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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 Analysis of Permafrost Bacteria—include identifying genes and proteins that enable Arctic and Antarctic permafrost bacteria to inhabit subfreezing environments. These studies include determining how gene expression in permafrost bacteria is affected by low temperature and other environmental conditions associated with the permafrost as well as 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 Analysis of Permafrost Bacteria now includes four interrelated research projects: “Genomes,” “Transcriptomes,” “Proteomes” and “Genetics.” 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. These studies include genome sequence analysis which is being carried out in collaboration with the Department of Energy Joint Genome Institute (JGI) and the Lawrence Livermore National Laboratory. We previously reported the complete genome sequence for Psychrobacter arcticus 273-4, which we had isolated from Siberian permafrost that had been frozen for some 40,000 years. Over the past year, the genome sequencing of Psychrobacter cryohaloentis K5 was completed and near full genome coverage has been obtained with Psychrobacter sp. PRw-f1. Psychrobacter cryohalolentis K5 and Psychrobacter sp. PRw-f1 were isolated from, respectively, a Siberian permafrost cryopeg (a pocket of cold brine water at -10°C having a salt content of 170-300 g/l) and a marine fish, Lutjanus vivanus, which inhabited the warm waters off the north coast of Puerto Rico. Psychrobacter sp. PRw-f1 has a temperature growth maximum of 15-20°C higher than P. arcticus 273-4 and P. cryohaloentis K5. Genome sequencing of these three Psychrobacter isolates from different environments having different temperature ranges provides a unique “genomic-gradient,” the study of which has the potential to provide a deeper understanding of bacterial cold adaptation mechanisms. To date, genome comparisons suggest that psychrobacter employ a variety of cold adaptation strategies. A comparison of the G+C content of the three psychrobacter genomes indicates that the average G+C content of the “cold” psychrobacters, 273-4 and K5, is about 2% lower than the “warm” psychrobacter PRw-f1 (which is 44.8%). A lower G+C content could facilitate DNA strand separation at lower temperatures. In addition, a comparison of each Psychrobacter arcticus ORF against five orthologous gene ORFs in the available dataset of mesophilic bacteria indicated an increase of the length of the disordered regions in P. arcticus proteins which could contribute to protein flexibility and function at low temperature.

The genome sequence of Exiguobacterium sibiricum 255-15, a psychrophilic bacterium that we isolated from permafrost soils thought to have been constantly frozen for some 3 million years, is also nearing completion. Results to date indicate that: 27% of Exiguobacterium ORFs are most similar to genes from Bacillus halodurans, while 25% and 24% were most similar to genes from B. anthracis and B. subtilis, respectively; that sugars and carbohydrate polymers are likely the preferred carbon sources; that both purine and pyrimidine biosynthesis pathways are complete; and that Exiguobacterium has both aerobic and anaerobic ribonucleoside diphosphate/triphosphate reductases and is capable of both aerobic (TCA cycle) and anaerobic (via fermentation) growth. In sum, these data suggest that Exiguobacterium survives in the permafrost by living aerobically or anaerobically on sugars or carbohydrate polymers that are produced from the degradation of plant materials.

Availability of the Psychrobacter arcticus 273-4 genome sequence has enabled us to construct an oligonucleotide microarray with probes for 95% of the P. arcticus genes. This microarray has been used to determine how gene expression is altered in response to temperature; in particular how it varies at 22°C, 17°C, 0°C, and -6°C in defined medium. The results indicate that genes of unknown function account for 32% of differentially expressed genes suggesting a potential role for them in cold acclimation. Significantly, several pairs of duplicate genes with homologous functions had inverse expression patterns over temperature raising the possibility that P. arcticus 273-4 has warm- and cold-adapted alleles of genes encoding isozymes adaptive for cold growth, a situation that has been well documented in higher eukaryotes, but not in bacteria. The stringent response was also found to be strongly activated in the cold indicating a possible metabolic imbalance at low temperature leading to carbon starvation and subsequent decreased growth rate. Indeed, supplementation of growth medium with proline, cystine, histidine and tryptophan increased the growth rates of P. arcticus at 0°C, but had no effect on growth rates at 17°C or 22°C. Future experimentation will be directed at determining the significance of this growth rate control in cold adaptation.

It is crucial to examine the physiological processes of psychrophiles at temperatures below 4°C to extrapolate laboratory results to in situ activity. Toward this end, two-dimensional electrophoresis was used to examine patterns of protein abundance during growth at 16, 4, and -4°C of the permafrost isolate Psychrobacter cryohalolentis K5. The results indicated that growth temperature substantially reprogrammed the proteome; the relative abundance of 303 of the 618 protein spots detected (~31% of the proteins at each growth temperature) varied significantly with temperature. The identities of 27 cold inducible proteins (CIPs) were determined by mass spectrometry, five of which, AtpF, EF-Ts, TolC, Pcryo_1988, and FecA, were present specifically at 4°C. The identities of these proteins suggested specific stress on energy production, protein synthesis, and transport during growth at subzero temperatures.

