nai

Executive Summary

The relevance of microbes to NASA’s exploration program.

The stage for life was set billions of years ago and we are on the verge of knowing what happened to its characters. Two convergent lines of evidence raise expectations that biological systems could occur beyond the confines of Earth. The first is the detection of microbial life forms in terrestrial environments that may be similar to those on other solar system bodies. The second is NASA’s spectacular discoveries of significant water reservoirs on Mars and Europa, and evidence of liquid organic environments on other solar system bodies such as Titan. Knowledge about the diversity and evolution of life on Earth will provide important inputs for the design of future astrobiology missions in NASA’s exploration program.

During the initial 2-3 billion years of Earth’s history, the only form of life was microbial and it generally consisted of complex consortia. The effects of the microbial biosphere, driven by metabolism and expressed as biogeochemical processes, have imposed an overwhelming force on planetary change and have shaped Earth’s habitability. They are the essential catalysts for all of the chemical reactions within the biogeochemical cycles. From an evolutionary perspective, microbes are important because they share an ancient common ancestry with lineages that ultimately evolved into more complex organisms such as fungi, plants and animals. The primary objective of the Astrobiology Program at the Marine Biological Laboratory is to study microbial diversity and evolution. We seek to understand the evolution of key enzymes in ancestral metabolic pathways and the diversity of microbial life in acidic environments, in hydrothermal vent environments, in the deep subsurface, and in anoxic, high-pressure environments. Whenever possible we integrate this information with studies of biogeochemical processes and interpretation of geological data from remote sensing activities. Several of our investigations target genomes in order to understand the evolution and diversity of organisms that carry out neutraphilic iron oxidation or which play a role in shaping the evolution of genomes in more complex organisms via formation of endosymbiotic relationships. We also participate in the development of technology and instruments for in situ life detection and planetary protection.

Microbially mediated iron metabolism.

Because iron plays a key role in all biological systems, the study of environments enriched in iron may provide insights into the biochemistry and metabolisms of the early Earth and putative life that might have existed on ancient Mars. We are currently investigating the microbial diversity and mineralogy of the iron-rich Rio Tinto of southwestern Spain, the metabolic diversity of neutraphilic iron oxidizers from the deep sea as revealed through genome-based investigations, and iron isotope investigations of ancient pyrites. The Rio Tinto studies include collaborations between Linda Amaral Zettler and Carmen Palacios (MBL), Jack Mustard and Aline Gendrin (Brown University and the MBL Astrobiology team) and Ricardo Amils and Elena Gonzalez-Toril (Centro de Astrobiología / Universidad Autónoma de Madrid). The iron and sulfate mineralogy of the Rio Tinto serves as an analogue for Mars. Dr. Mustard is building a database of field measurements that will provide a predictive framework for interpreting remote sensing measurements. The database includes diversity measurement of minerals and analysis of airborne hyperspectral data. It will ultimately be coupled to the biological and biogeochemical measurements made by Drs. Amaral Zettler and Amils. The biology measurements employ a novel technology (SARST-V6) to define microbial diversity at several sites (Origin, Anabel’s Garden and Berrocal) in the iron rich (22 mg ml^-1) and acidic (pH ranging from 1.7-2.3) Rio Tinto. Earlier studies based upon characterizations of ribosomal RNA genes isolated from the Rio Tinto suggested that eukaryotic diversity is much greater than prokaryotic diversity, which in this watershed includes fewer than a dozen kinds of bacteria. The SARST technology samples short homologous hyper-variable regions in rRNAs. It has the advantage of being at least an order of magnitude more efficient than traditional DNA sequence analyses of PCR amplicons from rRNAs in molecular characterizations of microbial population structures. This allows for more thorough sampling of microbial populations and the detection of minor components in microbial populations. Figure 1 is a rarefaction study of rRNAs based upon SARST analysis of several different sites in the Rio Tinto. It reveals that the microbial diversity of the Rio Tinto as measured by the presence of different rRNAs is much greater than previously appreciated. From one study of 4545 tags, Amaral Zettler and her colleagues identified 126 bacterial phylotypes. They also obtained many additional tags with less than 100% similarity to a reported sequence in GenBank for which we are designing primers to extract full-length sequence data. These sequences await further phylogenetic analyses. An analysis of a total of 11,951 tags is underway which is likely to significantly increase the total number of phylotypes known to date.

