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2007 Annual Science Report

Marine Biological Laboratory Reporting  |  JUL 2006 – JUN 2007

Executive Summary

Most of life’s genetic and metabolic diversity is microbial, from the beginning of life and throughout its history to the present day. These organisms form microbial communities of astounding genetic complexity. Their aggregate metabolic activities shape planetary habitability by maintaining the carbon cycle through remineralization of carbon with and without oxygen, controlling the global utilization of nitrogen and mediating biogeochemical cycles for sulfur, iron and manganese. In marine settings, between 50 and 98 percent of the total biomass is microbial.

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Figure 1. Microbes rule the ocean. Cultivation-based enumeration of microbes in the oceans generally reported 100 organisms/ml of sea water. In contrast, when viewed through the microscope, the number of cells that can be stained with the DNA binding dye DAPI, ranges from 105 to 106 microorganisms/ml. The number of viruses is at least ten fold greater. The large objects in this micrograph are protists. The intermediate size objects are microbial cells (~105/ml) and the faintly staining elements are viral particles. The total number of microbial cells in the oceans is estimated to be between 1028 and 1029 / ml. Photo courtesy of Jed Fhurman. University of Southern California.

Given their role in shaping habitability over billions of years and their pivotal role in the evolution of Earth’s biosphere, microbes are the most likely life forms to have either a history or a presence on other solar system objects. From an Earth-centric perspective, all of our biosphere’s diverse multi-cellular life forms including plants and animals are totally dependent upon the microbial world for their continued survival. Not only do they shape our environment, some microbial communities reside within multi-cellular hosts where they contribute intermediate metabolites, metabolic functions and microbial genes to the host organism. Finally, knowledge about microbial communities is important for understanding and monitoring global change. Global warming is exerting a profound impact on earth’s habitability and biodiversity including composition of the microbial world. It is now imperative to detect and monitor shifts in microbial populations because they, in effect, have become the proverbial “canary in the coal mine” by serving as exquisitely sensitive sentinels of environmental change.

This NAI team investigates the diversity and evolution of microbial communities in both nominal and extreme environments. We seek to understand their contribution to ecosystem processes and we use remote sensing techniques combined with field studies to facilitate NASA’s search for potential extraterrestrial biological worlds within our solar system. Finally we investigate the role of microbes as symbionts in the evolution of more complex life forms. Members of this team have participated in a number of mission related activities including Co-I of the OMEGA Imaging spectrometer experiment (Jack Mustard), Co-I of the HRSC imaging experiment (James Head), Deputy PI of the CRISM imaging spectrometer experiment (Jack Mustard), Co-I Preliminary Studies Towards a Microbial Genetic Inventory of Spacecraft rRNA V6-Tag Diversity Survey (Mitchell Sogin) and service on the Europa Explorer Science Definition Team. We also participated in two NRC studies; The Limits of Organic Life in Planetary Systems and Preventing the Forward Contamination of Mars. Finally, we organized the microbial exploration initiative (MSEI). This first MSEI workshop had a very significant impact on our strategy for exploring the population structure of microbial communities. It led to a strategy that allows us to evaluate the diversity (richness) and relative abundance (evenness) of microbial populations using a paradigm that led to discovery of low abundance taxa (the Rare Biosphere) that represent previously unappreciated diversity.

For nearly two decades microbiologists, have used gene cloning and/or polymerase chain reactions (PCR) to isolate and sequence DNA coding regions for ribosomal RNAs (rRNA) and other functional genes. This technology allows the interrogation of microbial communities without requiring the cultivation of individual taxa in the laboratory. Each sequence in these rapidly expanding data sets serves as a proxy for the presence of a taxon or operational taxonomic unit (OTU) in naturally occurring, complex microbial communities. Analysis of PCR products from DNA samples records information about both living and dead organisms whereas analyses of PCR products from RNA inform us about diversity of living organisms. The MBL NAI team has used this technology to document diversity of all three domains in hydrothermally heated sediments of Guaymas Basin, deep subsurface sediments, basalts from the Loihi seamount and the Rio Tinto of South Western Spain.

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Figure 2. Extreme microbial environments. The Loihi and Guaymas bacterial populations are complex whereas the acidic, iron rich environment of the Rio Tinto is dominated by five or six closely related taxa while ~300-400 low abundance OTUs account for the total bacterial richness. In contrast, eukaryotes in the Rio Tinto are very diverse and the community includes both photosynthetic and hetertrophic organisms.

Using rRNA templates to generate PCR amplicon libraries, the Teske laboratory has characterized active archaeal communities in the deep subsurface. His group has observed very large heterotrophic archaeal communities in the continental margins. Primers that target the methane coenzyme M reductase alpha subunit, revealed the presence of a novel lineage of methanogens and methane oxidizers , the “deep ANME-1 lineage. To date, these have only been found in the deep subsurface and in hydrothermal vents. These ANME-1 methane oxidizers occur below the methane/sulfate transition zone even in the absence of active methane oxidation. Futhermore, laboratory incubations show that these archaea tolerate very high pressures (>900 atmospheres) and therefore can tolerate an extended habitat range in the deep subsurface. Anaerobic methane-oxidation by Archaea was likely very important in the early carbon cycle of Earth because of their ability to transform methane to CO2.
Using similar technology, Katrina Edwards has been been studying microbial communities on the surface of basalts from the Loihi seamount. Her analysis of full length rRNA sequences suggests that some of these communities are surprisingly complex, possibly rivaling the diversity of soil microbial communities. The communities include anammox bacteria and they may play a role in nitrogen cycle and possibly in iron transformation. Her group is also exploring microbial communities in a new hydrothermal field “Ula Nui” at Loihi. This system is unusual in that its temperature is 0.2C above ambient bottom yet it is clearly a hydrothermal environment that is biological active and dominated by metal cycling bacteria.

