Bringing microbial diversity into focus: high-resolution analysis of iron mats from the Lo'ihi Seamount.

Thirty kilometers south of the island of Hawai'i lies the Lo'ihi Seamount, an active submarine volcano that hosts a network of low-temperature hydrothermal vents enriched in ferrous iron that supports extensive microbial mats. These mats, which can be a half a meter deep, are composed of ferric iron bound to organic polymers - the metabolic byproduct of iron-oxidizing Zetaproteobacteria. Though the role of Zetaproteobacteria in mat formation is well established, we have a limited understanding of how differences in diversity are related to mat morphology. We used Minimum Entropy Decomposition and ZetaOtu classification to demonstrate cryptic diversity between closely related Zetaproteobacteria while showing habitat and geographic specificity. Veiled mats, common structures at Lo'ihi, exhibit distinct community composition and contain diversity not detected in other mat types, including specific Zetaproteobacteria and an unclassified Gammaproteobacteria. Our analyses also indicate that diversity can change dramatically across small spatial transects from points of active venting, yet we found comparatively few differences between major sampling sites. This study provides a better picture of the microbiome responsible for iron mat production at Lo'ihi and has broad implications for our understanding of these globally distributed communities.

Refining real-time predictions of Vibrio vulnificus concentrations in a tropical urban estuary by incorporating dissolved organic matter dynamics.

The south shore of O'ahu, Hawai'i is one of the most visited coastal tourism areas in the United States with some of the highest instances of recreational waterborne disease. A population of the pathogenic bacterium Vibrio vulnificus lives in the estuarine Ala Wai Canal in Honolulu which surrounds the heavily populated tourism center of Waikiki. We developed a statistical model to predict V. vulnificus dynamics in this system using environmental measurements from moored oceanographic and atmospheric sensors in real time. During a year-long investigation, we analyzed water from 9 sampling events at 3 depths and 8 sites along the canal (n = 213) for 36 biogeochemical variables and V. vulnificus concentration using quantitative polymerase chain reaction (qPCR) of the hemolysin A gene (vvhA). The best multiple linear regression model of V. vulnificus concentration, explaining 80% of variation, included only six predictors: 5-day average rainfall preceding water sampling, daily maximum air temperature, water temperature, nitrate plus nitrite, and two metrics of humic dissolved organic matter (DOM). We show how real-time predictions of V. vulnificus concentration can be made using these models applied to the time series of water quality measurements from the Pacific Islands Ocean Observing System (PacIOOS) as well as the PacIOOS plume model based on the Waikiki Regional Ocean Modeling System (ROMS) products. These applications highlight the importance of including DOM variables in predictive modeling of V. vulnificus and the influence of rain events in elevating nearshore concentrations of V. vulnificus. Long-term climate model projections of locally downscaled monthly rainfall and air temperature were used to predict an overall increase in V. vulnificus concentration of approximately 2- to 3-fold by 2100. Improving these predictive models of microbial populations is critical for management of waterborne pathogen risk exposure, particularly in the wake of a changing global climate.

Validating the Cyc2 Neutrophilic Iron Oxidation Pathway Using Meta-omics of Zetaproteobacteria Iron Mats at Marine Hydrothermal Vents.

