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Project Progress

Task 1. Field Studies and Laboratory Characterization of Hydrothermal Vent Microbes (Baross)

During the past year Baross and his group continued their efforts to understand the phylogenetic, metabolic, and physiological diversity of microorganisms indigenous to hot sulfide chimneys and subsurface habitats associated with submarine hydrothermal systems. On the basis of the characteristics of “particle-attached” and “free-living” bacteria in subsurface fluids from Axial Volcano, Juan de Fuca Ridge, work complementary to an earlier assessment of the diversity of archaea from the same samples, there is a highly diverse community of bacteria and archaea that are unique to the subsurface. Included in this group are thermophilic and hyperthermophilic organisms that fix CO₂ and oxidize H₂ using elemental sulfur or polysulfides as the electron acceptors. This is a new group of bacteria that evidently use the reductive citric acid cycle to fix carbon dioxide. Other unique microorganisms isolated from subsurface fluids include thermophilic iron-reducing bacteria and archaea. The epsilon-proteobacteria were found to be the dominant bacteria identified in subsurface habitats on and off axis. While there are only limited data on the epsilon-proteobacteria from vent environments, they are clearly indigenous to vent habitats and are likely to be involved in sulfur cycling.

The group completed preliminary analysis of the diversity of microorganisms in the subsurface that harbor the nifH gene, which is involved in nitrogen fixation. This study was initiated because N₂ is the only abundant form of nitrogen in the hot, anerobic subsurface at many mid-ocean ridge sites. Earlier results suggested the presence in this habitat of a diverse community of potentially nitrogen-fixing archaea and bacteria. The latest work supports the hypothesis that a unique subsurface microbial community is not dependent on electron acceptors and nitrogen compounds produced by photosynthetic organisms.

Baross and colleagues also completed a preliminary study on the incidence and diversity of microorganisms in active sulfide chimneys. Results of the study show that microorganisms exist throughout these structures, even in zones that have experienced temperatures considerably greater than 100°C on the basis of mineral compositional constraints. Two observations from this study were particularly surprising. First, microbial communities, detected optically with deoxyribonucleic acid (DNA)-specific fluorescent stains and ribonucleic acid (RNA) probes, inhabit the hot interior zones of the sulfide chimney. All of these organisms belong to the Archaea, and most could be detected with RNA probes, indicating that they may well be viable and active. Second, and in all cases, the microbial communities exist by forming biofilms on mineral surfaces. These observations suggest that biofilm formation on minerals may be associated with microbial growth or survival at extremely high temperatures. The group completed preliminary phylogenetic characterization of the microbial community associated with the carbonate chimneys at Lost City Hydrothermal Field. Lost City is a novel off-axis hydrothermal system driven by serpentinization reactions between seawater and ultramafic oceanic crust. This reaction is exothermic and can produce high concentrations of hydrogen, methane, and low-molecular-weight organic compounds.

Task 2. Microbial Activity at Gigapascal Pressures (J. Scott, Fogel, Sharma, Steele)

In follow-on work to an initial demonstration that microbial activity can persist at pressures in excess of 1 GPa (Figure 1), Sharma, Scott, and colleagues have been working toward determining the pressure-expressed proteins and genetic elements of Escherichia coli and Shewanella oneidensis. The past year has been spent developing and optimizing techniques for recovering cells for manipulation in order ultimately to resolve the processes involved in prokaryotic stress response at the molecular level. Most living organisms survive under suboptimal conditions. It is therefore essential to understand how stress affects the formation of the molecular components of cells that make up the subpopulation of biomarkers and bio-indicators that potentially serve as evidence of biological activity.

One of the most important developments of the past year has been a move away from diamond anvil cells as the exclusive means for exposing microbial communities to excess pressure. The goal of these efforts is to optimize working volumes for performing basic microbiological, biochemical, and molecular-genetic manipulations on cells exposed to pressures approaching 1 GPa. To date the group has been able to expose E. coli cells to pressures in excess of 350 MPa in a hydrothermal pressure vessel and then recover cells aseptically in a volume of up to 2 milliliters for cultivation in a complex growth medium.

Together with Steele and Fogel, Scott has begun to identify key proteins and genetic components involved in the response of E. coli to pressure-induced stress. Steele’s laboratory provides an ideal battery of molecular approaches (from genome array technology to gene-amplification) for resolution of pressure-induced operons or regulons that play a key role in the the response to stress. A time-of-flight mass spectrometer system in Fogel’s laboratory is being optimized for identification of proteins found in a typical prokaryotic cell. Scott is also involved, with colleagues at The Institute for Genomic Research and the University of Southern California, in efforts to resolve the genome and proteome for S. oneidensis strain MR1. This work will provide an important second bacterium to determine whether the response of E. coli is common or unique among prokaryotes. Further, S. oneidensis is an environmentally important bacterium that should provide a good model for understanding the full range of environmental implications of prokaryotic stress responses.

Task 3. Iron-Oxidizing Lithotrophs (Emerson, Fogel, Sutka)

A major emphasis of the research by Emerson’s group during the past year was on C and N metabolism by lithotrophic Fe-oxidizing bacteria that grow at neutral pH. The guiding assumption, based on substantial circumstantial evidence, was that these organisms were autotrophic. Work led by postdoctoral fellow Robin Sutka, however, on isotopic analysis of ¹³C-labled bicarbonate, does not support this assumption. Instead, these bacteria appear to be obligate lithoheterotrophs, i.e., they obtain their energy from an inorganic energy source (Fe²⁺) and their C from organic matter. While this type of metabolism is unusual, what is particularly striking is that these organisms appear to use only certain amino acids as C-sources. Attempts to feed them on a number of more commonly used carbohydrates and organic acids were unsuccessful.

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