nai

Executive Summary

Low temperature is a predominant environmental characteristic of interstellar space, asteroids, comets, 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. The basic objectives of one line of investigation—Genomic and Proteomic Analysis of Permafrost Bacteria—include identifying genes and proteins that enable Arctic and Antarctic permafrost bacteria to inhabit subfreezing environments and determining how gene 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 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.

Our investigation on Genomic and Proteomic Analysis of Permafrost Bacteria now includes four interrelated research projects: Genomes, Transcriptomes, Proteomes, and Genetics. Over the past year, significant advances have been made in each area. The goal of the "Genomes" project is to determine the repertoire of genes encoded by representative permafrost bacteria with the long-range goal of identifying key genes necessary for bacteria to live in the permafrost environment. An initial objective is to determine, in collaboration with the Joint Genome Institute and the Lawrence Livermore National Laboratory, the complete genome sequences of two bacteria that we have isolated from Siberian permafrost: Psychrobacter strain 273-4 and Exiguobacterium strain 255-15, psychroactive bacteria isolated from permafrost soils thought to have been constantly frozen for some 40,000 and 3 million years, respectively. Sequencing of the Psychrobacter genome has been completed and now includes a complete “manual curation” of each open reading frame (ORF) yielding the first completely annotated genome of a psychroactive microorganism. Significant progress has also been made in sequencing the Exiguobacterium 255-15 genome, which is currently assembled into 70 DNA “contigs.” Results indicate that the Psychrobacter 273-4 genome is comprised of 2.64 Mb containing 2,147 ORFs while the Exiguobacterium 255-15 genome is approximately 2.9 Mb in size and includes 2,977 ORFs. About 75% of the Exiguobacterium 255-15 ORFs encode proteins that are putative homologs of Psychrobacter 273-4 proteins. Both genomes encode apparent homologs of stress-related proteins in other bacteria and encode many novel proteins that may have unique roles in adaptation to the permafrost environment. Two particularly striking features of the Psychrobacter 273-4 genome are a 20,145 base pair (bp) tandem repeat that encodes an extremely large novel hypothetical protein of 6,715 amino acids and a novel 139 bp sequence present 294 times in the genome that may have roles in cold-regulated gene expression.

Comparative genomic analysis has the potential to generate hypotheses regarding the importance of specific genes and molecular characteristics for life in the permafrost environment. Such analysis is aided by comparisons of closely related isolates originating from habitats with features that differ from those of the permafrost. In this regard, we are excited to have isolated at least three different Psychrobacter strains from fish in warm Puerto Rican marine waters (one isolate can grow up to 42°C). In addition, real-time polymerase chain reaction (PCR) analysis has established that Psychrobacter is present in warm Puerto Rican mangrove sediments. A comparison of these Psychrobacter strains at the genome level with those from the permafrost has the potential to reveal genes important to cryo-adaptation. In a related study, we are conducting physiological, biochemical, 16S rRNA gene and gyr B gene comparisons to resolve the taxonomy and phylogeny of 14 Psychrobacter strains that we have found have different temperature growth ranges. In addition, we have initiated polyphasic analysis of several Exiguobacterium strains including strain 255-15 and 7-3 (isolated from the permafrost) and four reference strains, E. acetylicum , E. aurantiacum, E. antarcticum and E. undae . 16S rDNA sequences, DNA-DNA similarities and BOX-PCR patterns demonstrated that the strains 255-15 and 7-3 are closely related and are related to E. antarcticum and E. undae . DNA-DNA hybridization and the 16S rDNA sequence data show that the strain 190-11 is probably the same species as E. undae , which is a strain isolated in a warmer environment (garden pond in Germany), and represents a strain for genomic comparison.

Another approach that can be used to identify candidate genes with roles in stress tolerance is the identification of genes responsive to stressful conditions. Thus, in the "Transcriptome and Proteome" projects, we are monitoring changes in the levels of transcripts and proteins, respectively, that occur in response to low temperature and low-water activity, two conditions that characterize the permafrost environment. Taking advantage of the results from our Genome project, we have developed a 70mer oligonucleotide array that probes expression of 1990 of the ~2100 genes encoded by Psychrobacter 273-4. Using the arrays, we have compared the transcriptomes of Psychrobacter 273-4 growing at 22 and 4ºC and at high and low water activities. In both cases, we have detected changes in gene expression. For instance, cold-induced changes in transcript levels were observed for about 22 genes involved in lipid biosynthesis, protein synthesis (ribosomal proteins), amino acid metabolism and transport. Most of the cold-regulated genes, however, were only up-regulated about 1.5 to 2 fold indicating that Psychrobacter 273-4 may not be physiologically stressed growing at temperatures above 0ºC. Experiments are, therefore, in progress to examine a wider range of growth conditions including subzero temperatures.

