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Project Progress

1. Life in deep-sea hydrothermal vents
The major research directions in Co-Investigator John Baross’s laboratory are (1) the identification and characterization of microbial communities from subseafloor, sulfide, and carbonate chimney hydrothermal vent environments and particularly those that have novel metabolic pathways for using carbon dioxide or low-molecular-weight organic compounds (formate and acetate) as carbon sources, and (2) microbial/mineral association, including high-temperature biofilm formation, mineral catalysis, and microbial dissolution of minerals as a mechanism for nutrient acquisition.
Baross and his research team’s astrobiology objectives continued to focus last year on the microbial and biogeochemical characterization of Earth environments that share geophysical and geochemical characteristics with other planetary systems. Baross’s emphasis is on magma-hosted and peridotite-hosted hydrothermal systems and subseafloor-rock-hosted ecosystems affected by hydrothermal activity. Baross and coworker’s hypothesis is that the predominant groups of microorganisms encountered in the extreme hydrothermal vent environments have metabolic pathways and other physiological properties that are ancient. The metabolic pathways of interest are those involving carbon dioxide reduction coupled with hydrogen oxidation, and iron- and sulfur-reduction coupled with the oxidation of simple organic compounds. They are also putting considerable emphasis on biofilms and particularly those associated with minerals, since it is likely that the earliest microbial communities were biofilms that relied on minerals rather than proteins as a source of catalysis.
Baross’s subseafloor research continues to focus on the microbial diversity and physiology at diffuse-flow vents at active hydrothermal vent environments and in ancient oceanic crust. They view sub-seafloor microbial ecosystems as potential analogs to environments on Mars and other planets/moons. Subseafloor hydrothermal vent environments are of particular interest since there is evidence that they produce carbon and energy sources that support microorganisms in the absence of input from photosynthesis, and they are ancient, becoming established as water accumulated. This implies that on Earth, subseafloor microbial communities may be important in global primary production and biogeochemical cycling while possibly resembling ancient microbial ecosystems. Baross and his students examined the subseafloor microbial community diversity from five geographically distinct diffuse-flow hydrothermal vent sites over the course of six years following the 1998 eruption at Axial Seamount and a similar suite of samples from the Main Endeavour Vent Field, Juan de Fuca Ridge, between 2000 and 2007. Terminal restriction fragment length polymorphism (tRFLP) and 16S rRNA gene sequence analyses were used to determine the bacterial and archaeal diversity, and the statistical software Primer was used to compare microbial diversity with temperature and fluid chemistry. At both Axial and Endeavour, individual vents harbored distinct community structures that were different from all other vents, and there were no obvious correlations between microbial diversity and chemical composition of vent fluids. Potential isolating or stabilizing mechanisms to explain our observed distribution patterns include biogeographic barriers preventing dispersal of microbes among vents, or environmental selection of ubiquitous, rare phylotypes that proliferate in response to specific environmental conditions.

