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2006 Annual Science Report

Marine Biological Laboratory Reporting  |  JUL 2005 – JUN 2006

Executive Summary

Microorganisms were the only forms of life for more than three-fourths of Earth’s history. 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. Microbial communities catalyze several significant chemical transformations within the biogeochemical cycles. Indeed, all macroscopic life completely depends upon processes mediated by complex microbial communities. Single-cell organisms are the most likely life forms to have either a history or a presence on other solar system objects. The Marine Biological Laboratory team investigates the diversity of microbial communities in sites that serve as analogues for past and present habitats on other solar system objects. We explore the evolution of microbial genomes, populations and communities in an effort to understand how biology shapes planetary processes. We investigate mechanisms such as those afforded by symbioses and phage infections in shaping microbial genomes, and we seek to understand the diversity and evolution of metabolisms that might resemble those carried out by ancestral microbial populations. Because biology has a profound effect on planetary environments we attempt to integrate our studies of ever changing microbial processes with remote sensing techniques that facilitate NASA’s search for potential extraterrestrial biological worlds within our solar system.

Our principle terrestrial study site is the Rio Tinto of southwestern Spain. Running through the largest pyritic belt on Earth, the Rio Tinto serves as an analogue for habitable zones on early Mars. In collaboration with Ricardo Amils of the Centro de Astrobiología /Universidad Autónoma de Madrid Spain, Dr. Amaral-Zettler has been using SARST-V6 (Serial Analysis of Ribosomal Sequence Tags) to characterize bacterial populations at the Origin, Anabel’s Garden and Berrocal sampling stations. They have collected over 10,000 SARST-V6 ribosomal RNA sequence tags. These short tag sequences describe the relative abundance of bacterial species estimates in which Acidithiobacillus and Leptospirillum spp. are the dominant taxa and with uncultured bacteria related to those described for Iron Mountain representing more than 17% of the total diversity. Dr. Amaral-Zettler has extended these studies to Archaea and Euakrya by developing a three-domain SARST strategy that focuses upon the V4-V8 region of rRNAs. Early analyses reveal the presences of phylotypes never reported from the Rio Tinto. These studies contribute toward understanding microbially mediated biogeochemical transformations that we are monitoring at the same sampling sites. Drs. Head and Mustard are characterizing the iron and sulfate mineralogy in the Rio Tinto. The combination of mineralogy and detailed molecular descriptions of the microbial populations will significantly enhance the interpretation of visible-near infrared spectroscopy remote sensing data. Initial results point towards a diverse range of sulfate and iron oxide-minerals. The combined data demonstrate that the most biologically active sites have the most complex mineralogic signatures. The remote sensing data analysis group is also examining the water history of Mars as recorded in glacial deposits. Initial results are compatible with a model in which frequent climate changes on Mars effect major redistribution of long-term water deposits over geological time scales.

In addition to the Rio Tinto system, marine environments including the deep subsurface, sediments, hydrothermal vents and the oxygen minimum zones serve as sampling sites for our investigations of metabolic and microbial diversity. We employ a combination of molecular surveys based upon PCR amplification and sequencing of functional genes and ribosomal RNAs or their coding regions. These studies provide information about microbial community composition, certain features of their metabolic activities and insights about evolutionary processes. For example, the reductive TCA cycle and anaerobic methane-oxidation may represent ancient metabolisms.Through PCR-based molecular surveys of complex microbial populations and cultivars, Dr. Sievert has discovered that two distinct modes of citrate cleavage within the reductive TCA cycle occur in hyperthermophiles; Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. Sievert also demonstrated that lateral gene transfer is the most parsimonious explanation for distribution of ATP citrate lyase from epsilon-proteobacteria into Hydrogenothermaceae and 'Desulfurobacteriacea'.

The Teske laboratory has explored the distribution of methyl coenzyme M reductase (mcrA) and dissimilatory sulfite reducatase (dsrAB) to characterize microbes in marine coastal, basin and deep subsurface sediments.

figure 1
Figure 1. Microbial life thrives in deeply buried marine sediments and oceanic basalt crust, and forms an extensive, well-diversified microbial subsurface biosphere, a counterpart to the photosynthetic surface biosphere of Earth.

The anaerobic methane-oxidizing archaea (ANME) targeted in the mcrA studies demonstrated existence of a diversified methanogen community that uses H2/CO2, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones to thermophilic and non-thermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas Basin sediments. Gulf of Mexico methane seep sites yield only ANME-1 type mrcA genes, and ANME-1 microbes appear to be much more widespread in nature than expected. Anaerobic methanogens uniquely complete the anaerobic carbon cycle in an oxidative direction from methane to CO2, an especially significant process in the early carbon cycle of Earth. Studies in the deep subsurface have relied upon analysis of rRNAs rather than their genes to demonstrate that the characterized organisms are active versus representing fossilized DNA. He has also shown that Archaeal communities in deep subsurface sediments from organic-rich continental margin sediments differ significantly from those in sediments from the organic-depleted, energy-starved central oceanic basins.