Two methods have been used to determine protein profiles in the Proteome project. One involves the development of a novel 2-dimensional liquid fractionation method that employs 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). Using the method, we have been able to detect about 500 proteins in Psychrobacter 273-4. A comparison of cells grown at 22 versus 14°C has revealed that over 40 proteins are differentially expressed at these two temperatures. The identity of these polypeptides has been determined by using maxtrix-assisted laser desorption/ionization-time-of-flight ( MALDI-TOF) mass spectrometry and matching the peptide mass maps against the DNA sequence database that we have generated for Psychrobacter 273-4 in the Genome project. Nearly a third of the proteins are conserved hypothetical polypeptides with unknown function, with most of the others being involved in translation or metabolism. Significantly, a comparison of the proteome results with the transcriptome results indicates that posttranslational regulation may play a major role in temperature-regulated gene expression in Psychrobacter 273-4.

Proteome analysis has also been conducted using two-dimensional gel separation followed by liquid chromatography-double mass spectrometry. Using this methodology we identified “cold acclimation proteins,” or CAPs, that were preferentially or uniquely expressed at temperatures below 0 ° C in Psychrobacter cryopegella , a bacterium isolated from a briny water lens (-12°C) within the Siberian permafrost. P. cryopegella was found to grow at temperatures between 28 and -10°C and to remain physiologically active down to at least -20°C. A total of 756 proteins were detected in the analysis, 24 of which were classified as CAPs having identities suggesting the importance of oxidative stress, increased energy needs, and compatible solutes in growth at subzero temperatures.

Identifying genes that enable bacteria to inhabit the permafrost environment will require the development of genetic systems to manipulate (including mutagenize) the genomes of the permafrost isolates, the goal of the Genetics project. Important progress has been made over the past year. Several factors that had been hampering transformation of Psychrobacter 273-4 have been identified; a conjugation system for Psychrobacter 273-4 has been developed; and conditions for natural competence of Psychrobacter 273-4 and Psychrobacter cryopegella have been identified. Over the coming year, these systems should enable us to test whether specific genes have important roles for Psychrobacter 273-4 and P. cryopegella in life at low temperature.

In the Bacterial Adaptation to Low Temperature project, we are using experimental evolution to adapt lineages of the bacterium Escherichia coli to low temperature and examining the genetic basis and functional consequences of the adaptations. We previously developed a primary set of six clonal lineages adapted to 20°C and are currently conducting a comparative proteomic analysis of the adapted lines with their common ancestor. The analysis is preliminary, but intriguing differences have been detected. In particular, the levels of two ribosomal proteins and two elongation factors have been altered, suggesting that the translational machinery may be a target of selection during cold adaptation. In addition, insights have been gained into evolutionary adaptation to freezing and thawing conditions in E. coli . We previously demonstrated substantial mortality during repeated freeze-thaw cycles in the absence of cryoprotectant, and that populations evolved at 37°C were more sensitive than were their ancestors. Based on these findings, we designed a 1,000-generation experiment to investigate the potential for evolutionary adaptation to alternating periods of freezing, thawing, and growth at 37°C. The evolution phase of this experiment was completed this past year, and work is now underway to analyze phenotypic and genetic changes in the freeze/thaw/growth evolved lines relative to their ancestors. Competition assays between the evolved lines and their ancestors indicate that significant genetic adaptation has occurred. Improved survival during the freeze/thaw phase, and a shorter lag prior to the start of exponential growth, are two major demographic changes underlying this adaptation.

In the Indigenous Bacteria of Arctic and Antarctic Permafrost project, we are exploring the microbial ecology of the permafrost environment and the physiological state of the resident microbial community. T he terrestrial permafrost provides an analogue of Martian subsurface cryogenic habitats and perhaps has signatures of preexisting life or of viable cryo-adapted life. We isolated and characterized viable microbes from water brines sandwiched between Arctic permafrost strata. These 120,000-yr-old communities living at -10°C could represent Earth analogues of subsurface water brines on Mars, the only possibility for liquid water on present day Mars. Because of the relevance of this habitat for Martian life, we plan to include high coverage sequencing of P. cryopegella , the isolate described above that was isolated from this environment, in our genome project. Over the past year, we also examined the phylogenetic composition of the bacterial community present in the 5millionyrold G laciogene Sirius Group permafrost deposits on Mt. Feather (Antarctic Dry Valleys). The most common sequences were of Proteobacteria , but about 1/4 of the sequences shared less than 80% similarity with those in the ribosomal database, suggesting the existence of novel genera. Finally, an important goal is to understand the strategy of biotic survival and adaptation in the permafrost. In this regard, we have detected methane production in Arctic permafrost below 0°C, and down to -28°C, that appears to result from microbial activity indicating a type of unknown, chemolithotrophic, psychrophilic energy-producing biota that might have the potential to exist cryogenic terrestrial planets free of oxygen.