Baross’s 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. The serpentinization reaction is one of the possible sources of methane detected in the Martian atmosphere. Since the reaction can occur anywhere ultramafic rock is exposed to liquid water, Europa and Titan also have the potential to support subsurface serpentinization. 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 fluids (10 to 11), a combination of extreme conditions never before seen in the marine environment. From their preliminary results from a physiological investigation of archaeal biofilms, they predicted that the single-species biofilm could mediate both methanogenesis and anaerobic methane oxidation. This was shown to be the case from experiments using 13C-methane and 13C-bicarbonate at 70 to 90°C during the August 2005 cruise to Lost City. Recently completed metagenomic sequencing of these biofilms was applied to test the hypothesis that the biofilm contains distinct methanogenic and methanotrophic ecotypes. The metagenomic data also revealed interesting features of the nitrogen and sulfur cycles of the Lost City microbial community. Recycling of methane within a single microbial consortium is likely to be difficult to detect by measuring bulk fluids. At the Lost City hydrothermal vent environment, the isotopic signature of methane indicates a purely abiogenic source, but thick methanogenic and/or methanotrophic biofilms are clearly thriving on serpentinization fluids. One of the group’s conclusions that are germane to astrobiology is that attempts to detect life in possible subsurface ecosystems on Mars, Europa, or Titan should be based on an awareness that isotopic signatures can’t disprove biogenicity. Doctoral student Billy Brazelton will be participating on a cruise to Lost City to conduct further experiments using stable isotopes to confirm that the single-species biofilm at Lost City can mediate both methanogenesis and anaerobic methane oxidation. Furthermore, in collaboration with Mitch Sogin (MBL) a new molecular method was applied to the Lost City biofilm confirming that there are multiple ecotypes within the single-species biofilm and that the ecotype composition changes depending on the age of the carbonate structure.
Baross and his team have completed a study of microbial colonization of newly formed hydrothermal vent sulfide chimneys. The microbial ecology of early succession in newly formed extreme environments is not well understood. A chimney less than one year old was sampled for co-registered mineralogical and microbiological analyses from the newly activated “Sasquatch” vent field on the Endeavour Segment of the Juan de Fuca Ridge. At the time of sampling, the anhydrite-dominated chimney was venting saline fluids at 280°C that were enriched in carbon dioxide, methane, and hydrogen above background seawater values. Phylogenic analyses indicated that the microbial assemblages in the interior of the chimney were dominated by the archaeal phylotypes Paleococcus, Methanothermus, and Geoglobus. The implication is that the microbial diversity in newly formed sulfide chimneys is extremely low and dominated by iron-reducing and methane-producing Archaea. Furthermore, our results suggest that the first colonizers may originate in the subseafloor biosphere associated with hydrothermal vent environments since similar species have been detected in subseafloor diffuse-flow fluids.

2. Life in cold environments
Cold environments, such as the Arctic, are excellent for studying life and its biosignatures in extreme habitats. As part of the Arctic Mars Analog Svalbard Expedition (AMASE), Co-Investigator Andrew Steele and his team study extremophiles and test life-detection instruments in extreme conditions on Svalbard, an island covered by artic tundra located at 77 to 80°N. To assess the diversity of microbial community structure and their biogeochemical processes in the arctic ecosystem, their group deploys an instrument suite, which includes (i) standard genetic techniques to identify and characterize microbial populations (polymerase chain reaction or PCR), (ii) protein microarrays for common microbial biomarkers as well as hand-held life detection instruments, such as (iii) an adenosine triphosphate (ATP) luminometer as an assay for overall metabolic activity, and (iv) a Limulus amebocyte lysate (LAL) assay that detects gram-negative bacterial cell wall materials (living or dead). In situ DNA sampling, PCR, and analysis performed in the field using gene-specific primers indicate differences in overall community composition and activity among different rock types. By characterizing organisms and their habitats, we characterize molecular structures that define their functionality, which in turn provides potential targets for biomarker investigations. When successful, biosignature correlations strengthen interpretations and may be particularly valuable in the search for life on Mars.
An impressive number of microorganisms and their genomic signatures, in particular their 16S or 18S rRNA gene, have been discovered and published all over the world. However, a vast majority of organisms, especially in extreme environments such as Svalbard, are still undetermined. Therefore, we are in the process of conducting sequencing analysis of collected sample material, such as ice from the ice caves, carbonates from Troll Springs and Jotun Springs, endolithic material, weathered olivines, and snow algae. Bacterial diversity is driven by different geological and mineralogical compositions of these materials, which establish different metabolic niches. Sequencing analysis is done by amplifying ribosomal gene sequences, in particular 16S and 18S rRNA, from extracted DNA samples analyzed for the occurrence of bacteria/Archaea and eukaryota, respectively. Most known genomic sequences are accessible in databases and can be used for sequence comparison. Correlation of phylogenetic data and physiological data with detailed chemistry, derived from other AMASE instruments, will lead to a comprehensive overview of specific areas in Svalbard.
Photosynthetic cyanobacteria have the capability to grow on minimal chemical components, utilizing the energy of sunlight and the ability to fix CO₂ (the “dark reaction” of photosynthesis). Additionally, cyanobacteria occur in this extreme environment and potentially have major contributions in biochemical processes. Cyanobacteria, collected during the AMASE expedition, are in the process of being cultured in our laboratories.