Dr. Edwards laboratory explores the molecular basis of microbes responsible for iron oxidation in seamount environments. She has developed the lithotrophic bacterium Marinobacter aquaeolei as a model to study neutrophilic iron oxidation. This organism is related to other deep-sea Fe(II)-oxidizing autotrophic bacteria and through the combination of genomics and protein expression she studies the influence of different Fe concentrations on growth and physiology of this organism. Given the availability of a cultivar, Edwards collaborated with the DOE Joint Genome Institute in generating a genome sequence for Marinobacter aquaeolei. This provides a basis for interpreting protein analyses of cultures subjected to different concentrations of Fe and therefore the genetic potential of the organism can be queried and exploited. In this study, batch cultures of the bacterium were inoculated in artificial seawater medium with various concentrations of Fe or “biologically unavailable” ferrozine chelated Fe. Under all conditions, the rate of Fe-oxidation was faster in the presence of the bacterium than in the abiotic controls. Protein extractions from organisms grown either in different concentrations of iron or with biologically unavailable iron reveal a 35-kDa protein located in the cell membrane which.contains a heme. Queries of the genome identified a heme containing protein in the 35-kda size range. A di-heme cytochrome c peroxidase occurs in a gene cluster with five other genes that encode for other proteins involved in electron transport. Q-PCR experiments will be used to test whether or not these six genes are up-regulated under Fe-replete conditions.

Despite informative molecular-based studies of microbial diversity, molecular microbial investigations have not comprehensively described microbial populations in terrestrial and marine settings. The evolution of microbes over billions of years predicts the composition of microbial communities should be much greater than published estimates of a few thousand distinct kinds of microbes in an analyzed sample. The cost of DNA sequencing constrains extensive characterization of microbial communities. Typical surveys include fewer than 1000 DNA sequences at a cost of 2-3 dollars per sequence read. Such surveys are sufficient only to identify the most abundant members in a microbial community. To address this problem we first developed the SARST technology described above to improve the extent of molecular sampling of a microbial community. In the case of the Rio Tinto, this technique revealed the presence of numerous, previously undocumented organisms. We extended this approach by developing a DNA tag sequencing strategy that takes advantage of the massively parallel sequencing capability of a Roche Genome Systems 20 DNA sequencer (454 technology). We were able to sample tens of thousands of sequence tags from the V6 hypervariable regions of bacterial small subunit rRNAs. These studies showed that microbial diversity in the oceans is at least two orders of magnitude greater than anticipated. There are at least 60,000 different kinds of microbes in a liter of sea water. Even more important from the perspective of understanding the ecology of microbial systems, a relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. We describe these low-abundance populations as members of the “rare biosphere”.

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Figure 2. Similarity of 454 sequence tags from FS396 to the V6RefDB database. “All tag distribution” plots the number of tag sequences for all samples versus the percentage difference from the best-matching sequence in V6RefDB. “Percent unique reads” from all samples shows the percentage difference between each distinct tag sequence and its best match in V6RefDB. “Percent total tags” plots the cumulative percentage of reads in all samples at or below a given percentage difference from best matches in V6RefDB.

Microbial oceanographers by necessity have focused their efforts on dominant components of microbial communities that mediate biogeochemical processes. What they have not tackled are very low-abundance members of microbial populations. The extreme phylogenetic diversity of the “rare biosphere” suggests these minor populations have persisted over geological time scales and they may episodically reshape planetary processes. This “rare biosphere” is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and at different times in earth’s history may have had a profound impact on shaping planetary processes.

Environmental shifts can influence microbial genome evolution but genome-genome interactions between two organisms can also shape genetic change. As an alternative to complex communities in open environments where a myriad of potential interactions makes it difficult to study this process, we take advantage of closed systems comprised of host-associated endosymbionts. These studies examine Proteobacterial species that represent both long-term, stable mutualisms and more dynamic, parasitic interactions. The Wernegreen and Bordenstein laboratories focus on characterization of genomes of endosymbionts for various insect species. This past year they showed that amino acid substitution rates for different coding regions in the endosymbiont’s genome can vary by 100 fold. These patterns of altered rates of substitution seem to be common among endosymbiont genes that carry out similar functions. Dr. Wernegreen has also been able to identify and predict patterns of gene loss in the symbiont that are a consequence of convergence among mutualist groups. Finally, working with Seth Bordenstein, it has been possible to characterize genomic changes caused by bacteriophage of Wolbachia, the most prevalent group of infectious bacteria in our biosphere. Bacteriophage regulation of in-host density and phenotype suggest that phage are significant players within the closed intracellular niches of some long-term bacterial symbionts of eukaryotes. Complex life can thus be shaped by the tripartite genomes of phage, bacteria and eukaryotes.