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Executive Summary

Low temperature is a predominant environmental characteristic of interstellar space, asteroids, meteors and of course, our solar system, including most of the planets and their satellites. An understanding of the impacts that low temperature has on the responses and evolution of biological organisms is, therefore, integral to our knowledge of Astrobiology. Toward this end, we are exploring multiple aspects of microbial adaptation to low temperature. One line of investigation — Genomic and Proteomic Analysis of Permafrost Bacteria — is to conduct genomic and proteomic analyses of bacteria that have been isolated from the Arctic and Antarctic permafrost. Our basic objectives include identifying genes and proteins that enable permafrost bacteria to inhabit subfreezing environments and determining how genome expression in the permafrost bacteria is affected by low temperature and other environmental conditions associated with the permafrost. We are also interested in conditions that “hitchhiker” bacteria might encounter during travel through space on natural objects or spacecraft. In a second line of investigation — Bacterial Adaptation to Low Temperature — we are directly examining, through “test-tube evolution” experiments, how bacteria genetically adapt to low temperatures. The fundamental objective here is to better understand how an organism, with a given complement of genes, can cross niche barriers that are defined by decreasing temperatures. Finally, in a series of “Field Truth” investigations — Indigenous Bacteria of Arctic and Antarctic Permafrost — we are exploring the microbial ecology of the permafrost environment and the physiological state of the resident microbial community. This is being accomplished by determining the phylogenetic diversity of the bacterial permafrost population and the metabolic activities present in permafrost soils.

An exciting advance in the Genomic and Proteomic Analysis project is the completion of the genome sequence for Psychrobacter 273-4, a bacterial strain that we isolated from 20-40 thousand year old permafrost soil. Completion of this bacterial genome sequence, which is being done in collaboration with the Department of Energy (DOE) Joint Genome Institute (JGI), will represent a milestone in bacterial genome sequencing as no complete genome sequence has been obtained for any psychoactive bacterium. Preliminary analysis of the Psychrobacter genome, in collaboration with JGI and the bioinformatics group at Oak Ridge National Laboratory, indicates that the isolate encodes approximately 2,140 predicted open reading frames (ORFs), with 11% of the ORFs being unique to Psychrobacter. Furthermore, 42% of the ORFs had a “hypothetical protein” as the best “hit” in the GenBank database, indicating that the function of a major portion of the Psychrobacter genome is yet unknown. A search of the Psychrobacter ORFs indicated the presence of potential orthologs for proteins previously shown to be involved in cold adaptation including four Csp cold shock proteins, but a number of genes known to be involved in growth of mesophilic bacteria at low temperature were not found in Psychrobacter 273-4 suggesting that it encodes novel cold adaptation genes. Interestingly, 40 transposases from other microorganisms were identified in the Psychrobacter isolate suggesting that horizontal gene transfer may have played a significant role in the evolution of its genome.

A fundamental component of the genomics/proteomics project is to determine how expression of the genomes of permafrost bacteria is affected by low temperature and other conditions associated with the permafrost environment. This work is currently focused on Psychrobacter 273-4 and Exiguobacterium 255-15, a bacterial strain that we isolated from 2-3 million year old permafrost soils. Work to date with the Psychrobacter isolate has directly demonstrated that the proteome responds to changes in temperature. In particular, using two dimensional polyacrylamide gel electrophoresis, we found that certain proteins were only present at either cold (4°C) or warm (24°C) temperature. Analysis of the low temperature specific proteins using mass spectrometry indicated that one was novel and that the other three were orthologs of the Escherichia coli Csp cold shock proteins. In addition, two of the high temperature specific proteins were found to be novel while a third belonged to the diene lactone hydrolase protein family. To obtain high resolution proteome information for the Psychrobacter and Exiguobacterium isolates, we have been developing a novel 2-dimensional liquid fractionation method that uses a pH column-based separation in the first dimension followed by separation of the proteins in each pH fraction using nonporous silica (NPS) reversed phase high-performance chromatography (HPLC). The result is a 2-dimensional image of the proteins in the cell as a function of pH versus hydrophobicity. After developing effective cell lysis conditions and appropriate temperatures for protein fractionation, we now have a powerful set of procedures and methods to produce high resolution protein “maps” for the permafrost isolates.