3. Survival of micro-organisms under high hydrostatic pressure
It is estimated that more life resides below the surface of Earth than on or above it. Because of this, understanding the effects of high pressure (HP) on microorganisms is necessary for fully understanding life in a planetary context. The limitations presented by HP inform our notions of habitability not only on Earth but also its potential existence elsewhere in the Solar System. HP treatment is also employed by food processors to limit microbial growth. All of these applications necessitate detailed investigation into the effects of HP on microbial systems.
The extant body of work on HP microbiology focuses on microbial inactivation, the cessation of organelle function, and alterations to cellular metabolism. Despite these great strides, the precise molecular-level processes responsible for the deleterious effects of HP exposure remain unknown. Genomic analyses of cellular stress responses and observations of the HP survival of mutant strains have provided several clues in this direction. However, such measurements have proven difficult to interpret. For instance, in Escherichia coli HP treatment induces both heat and cold shock proteins simultaneously and also invokes the SOS system although high pressure is not thought to be mutagenic. Further, very closely related organisms can exhibit highly disparate survival under HP conditions. The oxidative stress response has been observed after exposure to HP treatment, leading to the hypothesis that oxidative damage plays a significant role in microbial inactivation by HP, although such a process has never been directly observed. Work undertaken by Postdoctoral Fellow Andrienne Kish, Collaborator Patrick Griffin, Co-Investigators Steele and Marilyn Fogel seeks to test this hypothesis in order to determine the cytotoxic effects of exposure to HP at molecular-level.
Halobacterium sp. str. NRC-1 (Halobacterium), an extreme halophile and a member of the Archaea, was previously shown by Kish and others to withstand high doses of ionizing radiation, due in part to its intrinsic resistance to oxidative damage. High concentrations of intracellular Cl- ions, mainly involved in maintaining osmotic balance in the hypersaline environments inhabited by Halobacterium cells, were shown to scavenge hydroxyl radicals, thereby reducing the amount of oxidative damage to both nucleic acids and proteins. When exposed to HP conditions, Halobacterium cells showed no significant decrease in survival up to 300 MPa, and 80% survival at 400 MPa compared with control samples. To determine whether this barotolerance is the result of the resistance of Halobacterium to oxidative damage stemming from the use of halide ions as intracellular osmolytes, or from differences in membrane structure between archaeal cells and bacterial cells, two other halophiles, Salinibacter rubrum and Chromohalobacter salexigens, were selected for comparison. Both are gram-negative bacteria with membranes composed of ester-linked lipids in contrast to archaeal cell walls with an ether-linked lipid membrane surrounded by a surface layer (S-layer) of glycoprotein. The added structural rigidity of this composition may aid in barotolerance in Halobacterium. In addition, S. ruber cells sequester high KCl for osmoregulation as do Halobacterium cells, whereas C. salexigens cells utilize compatible solutes such as ectoine and glycine betaine, allowing for testing of benefits of free-radical scavenging by intracellular halides under HP conditions. Experiments are currently underway to determine the survival after exposure to HP of S. ruber and C. salexigens as a baseline for further molecular-level assays of oxidative stress in each species along with Escherichia coli as a non-halophilic control.
The team’s procedures have yielded reproducible survival data for Escherichia coli and Halobacterium. Escherichia coli exhibited a bifurcating survival curve, with some sub-populations capable of 100-fold greater survival at some pressures. Further, survival of Escherichia coli at pressures up to 400 MPa was measured to be higher than previously published data on Escherichia coli MG1655 with differences as great as 50-fold. The internal discrepancies may be partially explained by heterogeneity in RpoS, a gene that induces approximately 30 proteins, among them proteins associated with oxidative and osmotic stresses. This heterogeneity, however, would not explain the disagreement with previously published work on the same strain. Future work with the cultures used by other experimenters is planned in the future to determine the source of this discrepancy.
To determine the nature and extent of macromolecular damage for each of the three major macromolecules (proteins, lipids, and nucleic acids), immunodetection methods are being employed for detection of lipid peroxidation, oxidative damage to nucleic acids, and protein oxidation as evidenced by carbonyl group formation. Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (ToF) analysis of proteins of interest will provide further information into the identity and nature of the damaged proteins. Preliminary results indicate that lipid peroxidation does not increase with increasing pressure in Escherichia coli. This suggests that if membrane integrity is compromised by oxidation due to HP, the oxidation might be isolated to the protein fraction of the membrane. Similar work is currently underway to determine the extent of protein oxidation, and nucleic acid oxidation is planned for the near future, both for Escherichia coli as well as the three halophilic species.
This study will determine, for the first time, the role of oxidative stress and osmoregulation in the survival of micro-organisms under high-pressure conditions.

