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Executive Summary

Environmental Genomes and the Evolution of Complex Systems in Simple Organisms

Biology imposes an overwhelming force on planetary change through biogeochemical processes that originated in ~3.5 billion year old microbial communities. Through metabolic activities, microorganisms orchestrate key processes in carbon fixation, geochemical cycling, biodegradation and atmospheric change. For at least 80 percent of our evolutionary history, microbes were the only forms of life. By comparison, the multicellular world of plants, animals and fungi are derived forms whose continued existence is completely dependent upon a microbial world of uncharted diversity. Whether biological systems similar to those on Earth ever occurred or continue to function on other planets or large satellites is unresolved but if life occurs elsewhere in our solar system, it will be microbial in form.

The primary objectives of the Astrobiology Team at the Marine Biological Laboratory are rooted in delineating the evolutionary history of microbial life, exploring the nature of early metabolic processes, and defining the limits of the “habitable zone”. We seek to understand patterns and mechanisms of genome evolution and metabolic variation that allowed diverse microorganisms to adapt to new environments, to generate novel phenotypes, and to evolve processes that cause environmental changes on a global scale. Key questions include: When and where did life originate? How did biological complexity develop from emergent properties of living organisms? Which metabolic processes were significant drivers in modifying early Earth? What are the limits of extreme environments compatible with life? Answers to these questions will have a direct impact on how to organize and target exploration in search of living organisms beyond our own biosphere.

Our general strategy capitalizes on advanced molecular biological techniques and ranges from analyses of genomes to holistic studies of individual organisms within microbial communities. The physiological and microbial diversity studies focus on the hydrothermally altered sediments of Guaymas basin in the Gulf of California, the acidic, heavy metal laden Río Tinto of southwestern Spain, and photosynthetic cyanobacteria from microbial mats.

Evolution of Proteins: a genomic approach. The study of complete genome information from even a single organism can provide important insights about how metabolic diversity evolved from a relatively small number of catalytic centers that must have been present in early proteins. For example, fatty acid biosynthesis is an important pathway for every organism. It is a multi-step process and one of the key enzymes in the biosynthesis of lipids is fadB.

Figure 1. The Pathway (β oxidation of Fatty acids)

Using the N terminus as a probe for in silico studies, Dr. Riley and her colleagues mapped domains of the fadB sequence onto known structures of enzymes that catalyze reactions as diverse as epimerase,isomerase, hydratase and protease. They demonstrated that the C terminal part of the fadB protein has a dehydrogenase activity, whereas a single catalytic site is structurally related to epimerase, isomerase and hydratase.

Figure 2. The Reaction Mechanism

The proteins of the hydratase/isomerase superfamily catalyze a very diverse range of reaction types using a structurally conserved active site fold. This reinforces the idea that a relatively small number of catalytic domains in early proteins gave rise to metabolic complexity found in contemporary cells. The basic signatures of these seminal domains could provide the basis for design of life detection strategies.

Diversity and Evolution of MicroOrganisms.

Woese’s seminal work funded through NASA’s original Exobiology program unveiled a Universal Tree of Life that included three primary lines of descent; the Archaea, Bacteria and Eukarya. The MBL Astrobiology team continues to use and develop new molecular methodologies that can more efficiently describe microbial evolution, microbial diversity and how these organisms function in extreme environments. This information will expand our definition of habitable conditions and may suggest new targets for developing life detection capabilities. We now seek greater resolution in studies of microbial population structures as well as information about their association with specific metabolic functions. For example, we have explored the phylogenetic and functional diversity of sulfate-reducing bacteria in the sediments of Guaymas Basin (Gulf of California). In this hydrothermal vent site, thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material. These hydrocarbons are a significant carbon source to the vent microbial communities, which includes diverse sulfate reducing bacteria.

Figure 3. Microorganisms and their reactions (sulfate reduction, methanogenesis, methane oxidation, sulfide oxidation) in the methane and sulfur cyles in the Guaymas Basin hydrothermal vents.

Using conserved primers, we polymerase chain reaction (PCR) amplified dissimilatory sulfate reductase genes, the key gene for sulfate reduction (dsrAB), and 16S ribosomal ribonucleic acid (rRNA) genes from the upper 4 cm of Guaymas sediment. Figure 3 is a phylogenetic analysis of the nearly full length dsr sequences. In this analysis the dsrAB sequences revealed a major clade that branched with dsr sequences of the genus Desulfobacter, acetate oxidizers of the family Desulfobacteriaceae within the delta Proteobacteria, and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark). Two other clones showed similarity to dsr genes of gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the toluene degrader Desulfobacula toluolica, while one was related to Desulforhabdus amnigena and Thermodesulforhabdus norvegica.

Figure 4. Phylogenetic tree based on the translated amino acid sequences of PCR-amplified dsr AB genes from sulfate reducing prokaryotes.

The two-pronged approach of using 16S rRNA and dsr clone library sequencing has resulted in a more detailed picture of the sulfate-reducing bacterial community at Guaymasthan each of these approaches alone. At Guaymas, the 16S rRNA and the dsrAB datasets indicate the significance of members of the Desulfobacteriaceae, most likely the genus Desulfobacterium, in the Guaymas sediment. The dsrAB dataset also demonstrated the presence of uncultured and unknown major clades of sulfate reducing prokaryotes in the Guaymas sediments that could not be identified by 16S rRNA sequencing.