Figure 1. Rarefaction curves of microbial populations from three sites in the Rio Tinto based upon SARST-V6. DNA was extracted from three locations nicluding the Origin (OR), Anabel’s Garden (AG) and Berrocal (BE) from the Rio Tinto. Using conserved primers that flank hypervariable region V6 in Bacterial rRNA genes, we generated PCR amplicons approximately 65 bp in length. Concatemerized sequences of the short PCR amplicons (SARST-V6) were parsed into invidual tags and compared to rRNAs in GenBank. The number of different kinds of microbes (Organism Taxonomic Units – OTUs) in each sample are plotted vrs. the number of sampled tags.

Dr. Edwards, working with Ashita Dhillon in Dr. Sogin’s laboratory, has been investigating the genomics of Marinobacter aquaeolei, which is a cultured neutraphilic iron oxidizer from the deep sea. The genome sequence of this organism is nearly complete and we have constructed fosmid expression libraries. Using a simple plate-based assay we have identified fosmids that express genes required for Fe-oxidation in E. coli and production of HemA. Sequence analysis of these fosmids has allowed Edwards and Dhillon to identify an operon in M. aquaeolei that plays an important role in iron oxidation. The next phase of this project will explore the diversity of iron oxidation pathways in neutraphilic iron oxidizers from the sea. The traditional approach is to prepare large DNA insert libraries and employ molecular probes or PCR techniques to identify clones that contain a conserved protein associated with a metabolic activity. However screening strategies that rely upon conservation of nucleotide sequences frequently overlook highly diverged coding regions. Our solution is to use a functional assay such as the plate assay for Fe-oxidation. We will determine sequences for any fosmid that expresses genes required for Fe-oxidation. This will provide information about the genomic context and diversity of pathways responsible for iron oxidation under neutraphilic conditions. The final project related to iron is an iron isotope study of banded iron formations. The rise of atmospheric oxygen which began by ~2.3 Ga was a profound event in Earth’s history. The oxidation state of Fe, along with C and S, is linked to the redox state of the surface environment. It is very likely that the concentration and isotopic composition of Fe in seawater was linked to such global changes, yet deposition of banded iron formations (BIFs) indicates that the deep ocean remained anoxic until ~500 million years after the initial rise of atmospheric oxygen. Using Fe isotope evidence, Edwards provides evidence that the change in the ocean iron cycle occurred at the same time as the change in the atmospheric redox state (Figure 2). Variable and negative iron isotope values in pyrites older than about 2.3 Ga suggest that an iron-rich global ocean was strongly affected by the deposition of iron oxides. Between 2.3 and 1.8 Ga, positive iron isotope values of pyrite probably reflect an increase in the precipitation of iron sulfides relative to iron oxides in a redox stratified ocean. This data provides new insights into the Archean and Paleoproterozoic ocean chemistry and redox state. Fe isotopes suggest that the Archean oceans were globally Fe-rich and that their Fe isotope composition and Fe content were variable in response to the episodic establishment of an Fe-rich pool supplied by hydrothermal activity and the deposition of Fe oxides, either in BIFs or dispersed throughout sediments on continental shelves and in the deep sea. After the rise of atmospheric oxygen by about 2.3 Ga, the Paleoproterozoic Ocean became stratified and characterized by an increase of sulfide precipitation relative to Fe oxide precipitation. During this period, BIFs were likely deposited by upwelling of Fe(II)-rich plumes and rapid oxidation in the oxygenated layer of the ocean.

Figure 2. Plot of del 56Fe versus sample age for Fe-sulfides from black shales and Fe-oxides from banded iron formations (BIF) (Rouxel et al, 2005). Gray diamonds correspond to Fe isotope composition of pyrite from black shales and open squares and triangles correspond to Fe isotope composition of magnetite and hematite-rich samples from BIFs. Gray area corresponds to del 56Fe of Fe derived from igneous rocks (at 0.1 del) and hydrothermal sources (ca. -0.5 del). Dashed lines represent the contour lines of maximum and minimum Fe isotope compositions of sedimentary sulfides used to define Stages I to III.

Studies of microbial diversity and ancient metabolism in anoxic marine environments.