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 better how their physiology and ecology influences the mineralogy and geochemistry that are hallmarks of these organisms in the environment. Lately, they have focused on 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 lifeforms might exist that could use iron as an energy source on other planets or planetary bodies.
In late August 2007, Emerson moved his lab from the American Type Culture Collection (ATCC) at George Mason University in Virginia to the Bigelow Laboratory in Maine. Significant time had been devoted to setting up a new laboratory and hiring new staff, since none of the ATCC staff were able to make the move. In spring 2008, the new lab was fully operational, and a Postdoctoral Fellow, Emily Fleming, was hired to work on this project in May 2008, in addition to a technician who is funded through another project.
Work has been completed on identification of new freshwater and marine neutrophilic, oxygen-dependent Fe-oxidizing bacteria. In addition, Emerson and his team have isolated a new Fe-reducing bacterium, HR-1, a novel species of Geothermobacter from a hydrothermal vent at our study site at Loihi Seamount. This organism grows at temperatures upto 45°C and readily reduces iron with acetate. Overall, there is little evidence for Fe-reduction in the ecosystems within the iron-rich mats at Loihi. However, Fe-reducing bacteria are present. When the team incubates HR-1 with either natural mat samples from Loihi or biogenically produced oxides, HR-1 will readily carry out iron reduction if acetate is added to the medium; if not, then the rate of Fe-reduction is much lower (Figure 1). Together this evidence suggests that, while the potential for Fe-reduction exists in the iron-rich mats at Loihi, it is quite limited due to a shortage of labile C for Fe-reducing bacteria. The mats themselves contain about 108 bacterial cells/ml, thus while C is present, it is in a form that is not readily available for Fe-reducing bacteria. Emerson and coworkers 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 this is, not surpisingly, a specialized metabolism. Furthermore, it makes their identification in the environment easier, since detection probes can be designed more specifically. Finally, all these organisms have proven to be obligate Fe-oxidizers.

Likewise, Emerson and his team have identified a group of obligate Fe(II)-oxidizing bacteria in the marine environment that are even more phylogenetically distinct and represent a new candidates class, the zeta-proteobacteria. Interestingly, although these organisms have virtually the same physiology as the beta-proteobacteria, Emerson has not found members of the freshwater community in the marine environment, or vice versa, using either cultivation or cultivation-independent methods. To date they have discovered Mariprofundus ferrooxydans strains PV-1 and JV-1, both from Loihi Seamount. From a research cruise Emerson and his group completed in November 2007, they have 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 excretion products are directionally excreted from the cells, and the kinetics of excretion are proportional to the growth rates of the cells. Work done by collaborating postdoctoral researcher Clara Chan (WHOI), using scanning transmission X-ray microscopy (STXM), recently showed for the first time that C is a component of the stalk material and that the material 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. This inference would suggest that 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.

Emerson and his team have developed a rapid method for determining the genotypes of Archaea, including methanogens and halophiles. This method uses a kit-based repetitive-element 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.