Tiedje Lab - Projects

MICROBIAL LIFE IN PERMAFROST

Low temperature is a predominant environmental characteristic of interstellar space, our solar system, including most of the planets and their satellites, and asteroids and meteors. An understanding of the impact of low temperatures on the responses and evolution of biological organisms is, thus, integral to our knowledge of Astrobiology. Our research explores aspects of microbial adaptation to low temperatures. One major line of investigation is the structural and functional genomic and proteomic analyses of bacteria that have been isolated from the Arctic and Antarctic permafrost. What genes and proteins enable the permafrost bacteria to inhabit these subfreezing environments? Do they have special cryo-adaption genes and proteins, cryo-modifications of standard genes and proteins, or both? How is the expression of the bacterial genome affected by low temperatures and other conditions that "hitchhiker" bacteria might encounter during travel through space on natural objects or spacecraft? Finally, we hope to use the information gained to explore the potential development of signatures for the presence of life in cold environments including Earth and other bodies such as Mars and Europa. - Peter Bergholz

Functional and Comparative Genomics of Permafrost Psychrobacter Species (Peter Bergholz, Monica Ponder, Hector Ayala-del-Rio, Shannon Hinsa and Corien Bakermans)

The permafrost environment and its various niches are considered good Earth analogs for cryogenic environments elsewhere in the universe (2-4). Members of the genus Psychrobacter have repeatedly been isolated from Siberian permafrost (1, 4, 10). These isolates have been shown to grow under the major stresses in frozen environments: subzero temperatures (down to -10�C) and high salt (up to 2.7 osmolal). Due to their distribution and their well-adapted physiology, organisms from the genus Psychrobacter are good Gram (-) representative permafrost organisms. In cooperation with the DOE Joint Genome Institute, we have obtained the genome sequence of two Psychrobacter isolates from the Siberian permafrost. Psychrobacter 273-4 was isolated from 10,000-40,000 year old permafrost soils (10). Psychrobacter cryopegella was isolated from a cryopeg, an unfrozen brine pocket within the permafrost (1).

The Psychrobacter genus is also interesting from the perspective of genomic cold adaptation, because organisms throughout the genus exhibit different minimum growth temperatures, suggesting that genomic comparisons among members of Psychrobacter can reveal genes or alleles that allow the more cold-adapted members to push the limits of minimum growth temperature. A trade-off has been hypothesized such that cold-adapted Psychrobacter species must have given up some of their capacity to grow at warmer temperatures. This hypothesis is founded on the basis of data on thermal adaptation of cold-adapted enzymes and ribosomes (5, 8).

Our strategy to understand what makes these genomes cold and high salt adapted is to perform transposon mutagenesis (on Psychrobacter 273-4), and comparative genomic (gene sequence, genomic DNA microarray comparisons) and functional genomic (transcriptome and proteome) analyses on these organisms along with transposon mutagenesis. The transposon mutant library, generated at the optimum growth temperature of Psychrobacter, will be screened to identify genes important for growth at low temperatures and in high salt. Comparative genomic analysis are directed towards the identification of cold-adapted alleles present in the Psychrobacter genomes and also towards using bioinformatic and microarray approaches to identify gene content differences between genomes capable of growth at different temperatures within the Psychrobacter genus. Functional genomic approaches are targeted at formulating hypotheses about differences in physiology of Psychrobacter populations growing under different temperature and salt regimes. In the case of temperature, cell populations will be grown at different points on a temperature cline from well below zero (cold adapted growth) to above optimal (heat adapted growth). Comparisons of the transcription and protein profiles of Psychrobacter cultures at different temperatures will allow us to hypothesize about the metabolic demands and stress responses during growth across thermally defined metabolic states. Specific hypotheses regarding expected outcomes are numerous and range from hypotheses about changes in energy metabolism and membrane lipids to hypotheses about responses to chromosome structure changes at low temperature and changes in translational efficiency (5-7, 9, 11). Of course, we also hope to identify hypothetical proteins that respond to low temperature growth conditions.