Zetaproteobacteria create extensive iron (Fe) oxide mats at marine hydrothermal vents, making them an ideal model for microbial Fe oxidation at circumneutral pH. Comparison of neutrophilic Fe oxidizer isolate genomes has revealed a hypothetical Fe oxidation pathway, featuring a homolog of the Fe oxidase Cyc2 from Acidithiobacillus ferrooxidans However, Cyc2 function is not well verified in neutrophilic Fe oxidizers, particularly in Fe-oxidizing environments. Toward this, we analyzed genomes and metatranscriptomes of Zetaproteobacteria, using 53 new high-quality metagenome-assembled genomes reconstructed from Fe mats at Mid-Atlantic Ridge, Mariana Backarc, and Loihi Seamount (Hawaii) hydrothermal vents. Phylogenetic analysis demonstrated conservation of Cyc2 sequences among most neutrophilic Fe oxidizers, suggesting a common function. We confirmed the widespread distribution of cyc2 and other model Fe oxidation pathway genes across all represented Zetaproteobacteria lineages. High expression of these genes was observed in diverse Zetaproteobacteria under multiple environmental conditions and in incubations. The putative Fe oxidase gene cyc2 was highly expressed in situ, often as the top expressed gene. The cyc2 gene showed increased expression in Fe(II)-amended incubations, with corresponding increases in carbon fixation and central metabolism gene expression. These results substantiate the Cyc2-based Fe oxidation pathway in neutrophiles and demonstrate its significance in marine Fe-mineralizing environments.IMPORTANCE Iron oxides are important components of our soil, water supplies, and ecosystems, as they sequester nutrients, carbon, and metals. Microorganisms can form iron oxides, but it is unclear whether this is a significant mechanism in the environment. Unlike other major microbial energy metabolisms, there is no marker gene for iron oxidation, hindering our ability to track these microbes. Here, we investigate a promising possible iron oxidation gene, cyc2, in iron-rich hydrothermal vents, where iron-oxidizing microbes dominate. We pieced together diverse Zetaproteobacteria genomes, compared these genomes, and analyzed expression of cyc2 and other hypothetical iron oxidation genes. We show that cyc2 is widespread among iron oxidizers and is highly expressed and potentially regulated, making it a good marker for the capacity for iron oxidation and potentially a marker for activity. These findings will help us understand and potentially quantify the impacts of neutrophilic iron oxidizers in a wide variety of marine and terrestrial environments.

Author Correction: A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Architecture of Iron Microbial Mats Reflects the Adaptation of Chemolithotrophic Iron Oxidation in Freshwater and Marine Environments.

Microbes form mats with architectures that promote efficient metabolism within a particular physicochemical environment, thus studying mat structure helps us understand ecophysiology. Despite much research on chemolithotrophic Fe-oxidizing bacteria, Fe mat architecture has not been visualized because these delicate structures are easily disrupted. There are striking similarities between the biominerals that comprise freshwater and marine Fe mats, made by Beta- and Zetaproteobacteria, respectively. If these biominerals are assembled into mat structures with similar functional morphology, this would suggest that mat architecture is adapted to serve roles specific to Fe oxidation. To evaluate this, we combined light, confocal, and scanning electron microscopy of intact Fe microbial mats with experiments on sheath formation in culture, in order to understand mat developmental history and subsequently evaluate the connection between Fe oxidation and mat morphology. We sampled a freshwater sheath mat from Maine and marine stalk and sheath mats from Loihi Seamount hydrothermal vents, Hawaii. Mat morphology correlated to niche: stalks formed in steeper O2 gradients while sheaths were associated with low to undetectable O2 gradients. Fe-biomineralized filaments, twisted stalks or hollow sheaths, formed the highly porous framework of each mat. The mat-formers are keystone species, with nascent marine stalk-rich mats comprised of novel and uncommon Zetaproteobacteria. For all mats, filaments were locally highly parallel with similar morphologies, indicating that cells were synchronously tracking a chemical or physical cue. In the freshwater mat, cells inhabited sheath ends at the growing edge of the mat. Correspondingly, time lapse culture imaging showed that sheaths are made like stalks, with cells rapidly leaving behind an Fe oxide filament. The distinctive architecture common to all observed Fe mats appears to serve specific functions related to chemolithotrophic Fe oxidation, including (1) removing Fe oxyhydroxide waste without entombing cells or clogging flow paths through the mat and (2) colonizing niches where Fe(II) and O2 overlap. This work improves our understanding of Fe mat developmental history and how mat morphology links to metabolism. We can use these results to interpret biogenicity, metabolism, and paleoenvironmental conditions of Fe microfossil mats, which would give us insight into Earth's Fe and O2 history.

A distinct and active bacterial community in cold oxygenated fluids circulating beneath the western flank of the Mid-Atlantic ridge.

