Incorporation of DNA and Protein Precursors into Macromolecules by Bacteria at 15C

Bacteria can remain viable for hundreds of thousands of years when trapped in glacial ice (1, 2, 8 [http://www.ohiolink .edu/etd/send-pdf.cgi?osu1015965965], 9). In the absence of repair, macromolecular damage must accumulate through amino acid racemization, DNA depurination, and exposure to natural ionizing radiation (12, 13). However, it is known that temperature and the presence of water strongly influence macromolecular decay and that protein and nucleic acid decay rates are drastically reduced within materials with low water activity, such as amber (4) and perhaps ice as well. It also seems possible that such entrapped microbes might carry out slow rates of metabolism to repair the incurred macromolecular damage. Thin veins of liquid exist between ice crystals that could provide a habitat for microorganisms within apparently solid ice (15), and studies of permafrost (17) and South Pole snow (6) have detected low levels of metabolic activity at subzero temperatures. Therefore, to explore the concept that microorganisms trapped in glacial ice might also be metabolically active, reconstruction experiments were undertaken to determine if macromolecular synthesis occurs at 15°C when bacteria isolated from glacial ice core samples were refrozen. Bacterial strains and culture conditions. A -proteobacterial Psychrobacter species (8) (isolate Trans1; 16S ribosomal DNA [rDNA] GenBank accession no. AF479327) and Escherichia coli (Ohio State reference no. 422) were grown in LuriaBertani medium (18), and an actinobacterial Arthrobacter species (7) (isolate G200-C1; 16S rDNA GenBank accession no. AF479341) was cultured in R2 medium (16). Cultures (25 ml) were incubated aerobically with shaking (200 rpm) at 22°C in 125-ml Erlenmeyer flasks to the late exponential growth phase and were diluted to an initial A600 of 0.2 and allowed to grow to an A600 of 0.6. The cells present were then harvested by centrifugation at 17,000 g for 5 min, washed twice with distilled water, and resuspended at an A600 of 0.2 in distilled water (equivalent to 1.3 10 and 2 10 CFU ml 1 for Trans1 and G200-C1, respectively). Aliquots (500 l) of these cell suspensions were placed in 1.5-ml Eppendorf tubes and chilled to 4°C. Precursor incorporation assays. Cell suspensions were maintained on ice, and 100 l of a chilled working solution (10 Ci ml ; diluted 1:100 from stock) of either [H]thymidine (ICN Biomedicals, catalog no. 24060; 60 to 90 Ci/mmol in sterile water) or [H]leucine (ICN Biomedicals, catalog no. 20036E; 40 to 60 Ci/mmol in a sterile 2:98 ethanol-water mixture) was added to each sample, and the mixture was rapidly frozen by incubation at 70°C. After 1 h at 70°C, tubes were transferred to a 15°C freezer, except for control cell suspensions that were maintained at 70°C. At designated experimental time points, 100 l of 50% trichloroacetic acid (TCA) was added to a frozen mixture, which was then allowed to melt at 4°C. After 30 min at 4°C, the TCA-insoluble macromolecules were sedimented by centrifugation at 18,000 g for 15 min. The pellet was washed with 500 l of 5% TCA, recentrifuged for 10 min at 18,000 g, washed with 500 l of 70% ethanol, and suspended in 1 ml of Ecoscint H scintillation fluid (Life Sciences, Inc., catalog no. LS-275). The Eppendorf tube was placed into a scintillation vial, and the radioactivity present was quantitated by liquid scintillation counting (Beckman model LS-7500 scintillation counter). Incorporation into TCA-precipitable material at 15°C. [H]thymidine and [H]leucine were incorporated into TCAprecipitated material by cells frozen in distilled water during incubation at 15°C (Fig. 1). The majority of the incorporation occurred within the first 50 days after freezing. Incorporation did not occur in identical samples incubated at 70°C, a temperature below that predicted for liquid water to exist in ice (14), or by controls pretreated with 5% TCA before freezing. The radioactive counts observed in TCA-treated controls were similar in samples incubated at 15 and 22°C (1,000 to 5,000 dpm), indicating that 0.2% of the radiolabel persisted following 1:1,000 dilution of the mixture during precipitation and washing. Since there is not a significant difference between the radioactive counts obtained in samples prefixed in TCA and those incubated at 70°C in distilled water, this suggests that the background in these experiments results from persistence of residual radiolabel and is not due to acid conditions effecting chemical binding. No precursors were incorporated after 5 months at 15°C by cells frozen in their respective growth media (data not shown). The presence of substrate in these undefined media may have diluted the radiolabel and prevented detection of a * Present address: Land Resources & Environmental Sciences, Montana State University, 334 Leon Johnson Hall, Bozeman, MT 59717. Phone: (406) 994-2733. Fax: (406) 994-5863. E-mail: bchristner @montana.edu.