nai

Project Progress

1. Life in Deep-Sea Hydrothermal Vents

The research objectives of Co-Investigator John Baross continue to focus on the microbial and biogeochemical characterization of Earth environments that share geophysical and geochemical characteristics with other planetary systems. The emphasis of Baross and his team is on magma-hosted and peridotite-hosted hydrothermal systems and subseafloor rock-hosted ecosystems affected by hydrothermal activity. These hydrothermal systems produce biologically important carbon and energy sources that can support microbial ecosystems without dependence on chemical input from photosynthesis. These environments are also primordial and may be the sites for key chemical reactions that led to the origin of life on Earth and for the earliest microbial ecosystems. Baross and his colleagues hypothesize that these commonly encountered metabolic pathways in hydrothermal vent environments are ancient and may have developed before the evolution of catalytic proteins that instead use minerals as the source of catalysis.

During the past few years, Baross’s research has focused on microorganisms and microbial communities that are involved in carbon dioxide reduction (primary producers) and particularly those organisms that are hydrogen, sulfur, and methane metabolizers. They have also begun a new focus on hydrothermal vent microbes that can oxidize simple organic compounds such as formate and acetate using iron as the electron acceptor. A component of the research on iron reducers will be to measure the fractionation of iron isotopes at different temperatures and pressures.

Baross’s subseafloor research continued to focus on the microbial diversity and physiology at diffuse-flow vents in active hydrothermal vent environments and in ancient oceanic crust. Baross, former Doctoral Student Julie Huber (now a Postdoctoral Research Associate at the Marine Biological Laboratory), and his team have recently demonstrated that the microbial diversity is different at different diffuse vent sites and also changes with time at any given vent site. Fluid temperature and the degree of mixing of hydrothermal vent fluid with seawater are key factors that affect microbial community composition. Their research at the Lost City Hydrothermal Field continued to focus on the role of serpentinization reactions in producing carbon and energy sources that support microbial ecosystems. A focus of this research is on the possible importance of serpentinization reactions as a source of methane and other organic compounds, and hydrogen, which could support life on other planets and moons where water and ultramafic rock are present.

The Lost City Hydrothermal Field is driven by a water/rock interaction that results in the production of high-temperature (>90°C) and high-pH (10 to 11) fluids, a combination of extreme conditions never before seen in the marine environment. Initial studies by the Baross group using molecular methods showed that a species related to the Methanosarcinales order of methanogens is the only Archaea found in high-temperature, high-pH carbonate structures. Lost City Methanosarcinales (LCMS) is present as a dense biofilm coating every available surface throughout the porous carbonate chimneys. Results from microcosm experiments using samples of carbonate incubated with 13C-methane and 13C-bicarbonate at 70 to 90°C during the August 2005 cruise to Lost City showed that both methane oxidation and methane production occurred in the highest temperature, highest pH chimneys where LCMS dominates. Anaerobic oxidation of methane is thermodynamically favorable for energy production when in co-culture with another microbe that can remove protons. The best example involves the syntrophic association between Methanosarcinales-related Archaea and sulfate-reducing delta-proteobacteria, sulfate-reducing microbes similar to those that interact with anaerobic methane oxidizers in methane hydrate environments. However, sulfate-reducing bacteria were not detected in hot carbonates either from 16S rRNA or dsrAB clone libraries. The team’s current hypothesis is that this single species of Methanosarcinales (based on >97% identical 16S rRNA sequences) has evolved into different metabolic groups that interact syntrophically so that some strains produce methane and others oxidize methane anaerobically. If correct, this hypothesis would profoundly affect paradigms in microbial ecology and microbial evolution: metabolically diverse cells existing within a community of a single species are novel. Moreover, the association between cells that make methane and those that oxidize it appears to be obligatory, with neither process occurring without the other. Such a symbiotic association would be the first between two metabolically distinct strains of the same species.

Ongoing research is focused on testing this hypotheses using molecular and biochemical approaches. Baross and his group have a proposal approved by the Department of Energy’s Joint Genome Institute to undertake the environmental metagenomic sequencing of LCMS-dominated carbonate chimneys. These analyses should reveal the extent of micro-evolutionary diversity within metabolic as well as other physiological genes and thus prove or disprove their hypothesis.

During this past year, Mausmi Metha completed her Ph.D. thesis on studies of nitrogen fixation in hydrothermal vent environments. A publication in Science in December 2As part of her dissertation research, she reported on the isolation and characterization of a deep subseafloor methanogen that is capable of fixing nitrogen at 92°C (the high temperature for growth of the isolate). This temperature is almost 30°C higher than has ever been observed for nitrogen fixation. Baross and colleagues have been able to express the nitrogen fixation gene (nifH) at 92°C, genetically map most of the nitrogen fixation operon, and demonstrate nitrogen assimilation into proteins using15N2 over the temperature growth range of the isolate. This work has two important implications: it expands nitrogen fixation into hyperthermophilic environments that have no form of nitrogen other than N2, and it shows that nitrogen fixation genes are ancient and preceded the evolution of iron-bearing proteins associated with early photosynthesis. Moreover, phylogenetic models of these hyperthermophilic nitrogen fixation genes indicate that they evolved before the separation of the three domains of life. Current work involves further characterization of the proteins involved in nitrogen fixation including the metals associated with the protein, and the completion of a genome sequence of the isolate.