The MBL NAI team’s work in the Rio Tinto is interdisciplinary. Dr. Amaral Zettler has been studying the microbial evolutionary ecology of this system in collaboration with Ricardo Amils of the CAB. Drs Mustard and Head have been working on techniques that will potentially allow for the identification of habitable environments using remote sensing data. The goal is to use this system as an analogue for remote sensing on Mars from the CRISM data. Their focus has been to use aerial VNIR data from the Rio Tinto to characterize deposits of sulfate and iron oxide minerals. The interpretation of these remote sensing data sets was verified by field work at the Rio Tinto which discovered greater than anticipated mineral diversity. The microbial work uses a new technology Serial Analysis of Ribosomal Sequence Tags (SARST-V6 originally developed for studying microbial diversity in Guaymas basin. This molecular biological strategy allows us to sequence concatomers of short hypervariable regions from rRNA genes. Because a larger number of sequences from an environment can be sampled, this technology allows for a more detailed description of microbial diversity from complex communities. Dr. Amaral-Zettler has shown that bacterial diversity is represented by a handful of dominant taxa plus nearly 400 diverse, low abundant OTUs. In contrast, the protist populations of the Rio Tinto are extremely diverse. This study demonstrates the similarities and differences of discrete sites in this acidic, iron-rich drainage system and ties these microbial population studies into information about biogeochemical parameters that influence microbial growth.

An extension of the SARST technology was the development of tag sequencing strategies using massively parallel pyrosequencing technology. The strategy takes advantage of the parallel DNA pyrosequencing capability of a Genome System 20 454 machine to sequence thousands of short V6 hypervariable regions from rRNA genes.
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Figure 3. Secondary structure of rRNA gene. Red regions correspond to rapidly evolving / hypervariable regions in small subunit rRNAs. The V6 region is flanked by conserved primers that direct the generation of PCR amplicon libraries for massively parallel pyrosequence analysis.

This past year, we used tag sequencing to document the microbial population structures of communities at different locations in the Deep Water North Atlantic Flow and in diffuse flows of seamount environments. We have now extended those studies to include the Archaea in diffuse flows and to examine Bacterial compositions on surfaces of basalts. The studies of Archaea show that with this technology we can obtain a nearly exhaustive description of archaeal communities in diffuse flows. The non-parametric estimates of diversity suggest that despite their dominant biomass, the archaeal diversity is on the order of 2700 different OTUs. This contrasts to Bacterial diversity that remains unknown despite collection of nearly 750,000 sequence tags. The estimates of bacterial diversity in this system exceeds 40,000 OTUs in a liter of sea water. Furthermore, microbial compositions of different diffuse flows (16 have been sampled to date) are remarkably different from each other and very complex. Most of the diversity in all of these sites correspond to low abundance taxa that we refer to as the “Rare Biosphere”. Most recently we have applied the technology to determining inventories of microbial taxa on spacecraft and clean room assembly facilities. The analysis of microbial compositions on spacecraft is at a very early stage of development and a report on this project will be deferred until our next progress report. However we already know that the diversity of sequence tags isolated from the spacecraft is much greater than anticipated. Rarefaction analysis suggests there are at least 2000 OTUs on the spacecraft and that the number of OTUs in the clean room is even greater. These are total estimate of microbial diversity for both living and dead microbes

Our final project is tied to the role of symbionts and the evolution of more complex life forms. Using Wolbachia as a model system, Wernegren and Bordenstein have shown that parasitic forms arose earlier in the endosmbiotic than the mutualistic strains. This suggests that organelles might have arisen from parasites that evolved to become endosymbiotic or mutualistic with their hosts. The key is establishing vertical transmission in new host taxa.

Our EPO activity continues to focus on training students in phylogenetic theory through the Workshop in Molecular Evolution at the MBL and training of K-12 educators in our workshop “Living in the Microbial World”. There has also been a very major development that extends our work on the NAI supported web site micro*scope developed by David Patterson. This past year, the MacArthur and Sloan Foundations have funded a new initiative, the Encyclopedia of Life (EOL) The goal of this 50 million dollar initiative is to establish web page for every known organism on Earth and to link all known information about those organisms to user configurable web pages. The organization of EOL is a direct outgrowth of micro*scope and it involves participation by five major institutions including but not limited to the MBL, the Smithsonian, The American Museum, The Field Museum and Harvard University. The software development is taking place at the MBL and it takes full advantage of the development work that underlies the NAI’s micro*scope initiative.