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Project Progress

A major objective of our research team is to determine the repertoire of genes necessary for bacteria to live in the permafrost environment. Central to this effort is determining (in collaboration with the Joint Genome Institute and the Lawrence Livermore National Laboratory) the complete genome sequences of bacteria that we have isolated from Siberian permafrost including Exiguobacterium sibiricum 255-15, a psychrophilic bacterium isolated from permafrost soils thought to have been constantly frozen for some 3 million years. The fact that E. sibiricum 255-15 was isolated from an old geological stratum, has a wide growth temperature range (-2.5°C to 41°C), has survived long-term freezing, grows rapidly at low temperatures, and is halotolerant made this microorganism an excellent candidate for genome analysis.

Genome sequence data were analyzed by identifying putative genes, also referred to as open reading frames (ORFs), and comparing the ORFs of E. sibiricum 255-15 with gene sequences from other microorganisms. Also, the pathways and types of carbohydrate metabolism, amino acid biosynthesis, amino acid catabolism, coenzyme and cofactor biosynthesis, nucleotide biosynthesis and energy metabolism of this microorganism were inferred from the genome. The results indicate that 27% of Exiguobacterium ORFs were most similar to genes from Bacillus halodurans, while 25% and 24% were most similar to genes from B. anthracis and B. subtilis, respectively. Sugars and carbohydrate polymers are likely the preferred carbon sources. Exiguobacterium is auxotrophic for biotin and branched-chain amino acids and threonine due to the absence of any enzymes for both biosynthetic pathways. Exiguobacterium has several amino acid degradation pathways; some of which can be used as energy sources. Both purine and pyrimidine biosynthesis pathways are complete. Exiguobacterium has both aerobic and anaerobic ribonucleoside diphosphate/triphosphate reductases and is capable of both aerobic (TCA cycle) and anaerobic growth (via fermentation). 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. Further analysis will determine if Exiguobacterium contains specific genomic modifications indicative of low-temperature adaptation.

Exiguobacterium has been recovered from multiple permafrost samples, of varying depths and ages, as well as from an amazingly diverse array of habitats, including mats in Lake Fryxell (Antarctic), glacial ice, various types of food processing plants, marine water, and warm pools. To estimate total genomic size of the organisms and genomic diversity among isolates, pulsed field gel electrophoresis (PFGE) was used. The results indicated that organisms from all these diverse sources had similar genome size, ca. 2,500 kb, which was similar to that of Exiguobacterium sibiricum 255-15. Utilizing the genome sequence of E. sibiricum 255-15, we constructed a panel of 82 probes, chosen so as to represent genes involved in nucleic acid metabolism, translation, lipid metabolism, signal transduction, and stress responses. Macroarray hybridizations utilizing this panel identified ten probes that were highly conserved among all tested isolates, regardless of the source. In addition, the macroarray hybridizations revealed that the isolates could be generally classified in two divisions: Division I included isolates from permafrost and other low-temperature environments, whereas Division II included isolates from high-temperature habitats. Interestingly, Exiguobacterium isolates from habitats characterized by marked differences in temperature (e.g. Arctic/ Antarctic vs. hot springs) exhibited noticeable differences in optimal temperature for growth. Conserved probes were utilized to assist the identification of Exiguobacterium in additional habitats, including food processing plant environments. Exiguobacterium isolates with different temperature optima may serve as model systems for the identification and characterization of genomic adaptations to distinct temperature regimes.