One of the remarkable discoveries of the last decade is the occurrence of deep subsurface life. Microbial communities exist in habitats that support exclusively chemotrophic communities. In a study carried out in collaboration with the URI Astrobiology team, Teske has been characterizing the rRNAs of deep subsurface sediments from sites containing very low levels of organic matter. The archaeal lineages represented in the PCR amplicon libraries from rRNA molecules are related to rDNA genes amplified from other deep subsurface sites. There is no evidence of remineralization, sulfate reduction or methanogenesis in this environment, but we infer that these sequences correspond to active organisms. Since the rRNA content of microbes is to the first approximation proportional to metabolic activity, detection of PCR amplicons templated from rRNA molecules provides presumptive evidence of active microbial communities.

Two other studies of microbes from deep sea, anoxic sediments address questions about the evolution of pathways that may be very ancient. The first is a comparative study by Teske, Dhillon, and Sogin of dissimilatory (dsr) and assimilatory sulfite reductases (asr) including new environmental sequences from Guaymas Basin. Sulfite reductases play a key role in the global sulfur cycle. Comparative amino acid sequence analyses of the dissimilatory sulfite reductase genes (dsrAB) from cultured isolates and environmental PCR amplicons have provided insights about the evolutionary history of anaerobic sulfate (sulfite) respiration. Our more recent data allowed us to infer that a gene duplication of ancestral dsr genes gave rise to ars before the archaeal- bacterial divergence. In a related investigation based upon key genes of methanogenesis, mcrA, we showed that the populations of Guaymas are phylogenetically and physiologically quite diverse. The sequences include novel lineages within the Methanosarcinales that are distinct from all other cultured and cloned Methanosarcinales, and from ANME-2 groups. Methanogens and sulfate-reducing bacteria competing for the same carbon substrates coexist in the same sediment horizons, an unusual occurrence in marine sediments. The second investigation seeks to define the distribution and phylogeny of enzymes in the reductive TCA cycle (rTCA). This pathway is used by chemolithoautrophs for carbon fixation. Sievert and Edgcomb have amplified portions of the ATP-citrate lyase gene and flanking regions from several epsilon-protobacteria and members of the Aquificales. This is the first report of reductive TCA cycle carbon fixation in epsilon-proteobacteria. Initial phylogenetic analyses support the view that citrate lyase is an early evolved metabolic activity. The next phase will be to analyze PCR amplicons from environmental DNA samples from the deep sea. Seivert is also working towards the complete genome sequence of Thiomicrospira denitrificans, a chemolithoautotrophic sulfur-oxidizing epsilon-proteobacterium.

Genome reduction in microbial symbionts.

Phylogenetic analyses of microbial populations from diverse environments provide information about patterns of evolution but few details about underlying mechanisms. An important contribution from genome sequence studies is elucidation of major changes in genome architecture that we can associate with specific mechanisms. As part of our interest in microbial evolution, Wernegren and Bordenstein are exploring changes in genome organization in endosymbiotic bacteria. They are studying the genome of Blochmannia pennsylvanicus, a proteobacterial, intracellular mutualist of carpenter ants. They have completed the 792 kb genome sequence of B. pennsylvanicus using facilities in the Bay Paul Center at the MBL. Through bioinformatics techniques they are exploring how severe genome reduction has occurred in this microbe. They have determined that the mutualist has lost many genes that would normally utilize environmental substrates and that the genome is evolving very rapidly. Surprisingly, there is little evidence of gene transfer, inversions or translocations. This is counter to mechanisms that drive major changes in genome structure in free- living microbes. They are also investigating evolution of the genome and associated viruses of Wolbachia, a genus of obligate intracellular α—Proteobacteria. One of the major changes in earth’s history was ushered in by the rise of oxygen. This may have lead to the evolution of α—Proteobacterial endosymbionts into mitochondria, the site of aerobic energy production in eukaryotic organisms. The Wolbachia species parasitize a broad range of arthropods and they are infected with phage that can move between different intracellular bacteria. Wernegreen and Bordenstein demonstrated through microarray analyses that most of the divergent genes between Wolbachia strains reside within prophage rather than within other mobile genetic elements, thus offering an explanation for how genes have moved between different Wolbachia species.