References:

1. Bakermans, C., Tsapin, A, Souza-Eglpsy, V, Gilichinsky, D, Nealson, K. 2003. Reproduction and Metabolism at -10�C of bacteria isolated from Siberian permafrost. Environmental Microbiology 5:321-326.
2. Brinton, K. L., A. I. Tsapin, D. Gilichinsky, and G. D. McDonald. 2002. Aspartic acid racemization and age-depth relationships for organic carbon in Siberian permafrost. Astrobiology 2:77-82.
3. Gilichinsky, D. 2001. Permafrost model of extraterrestrial habitat, p. 271-295. In G. Horneck (ed.), Astrobiology. Springer-Verlag, New York.
4. Gilichinsky, D., E. Rivkina, V. Shcherbakova, K. Laurinavichuis, and J. M. Tiedje. 2003. Supercooled water brines within permafrost- An unknown ecological niche for microorganisms: A model for astrobiology. Astrobiology 3:331-341.
5. Miller, A. J., D. O. Bayles, and B. S. Eblen. 2000. Cold shock induction of thermal sensitivity in Listeria monocytogenes. Appl Environ Microbiol 66:4345-50.
6. Rivkina, E. M., E. I. Friedmann, C. P. McKay, and D. A. Gilichinsky. 2000. Metabolic activity of permafrost bacteria below the freezing point. Appl Environ Microbiol 66:3230-3.
7. Russell, N. J. 1983. Adaptation to temperature in bacterial membranes. Biochem Soc Trans 11:333-5.
8. Russell, N. J. 2000. Toward a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles 4:83-90.
9. Scherer, S., and K. Neuhaus. 2002. Life at Low Temperatures. In M. Dworkin (ed.), The Prokaryotes: An Evolving Electronic Resource for the Microbiological Community, 3rd, Release 3.9 ed. Springer-Verlag, New York.
10. Vishnivetskaya, T., S. Kathariou, J. McGrath, D. Gilichinsky, and J. M. Tiedje. 2000. Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles 4:165-73.
11. Whyte, L. G., and W. E. Iniss. 1992. Cold shock proteins and cold acclimation proteins in a psychrotrophic bacterium. Can. J. Microbiol. 38:1281-1285

Biofilm formation by Psychrobacter (Shannon Hinsa)

Psychrobacter arcticum str. 273-4 was isolated from the Siberian permafrost, which has a very limited amount of unfrozen water. The unfrozen water is found a thin films coating the surfaces of mineral and organic particles. We propose that the ability of Psychrobacter to attach to these surfaces and have access to unfrozen water is key for survival and growth in this environment. Initial biofilm studies done in the 96-well plate assay have shown that P. arcticum is able to attach to plastic at 22^oC, 4^oC and 0^oC when grown in minimal media but not when grown in rich media. Future studies will study biofilm formation in a variety of medium and on a variety of surfaces.

Acclimation of ancient Siberian permafrost bacterium to extreme temperature conditions (Debora Rodrigues)

Among the environmental conditions that affect microbial growth, temperature is a major player in controlling cell physiology and growth. Acclimation to stressful temperatures permits the growth and survival of microorganisms in extreme environments. Microbial responses to temperature stress are manifested by physiological and metabolic changes which are reflected at the molecular level. However, little is known about the extent of mechanisms related to heat and cold acclimation which permit their survival and growth. In the present work, a microorganism ( Exiguobacterium 255-15) isolated in an ancient Siberian permafrost dated from 2 to 3 million years old, will be investigated for the network of genes involved in acclimation to extreme temperatures. The main goals for this proposal are: 1) Determine the changes in the gene expression when exposing Exiguobacterium 255-15 to different temperatures; 2) Determine the variations in protein expression when exposing Exiguobacterium 255-15 to different temperatures and verify the differences between gene expression and protein expression; 3) Determine if an uniquely expressed gene at a specific temperature affects the cell growth and can affect other genes at this temperature.