We also seek to establish linkages between patterns of gene expression that underlie metabolic activity and the formation of biogeochemical gradients. To meet this objective, we must obtain information about global gene expression patterns and detailed information about microbial population structures. We have constructed a custom microarray to examine how the cyanobacterium Microcoleus chthonoplastes responds to changes in environmental conditions. The array contains 1090 unique sequences and it has provided information about changing gene expression patterns in response to diel cycling and salinity changes of Microcoleus cultures. High and low salt shocks induced quite distinct expression signatures; genes involved in metabolism, signal transduction, transportation of compounds across membranes, and other cellular processes appear to change their expression levels in response to this environmental stimulus. With knowledge about coordinate gene expression patterns associated with changing metabolic patterns, we will be able to faithfully model fluxes in microbial ecosystems and identify biomarkers for detecting and monitoring biological activity.

Figure 5. Coordinate gene expression during high and low shock treatments of 1 to 3 hours.

In order to interpret the gene expression data it is necessary to identify the diversity and relative numbers of different kinds of taxa in a microbial setting. Analyses of PCR products from ribosomal RNA (rRNA) coding regions provides a window on the microbial world that has revealed new levels of largely unexplored microbial diversity not represented in laboratory cultures. Unfortunately this low throughput methodology is expensive (typically $1-2/sequenced template) and sometimes inefficient when a limited number of organisms dominate a microbial population. Deoxyribonucleic acid (DNA) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) offer higher throughput but little or no taxonomic information, and many fingerprinting studies ultimately rely on DNA sequencing to characterize phylotypes.

We have developed a new, high-throughput method Serial Analysis of Gene Tags (SAGT) that provides estimates of relative frequencies and sufficient phylogenetic information to identify the phylotype of most taxa in an environmental sample. SAGT ligates together the PCR products from orthologous hypervariable regions in rRNA genes to form large concatemers. A single DNA sequencing reaction of a cloned concatemer can include as many as 20-30 orthologous hypervariable regions represented in a population of nucleic acid molecules. In this way, samples loaded onto a 96-channel capillary sequencing machine can provide information about thousands of microorganisms in an analyzed sample. Comparison against a comprehensive rRNA gene database identifies the taxonomic assignment of individual amplicons in the concatemers. SAGT analyses of bacterial community composition in hydrothermal marine sediments from Guaymas Basin (Gulf of California) are comparable to results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identify the same major bacterial groups. The application of this technology will allow us to deeply sample the diversity and relative abundance of microbial populations in any environmental setting.

Education and Outreach.

Our EPO program includes a rich mixture of workshops and courses in disciplines that are relevant to NASA’s Astrobiology initiative. Our workshop series includes “Living in the Microbial World”, a one-week intensive summer workshop for middle and high school students, focusing on microbial diversity and evolution. “Life and Living in Space”, a four-day workshop for high school and community college teachers, convenes in the fall and is offered in conjunction with the Center for Advanced Studies of Space Life Sciences (CASSLS) at the MBL. This workshop combines NASA Life Science topics and areas of Astrobiology content including planetary protection and life detection. We also offered two advanced workshops that target investigators who work in astrobiology related disciplines. The first is the Workshop on Molecular Evolution at the Marine Biological Laboratory directed by Michael Cummings. The Workshop includes a series of lectures, demonstrations and computer labs that span the field of molecular evolution. A distinguishing feature of the course is a state-of-the-art computer laboratory furnished with Linux and Unix servers and workstations for comparative analysis of molecular data. Topics covered in the course include: databases and sequence matching, phylogenetic analysis including Bayesian analysis and maximum likelihood theory, molecular evolution at organismal and higher levels, molecular evolution and development, gene duplication and divergence, gene family organization, evolution of large multigene families, molecular evolution in bioinformatics and comparative genomics.

Our new course Advances in Genome Technology and Bioinformatics, directed by Mitchell L. Sogin and Claire Fraser (President of The Institute for Genomic Research (TIGR)), is a comprehensive, four-week course in genome science that integrates bioinformatics with the latest laboratory techniques for genome sequencing, genome analysis, and high throughput gene expression (DNA microarrays). With a distinguished faculty from major universities and bioinformatics centers, TIGR and the MBL provide instruction that integrates lectures with laboratory exercises both at the computer and in a high technology, high throughput facility.

Figure 6. The computational biology laboratory for Advances in Genome Sciences and Bioinformatics.

The major laboratory modules include 1) Genome Sequencing (vector development, library construction, high throughput sequencing technologies, principles of automation using advanced robotic liquid handlers, genome assembly algorithms and closure strategies); 2) Bioinformatics (Gene prediction algorithms, annotation, database construction and searching, phylogenetics and molecular evolution); and 3) Functional Genomics (DNA microarrays, data analysis). Symposia focusing on Environmental and Evolutionary Genomics, Eukaryotic Microbial Genome Projects, Organelle Evolution and other topics are also part of the program.

In addition to our MBL Astrobiology web site (which is under total revision) we have expanded micro*scope to include a broader range of microbial habitats, more images, more text, enhanced functionality in the form of outlinks and input fields for user contributions. The micro*scope web site (http://www.mbl.edu/microscope) is an image-rich resource providing descriptions and pictures of all categories of microorganisms, currently containing thousands of downloadable high-resolution images. The images and accompanying information can be accessed by a number of methods, including by habitat, cell shape, alphabetically by genus name and by hierarchical taxonomic classification. We are developing micro*scope into a central educational repository for students and teachers interested in astrobiology and microbial diversity. The micro*scope website fills the gap in available resources and provides students, teachers and researchers with easy access to high quality digital micrographs and information about diverse microbes, both prokaryotic and eukaryotic. Micro*scope is designed to bring together information distributed at other authoritative sites. It uses software to track the taxonomic location of the user and to initiate searches into remotely located databases or other sites generally available on the web, based on the genus being examined. The list of databases and remote sites to be searched can be customized to return results from only certain selected databases. Our goal is to maintain micro*scope as a resource to assist those interested in gathering information about microbial taxa and in understanding better the diversity, complexity and evolution of the microbial world.