The rock-hosted, oceanic crustal aquifer is one of the largest ecosystems on Earth, yet little is known about its indigenous microorganisms. Here we provide the first phylogenetic and functional description of an active microbial community residing in the cold oxic crustal aquifer. Using subseafloor observatories, we recovered crustal fluids and found that the geochemical composition is similar to bottom seawater, as are cell abundances. However, based on relative abundances and functional potential of key bacterial groups, the crustal fluid microbial community is heterogeneous and markedly distinct from seawater. Potential rates of autotrophy and heterotrophy in the crust exceeded those of seawater, especially at elevated temperatures (25 °C) and deeper in the crust. Together, these results reveal an active, distinct, and diverse bacterial community engaged in both heterotrophy and autotrophy in the oxygenated crustal aquifer, providing key insight into the role of microbial communities in the ubiquitous cold dark subseafloor biosphere.

Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank.

To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.

Microbial activity in the marine deep biosphere: progress and prospects.

The vast marine deep biosphere consists of microbial habitats within sediment, pore waters, upper basaltic crust and the fluids that circulate throughout it. A wide range of temperature, pressure, pH, and electron donor and acceptor conditions exists-all of which can combine to affect carbon and nutrient cycling and result in gradients on spatial scales ranging from millimeters to kilometers. Diverse and mostly uncharacterized microorganisms live in these habitats, and potentially play a role in mediating global scale biogeochemical processes. Quantifying the rates at which microbial activity in the subsurface occurs is a challenging endeavor, yet developing an understanding of these rates is essential to determine the impact of subsurface life on Earth's global biogeochemical cycles, and for understanding how microorganisms in these "extreme" environments survive (or even thrive). Here, we synthesize recent advances and discoveries pertaining to microbial activity in the marine deep subsurface, and we highlight topics about which there is still little understanding and suggest potential paths forward to address them. This publication is the result of a workshop held in August 2012 by the NSF-funded Center for Dark Energy Biosphere Investigations (C-DEBI) "theme team" on microbial activity (www.darkenergybiosphere.org).

An oligarchic microbial assemblage in the anoxic bottom waters of a volcanic subglacial lake.

In 2006, we sampled the anoxic bottom waters of a volcanic lake beneath the Vatnajökull ice cap (Iceland). The sample contained 5 x 10(5) cells per ml, and whole-cell fluorescent in situ hybridization (FISH) and PCR with domain-specific probes showed these to be essentially all bacteria, with no detectable archaea. Pyrosequencing of the V6 hypervariable region of the 16S ribosomal RNA gene, Sanger sequencing of a clone library and FISH-based enumeration of four major phylotypes revealed that the assemblage was dominated by a few groups of putative chemotrophic bacteria whose closest cultivated relatives use sulfide, sulfur or hydrogen as electron donors, and oxygen, sulfate or CO(2) as electron acceptors. Hundreds of other phylotypes are present at lower abundance in our V6 tag libraries and a rarefaction analysis indicates that sampling did not reach saturation, but FISH data limit the remaining biome to <10-20% of all cells. The composition of this oligarchy can be understood in the context of the chemical disequilibrium created by the mixing of sulfidic lake water and oxygenated glacial meltwater.

Soluble Mn(III) in suboxic zones.

Soluble manganese(III) [Mn(III)] has been thought to disproportionate to soluble Mn(II) and particulate Mn(IV)O2 in natural waters, although it persists as complexes in laboratory solutions. We report that, in the Black Sea, soluble Mn(III) concentrations were as high as 5 micromolar and constituted up to 100% of the total dissolved Mn pool. Depth profiles indicated that soluble Mn(III) was produced at the top of the suboxic zone by Mn(II) oxidation and at the bottom of the suboxic zone by Mn(IV)O2 reduction, then stabilized in each case by unknown natural ligands. We also found micromolar concentrations of dissolved Mn(III) in the Chesapeake Bay. Dissolved Mn(III) can maintain the existence of suboxic zones because it can act as either an electron acceptor or donor. Our data indicate that Mn(III) should be ubiquitous at all water column and sediment oxic/anoxic interfaces in the environment.