As noted above, a major goal of our research team is to identify genes that enable bacteria to inhabit the permafrost environment. The ability to mutagenize and manipulate the genomes of our permafrost isolates would be a of great aid in this regard. Thus, we have put significant effort into developing genetic systems for the permafrost bacteria that we have isolated. Over the past year, we have had success with Psychrobacter arcticus. One advance is that we have identified a number of factors that were hampering genetic manipulation of Psychrobacter arcticus. It was found that P. arcticus has a methylation sensitive restriction system that destroys “foreign” methylated deoxyribonucleic acid (DNA); that successful electroporation requires freshly prepared competent cells and long recovery times after electroporation (between 16-20 h); and that plasmids anticipated to serve as “suicide vectors” (RSF1010, p15a, and ColE1), in fact replicate in P. arcticus. But even more importantly, we have had success using the allelic exchange vector pJK100 (with a Pir-dependent origin of replication) to create targeted gene mutations and the mariner transposon system to create random mutations in P. arcticus. These advances now enable us to test specific genes, and to screen “globally” for genes, that enable P. arcticus to inhabit the permafrost environment.

In our investigations of Bacterial Adaptation to Low Temperature, we are using experimental evolution to adapt lineages of the bacterium Escherichia coli to low temperature and examine the genetic basis and functional consequences of the adaptations. We previously developed clonal lineages adapted to 20°C. Recent efforts include the development of 12 new lineages adapted to 14°C which were derived from 4 different ancestors. These new lineages, which have undergone over 1800 generations, exhibit increased competitive fitness of 22% on average at 14°C. However, there is highly significant heterogeneity among the lines derived from the different progenitors. These data indicate that subtle differences in the starting genotype, even within the same species, can strongly constrain subsequent adaptation to low temperature.

In addition, we have evolved a set of 14 experimental populations of E. coli for 1000 generations under freeze-thaw-growth cycles. All populations achieved greater fitness under this regime owing to improvements in two aspects of their performance: greater survival during freeze-thaw cycles, and a shorter lag phase after they thaw that allows the bacteria to commence growth sooner. Multiple approaches are now being taken to determine the genetic changes responsible for these adaptations. In one, we screened the entire genome of several evolved lines for changes that involve insertion sequence (IS) elements and found, in 8 independent populations, IS150 elements inserted into the uspA-uspB intergenic region encoding universal stress proteins A and B. These results strongly implicate this locus as having a role in the improved freezing tolerance phenotype. In addition, in several of the populations, IS150 and IS186 elements had inserted into cls, which encodes cardiolipin synthase. Results to date indicate that inactivation of cls is a beneficial mutation which improves freeze-thaw survival more than it affects growth after thawing. Experiments are now underway to further detail the roles of the uspA-uspB and cls genes in adaptation to cold and freezing temperatures.

In our studies on Indigenous Bacteria of Arctic and Antarctic Permafrost, we are exploring the microbial ecology of the permafrost environment. Our previous studies resulted in the isolation of several strains of Exiguobacterium and Psychrobacter from Siberian permafrost, suggesting that these species are abundant in this environment. To test this hypothesis further, 29 soil samples from 16 Siberian permafrost sites were collected and the DNA was extracted and subjected to quantitative real-time PCR with primers designed to detect Exiguobacterium spp. and Psychrobacter spp. The results indicated that Exiguobacterium and Psychrobacter were present in, respectively%, 27 of 29 and 21 of 24 permafrost soil samples and indicate that they can account for up to 1% of the microbial community at certain locations. The composition and diversity of microbial communities in low-temperature sediments were also explored more generally in Siberian permafrost (300-400 years old and 9 m depth; 100-120 years old and 17 m depth, marine horizon) and Antarctic sediments (sediments from fresh and brackish ponds on Bratina Island) by isolating DNA from soil samples and sequencing 16S ribosomal RNA genes. The results indicate that communities clustered based on geographic location with Siberian communities being less diverse than Antarctic communities. Whether this difference in diversity is due to the temperature difference between Siberian and Antarctica samples (-10 to -12 °C versus 1.5 to 9.5°C, respectively) is a question that now needs to be addressed.

A number of additional results point to the diversity of the microbial permafrost communities. Mycelial filamentous fungi (mostly of genus Geomyces), yeast and a new species of strict anaerobic halopsychrotolerant bacteria, Clostridium algidum, were isolated from 10°C sodium-chloride water brines sandwiched within permafrost marine sediments of ~100,000 years old and main groups of Protozoa (nude amoebas, heterotrophic flagellates, and ciliates) were isolated from late Pleistocene and Holocene permafrost sediments. And finally, experiments were performed that confirmed methane formation within the permafrost and for the first time, cultures of methane-forming archaea, Methanosarcina and Methanobacterium, were isolated from this environment.