Significant insights have also emerged from the Bacterial Adaptation to Low Temperature project. In studying adaptation of the mesophilic bacterium E. coli to low temperature (20°C), it has been found that the rate of adaptation is independent of previous thermal adaptive history; that is, change in relative fitness or fitness after 2000 generations is no different if the bacteria were previously adapted to 32, 37, or 42°C or an alternation of 32 & 42°C. We have further found that adaptation to 20°C is associated with a significant decrease in fitness at high (40°C) temperature. Genomic analysis of the 20°C adapted lineages has revealed the existence of several small deletions of the chromosome. The deletions appear to have occurred independently in many of the lineages. Most remarkably, genes at approximately 1.85Mb on the circular chromosome were independently deleted in 6 of the 30 lines examined. Thus, one mechanism of evolutionary adaptation to low temperature appears to involve gene deletion. Moreover, with E. coli, the occurrence of deletions in the same region of the genome suggests a functional significance for elimination of a few particular genes. We are in the process of mapping exactly which genes were involved in each lineage and will determine the genes in common that were deleted.

A fundamental objective of the “Field Truth” investigations is to increase our understanding of the phylogenetic diversity of the bacteria that inhabit the Arctic and Antarctic permafrost. In the past, we have examined permafrost under typical subarctic tundra, near the East Siberian Sea, Russia and permafrost of the Beacon Dry Valley, Antarctica. This year we extended our work to the slopes of the active volcano Ploskii Tolbachik (Fig. 1), situated on Kamchatka peninsula in the far-east of Russia. It is one of the largest modern volcanic regions with widespread glaciers and permafrost at high altitudes. This site was chosen because one possibility for liquid water on Mars at shallow depths would be in proximity to subglacial volcanism. Such volcano-ice interfaces could occur beneath the polar caps of Mars today, or even within the adjacent permafrost around the margins of the ice caps.

To examine the microbial communities in this niche, we drilled a series of boreholes at different landscapes and altitudes between 800 and 2600 m a.s.l. (Fig. 2). Frozen (-1 to -2°C) samples extracted from a borehole at 1100m, representing young volcanic interstratified ash, sand and scoria 12 to 16 m thick (from the eruption of 1975-76), contained viable microorganisms and methane up to 1100-1900 µlCH4/kg soil. We found both psychrophilic and thermophilic microbes in the samples, with the psychrophilic heterotrophs the most numerous followed by psychrophilic methanogens and sulfate reducers. We have obtained enrichment cultures of psychrophilic, mesophilic and thermophilic bacteria of different metabolic types, including acetogens, methanogens, sulfate reducers, ferroreducers and spore-formers. The scoria texture of these sites is presumably close to Martian regolith. Such terrestrial volcanic microbial communities in permafrost potentially serve as good exobiological models for testing hypotheses on existing ancient microbial communities.

Last year we reported initial results of determining the diversity of Arctic and Antarctic permafrost bacteria using high throughput 16S ribosomal deoxyribonucleic acid (rDNA) gene sequencing. During the past year, we continued this analysis and determined whether new phylotypes could be isolated after incubating permafrost samples at low temperature (4°C) under both aerobic and anaerobic conditions. The analysis indicated that Arctic surface soils had considerably more phylotype diversity than did Antarctic surface soils and that incubating soil samples at 4°C resulted in significant changes in the microbial profile in all soil samples tested, indicating that less numerous microbes were alive and could be recovered.

Finally, we have sought to isolate bacterial strains that are related to permafrost bacteria from tropical sites in Puerto Rico. In particular, soil samples obtained from the El Yunque National Forest (hot and moist ecosystem), the Las Cabezas de San Juan Reserve (hot, dry and salty ecosystem) the Guánica State Forest (hot and dry ecosystem) and the Las Carmelitas Cave System were processed for the isolation of Psychrobacter, Exiguobacterium and Arthrobacter strains (we previously isolated Arthrobacter strains from ancient permafrost). Preliminary results indicate that we potentially have isolated relatives of Exiguobacterium and Arthrobacter. The isolation of such strains will provide powerful tools for comparative genomic studies to help identify cold adaptive genes in the permafrost microbes.

Figure 1. View of volcano Ploskii Tolbachik which has surrounding permafrost

Figure 2. A schematic cross-section of the volcano Ploskii Tolbachik with the drilled boreholes