtain Fe and S from dissolution products. The addition of a synthetic electron shuttle, anthraquinone-2,6-disulfonate, allowed for biological reduction of FeS2 when physical contact between cells and FeS2 was prohibited, indicating that exogenous electron shuttles can mediate FeS2 reduction. Transcriptomics experiments revealed upregulation of several cytoplasmic oxidoreductases during growth of M. barkeri on FeS2, which may indicate involvement in provisioning low potential electrons for EET to FeS2. Collectively, the data presented herein indicate that reduction of insoluble FeS2 by M. barkeri occurred via electron transfer from the cell surface to the mineral surface resulting in the generation of soluble HS- and mineral-associated Fe1- x S. Solubilized Fe(II), but not HS-, from mineral-associated Fe1- x S reacts with aqueous HS- yielding aqueous iron sulfur clusters (FeS aq ) that likely serve as the Fe and S source for methanogen growth and activity. FeS aq nucleation and subsequent precipitation on the surface of cells may result in accelerated EET to FeS2, resulting in positive feedback between cell activity and FeS2 reduction.

The Physiology and Biogeochemistry of SUP05.

The SUP05 clade of gammaproteobacteria (Thioglobaceae) comprises both primary producers and primary consumers of organic carbon in the oceans. Host-associated autotrophs are a principal source of carbon and other nutrients for deep-sea eukaryotes at hydrothermal vents, and their free-living relatives are a primary source of organic matter in seawater at vents and in marine oxygen minimum zones. Similar to other abundant marine heterotrophs, such as SAR11 and Roseobacter, heterotrophic Thioglobaceae use the dilute pool of osmolytes produced by phytoplankton for growth, including methylated amines and sulfonates. Heterotrophic members are common throughout the ocean, and autotrophic members are abundant at hydrothermal vents and in anoxic waters; combined, they can account for more than 50% of the total bacterial community. Studies of both cultured and uncultured representatives from this diverse family are providing novel insights into the shifting biogeochemical roles of autotrophic and heterotrophic bacteria that cross oxic-anoxic boundary layers in the ocean.

Microbial Community Response to Polysaccharide Amendment in Anoxic Hydrothermal Sediments of the Guaymas Basin.

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community's metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach.

Spatially resolved correlative microscopy and microbial identification reveal dynamic depth- and mineral-dependent anabolic activity in salt marsh sediment.

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were resin-embedded and analysed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from ~60% in the top centimetre to 9.4%-22.4% between 2 and 10 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to putative sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling.

Examining Pathways of Iron and Sulfur Acquisition, Trafficking, Deployment, and Storage in Mineral-Grown Methanogen Cells.

Methanogens have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, deploy, and store these elements and how this, in turn, affects their physiology. Methanogens were recently shown to reduce pyrite (FeS2), generating aqueous iron sulfide (FeSaq) clusters that are likely assimilated as a source of Fe and S. Here, we compared the phenotypes of Methanococcus voltae grown with FeS2 or ferrous iron [Fe(II)] and sulfide (HS-). FeS2-grown cells are 33% smaller yet have 193% more Fe than Fe(II)/HS--grown cells. Whole-cell electron paramagnetic resonance revealed similar distributions of paramagnetic Fe, although FeS2-grown cells showed a broad spectral feature attributed to intracellular thioferrate-like nanoparticles. Differential proteomic analyses showed similar expression of core methanogenesis enzymes, indicating that Fe and S source does not substantively alter the energy metabolism of cells. However, a homolog of the Fe(II) transporter FeoB and its putative transcriptional regulator DtxR were up-expressed in FeS2-grown cells, suggesting that cells sense Fe(II) limitation. Two homologs of IssA, a protein putatively involved in coordinating thioferrate nanoparticles, were also up-expressed in FeS2-grown cells. We interpret these data to indicate that, in FeS2-grown cells, DtxR cannot sense Fe(II) and therefore cannot downregulate FeoB. We suggest this is due to the transport of Fe(II) complexed with sulfide (FeSaq), leading to excess Fe that is sequestered by IssA as a thioferrate-like species. This model provides a framework for the design of targeted experiments aimed at further characterizing Fe acquisition and homeostasis in M. voltae and other methanogens. IMPORTANCE FeS2 is the most abundant sulfide mineral in the Earth's crust and is common in environments inhabited by methanogenic archaea. FeS2 can be reduced by methanogens, yielding aqueous FeSaq clusters that are thought to be a source of Fe and S. Here, we show that growth of Methanococcus voltae on FeS2 results in smaller cell size and higher Fe content per cell, with Fe likely stored intracellularly as thioferrate-like nanoparticles. Fe(II) transporters and storage proteins were upregulated in FeS2-grown cells. These responses are interpreted to result from cells incorrectly sensing Fe(II) limitation due to assimilation of Fe(II) as FeSaq. These findings have implications for our understanding of how Fe/S availability influences methanogen physiology and the biogeochemical cycling of these elements.

