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Project Progress

The permafrost environment is extremely nutrient-limited due in large part to its frozen state. Thus, it would be predicted that permafrost bacteria should be able to synthesize most of their cellular constituents, vitamins and cofactors from a limited array of available substrates. Indeed, a metabolic pathway reconstruction for Psychrobacter sp. 273-4 performed by Natalia Ivanova (Lawrence Berkeley National Labs), based on our genome sequencing of this organism, indicated that this bacterium has a gene complement that would enable it to synthesize all of the amino acids, most vitamins and cofactors from a limited set of carbon sources including glycine, proline, serine and TCA cycle intermediates. The analysis also indicated that Psychrobacter sp. 273-4 would not be capable of growth on sugars due to a lack of genes encoding glycolytic enzymes, but that it should be able to utilize acetate and fatty acids as energy and carbon sources. Overall, the results suggested that Psychrobacter sp. 273-4 should be able to grow on a minimal sea salts based medium that included acetate as the carbon and energy source and ammonium chloride as the sole nitrogen source. This hypothesis was confirmed experimentally establishing the presence of the predicted metabolic pathways and providing a valuable medium that can be used to conduct a wide range of physiological and genetic studies on this bacterium.

In addition to isolating Psychrobacter sp. 273-4 from the Siberian permafrost, we have isolated Psychrobacter sp. K5. Both isolates were thoroughly characterized phenotypically and genetically and shown to be distinct species within the genus Psychrobacter. These species represent the first fully described novel species of Psychrobacter isolated from permafrost. Significantly, species K5, which was isolated from a salt lens within the permafrost, was tolerant to higher salt concentrations than species 273-4, which was isolated from permafrost sediments. In addition, reflective of the anaerobic nature of some permafrost environments, the membrane lipids of species K5 were dominated by lipids that do not require the presence of O₂ for their formation.

We previously described the isolation of three Exiguobacterium strains from different layers of the Siberian permafrost with ages ranging from 20 thousand to 3 million years old. These results suggested that members of this bacterial genus might be widely distributed and abundant in the Siberian permafrost environment. To test this, 29 Siberian permafrost soil samples with ages between 5 thousand and 3 millions years were collected and analyzed through quantitative real time PCR (Q-RT-PCR) using specific Exiguobacterium primer sets for the genes gyr B (gyrase B), rpoB (RNA polymerase beta subunit) and a hypothetical gene found in Exiguobacterium isolates from the Siberian permafrost. The results were consistent with Exiguobacterium species being widely distributed in the Siberian permafrost and accounting for up to 1% of the microbial community at certain locations.