2. Water at Gigapascal Pressures

The behavior of H₂O under pressure is central to the work of Co-Investigator Russell Hemley and his colleagues. New transformations in ice were documented by X-ray diffraction and Raman spectroscopy, and the equations of state and optical properties of ice were measured to very high pressures. Studies of metastable transformations in H2O continued as they could be important for characterizing cold, water-rich environments. In addition, Hemley and his team have found evidence for the facile dissociation of H₂O to form H₂ and O₂ under pressure. These observations may limit the stability range of H₂O in planetary interiors. A related reaction was observed in NH₃.

Various clathrate hydrates were also investigated over a broad range of planetary environments. Novel compounds that may be stable in the satellites of the outer planets such as H₂O-Hsub>2 and CH₄(H₂)₄ were found to be stable over a wide range of pressure and temperature. Neutron scattering measurements on clathrate hydrates under pressure were begun in order to understand the structure, dynamics, and reactivity of these systems. The stability and formation of reduced species such as methane and higher hydrocarbons continue to be explored.

3. Microbial Adaptation at Gigapascal Pressures

Research continued on the response of microbial life to elevated hydrostatic pressure. Recent work focused on obtaining in situ optical observations of specific cellular mechanisms at high pressure both in non-piezophiles, such as Escherichia coli, and more exotic organisms, such as Ax99. Traditional fluorescence microscopy combined with a custom diamond anvil cell designed for improved imaging allow for the study of such phenomena as membrane transport of ions, cellular morphology, and cell viability in vivo. Preparation is underway to incorporate large-volume techniques, which will permit the collection of bulk material for nucleic acid microarray analysis as well the initiation of studies of long-term adaptations to high pressure rather than simply the immediate response to short-term stress. This approach takes full advantage of the institution’s capabilities to answer key questions about life in extreme environments.

{{ 1 }}

4. Iron-based Metabolic Strategies for Microbial Life

Co-Investigator David Emerson’s group is investigating both natural populations and pure cultures of iron-oxidizing bacteria (FeOB) in an attempt to understand how their physiology and ecology influence the mineralogy and geochemistry that are hallmarks of these organisms in the environment. Lately, Emerson and colleagues have focused on the description of novel FeOB, their biological and mineralogical properties, and their habitats. By developing a more thorough understanding of iron-metabolizing bacteria in Earth environments, they hope to constrain the conditions under which life forms might exist that could use iron as an energy source on other planets or planetary bodies.

Work has focused on completing taxonomic descriptions of a group of freshwater and marine neutrophilic, oxygen-dependent, Fe-oxidizing bacteria. Emerson and colleagues are beginning to understand the coherence of these groups. They have identified a new order of freshwater beta-proteobacteria that appear to be specially adapted to growing on Fe(II) as a sole energy source. These include two new genera, Ferritrophicum radicicola and Sideroxydans lithotrophicus, as well as one new species, Gallionella capsiferriformans. The fact that these organisms show some phlyogenetic continuity is interesting since it suggests that this is, not surpisingly, a specialized metabolism. Furthermore, it makes their identification in the environment easier, because detection probes can be designed more specifically. Finally, all these organisms have proven to be obligate Fe-oxidizers.

Likewise, in the marine environment Emerson and colleagues have identified a group of obligate Fe(II)-oxidizing bacteria that are even more phylogenetically distinct and represent a new candidates class, the zeta-proteobacteria. Although these organisms have virtually the same physiology as the beta-proteobacteria, they have not found members of the freshwater community in the marine environment or vice versa, using either cultivation or cultivation-independent methods. To date Emerson and his team have discovered Mariprofundus ferrooxydans strains PV-1 and JV-1 from Loihi Seamount, an undersea volcano located near Hawaii. From samples collected on a research cruise completed in November 2007, they isolated an additional strain of this species. The most striking characteristic of all these strains is that they form a filamentous iron-oxide structure that is composed of approximately 50-nm-diameter nanofibrils of Fe-oxides. These nanofibrils are directionally excreted from the cells, and the kinetics of excretion are proportional to the growth rates of the cells. Work done by Collaborator Clara Chan, using scanning transmission X-ray microscopy (STXM), recently showed for the first time that C is a component of the stalk material and that it has characteristics of an acidic polysaccharide. Further work done using an innovative microslide culture shows that the organisms may be able to orient themselves in gradients of Fe(II) and oxygen. Emerson and colleagues are still in the process of investigating this result; it would suggest these organisms are capable of a form of mechanotaxis not previously described in the prokaryotic world. Furthermore, these types of structures with similar orientations have been documented in the fossil record.

{{ 2 }}

{{ 3 }}

In other work, Emerson’s group has developed a rapid method for determining the genotypes of Archaea, including methanogens and halophiles. This method uses a kit-based repetitive element polymerase chain reaction (PCR) assay to distinguish strain-level differences among strains of these organisms. This technique could be useful for understanding the population dynamics of these organisms, as well as being an aid in their identification.