Reductive dissolution of pyrite by methanogenic archaea.

The formation and fate of pyrite (FeS2) modulates global iron, sulfur, carbon, and oxygen biogeochemical cycles and has done so since early in Earth's geological history. A longstanding paradigm is that FeS2 is stable at low temperature and is unavailable to microorganisms in the absence of oxygen and oxidative weathering. Here, we show that methanogens can catalyze the reductive dissolution of FeS2 at low temperature (≤38 °C) and utilize dissolution products to meet cellular iron and sulfur demands associated with the biosynthesis of simple and complex co-factors. Direct access to FeS2 is required to catalyze its reduction and/or to assimilate iron monosulfide that likely forms through coupled reductive dissolution and precipitation, consistent with close associations observed between cells and FeS2. These findings demonstrate that FeS2 is bioavailable to anaerobic methanogens and can be mobilized in low temperature anoxic environments. Given that methanogens evolved at least 3.46 Gya, these data indicate that the microbial contribution to the iron and sulfur cycles in ancient and contemporary anoxic environments may be more complex and robust than previously recognized, with impacts on the sources and sinks of iron and sulfur and other bio-essential and thiophilic elements such as nickel and cobalt.

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment.

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

Next-generation physiology approaches to study microbiome function at single cell level.

The function of cells in their native habitat often cannot be reliably predicted from genomic data or from physiology studies of isolates. Traditional experimental approaches to study the function of taxonomically and metabolically diverse microbiomes are limited by their destructive nature, low spatial resolution or low throughput. Recently developed technologies can offer new insights into cellular function in natural and human-made systems and how microorganisms interact with and shape the environments that they inhabit. In this Review, we provide an overview of these next-generation physiology approaches and discuss how the non-destructive analysis of cellular phenotypes, in combination with the separation of the target cells for downstream analyses, provide powerful new, complementary ways to study microbiome function. We anticipate that the widespread application of next-generation physiology approaches will transform the field of microbial ecology and dramatically improve our understanding of how microorganisms function in their native environment.

Complete Genome Sequence of "Candidatus Thioglobus sp." Strain NP1, an Open-Ocean Isolate from the SUP05 Clade of Marine Gammaproteobacteria.

"Candidatus Thioglobus sp." strain NP1 is an open-ocean isolate from the SUP05 clade of Gammaproteobacteria Whole-genome comparisons of strain NP1 to other sequenced isolates from the SUP05 clade indicate that it represents a new species of SUP05 that lacks the ability to fix inorganic carbon using the Calvin-Benson-Bassham cycle.

Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria.

A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 µm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.

A Dissolved Oxygen Threshold for Shifts in Bacterial Community Structure in a Seasonally Hypoxic Estuary.

Pelagic ecosystems can become depleted of dissolved oxygen as a result of both natural processes and anthropogenic effects. As dissolved oxygen concentration decreases, energy shifts from macrofauna to microorganisms, which persist in these hypoxic zones. Oxygen-limited regions are rapidly expanding globally; however, patterns of microbial communities associated with dissolved oxygen gradients are not yet well understood. To assess the effects of decreasing dissolved oxygen on bacteria, we examined shifts in bacterial community structure over space and time in Hood Canal, Washington, USA-a glacial fjord-like water body that experiences seasonal low dissolved oxygen levels known to be detrimental to fish and other marine organisms. We found a strong negative association between bacterial richness and dissolved oxygen. Bacterial community composition across all samples was also strongly associated with the dissolved oxygen gradient, and significant changes in bacterial community composition occurred at a dissolved oxygen concentration between 5.18 and 7.12 mg O2 L(-1). This threshold value of dissolved oxygen is higher than classic definitions of hypoxia (<2.0 mg O2 L(-1)), suggesting that changes in bacterial communities may precede the detrimental effects on ecologically and economically important macrofauna. Furthermore, bacterial taxa responsible for driving whole community changes across the oxygen gradient are commonly detected in other oxygen-stressed ecosystems, suggesting that the patterns we uncovered in Hood Canal may be relevant in other low oxygen ecosystems.