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There are no reports which document active metabolism of bacteria in the surface snow of the interior of the Antarctic continent. At the south pole, temperatures are extreme and austral winter air temperatures reach about 85°C, while in summer it can warm to about 13°C (mean monthly air temperature in December is 26°C). Bacteria have previously been cultured from samples taken from Antarctic ice cores (1), and deep cores from the accreted ice above subglacial Lake Vostok revealed a high diversity (24) of species that were reported to be metabolically active when warmed to 3°C (16). We report here bacterial populations and associated metabolic activity in surface (upper 20 cm) snow and firn collected at the south pole in the austral summer.

Sampling. Snow samples were collected on 9 and 18 January 1999 and 10 January 2000 at the Amundsen-Scott South Pole Station. Care was taken to sample at the edge of the Clean Sector of the National Oceanic and Atmospheric Administration Clean Air Laboratory upwind of the Station (grid 115 to 120), so that contaminating bacteria from the human habitat would not be collected. Snow was sampled using sterile procedures, and Snowpak containers, which hold ca. 60 to 80 liters of snow, were returned frozen to the Crary laboratory at McMurdo Station for analysis within 24 h of collection.

Microscopy. Microbes were concentrated by filtering 20 to 50 ml of snowmelt onto a 0.02-µm-pore-size Anodisc filter, and bacteria were stained with the DNA fluorescent dye SYBR green (22). Counts were done using a Zeiss Axioskop microscope at ×1,000. Scanning electron microscopy (SEM) was done on samples preserved in 4% glutaraldehyde-1.8% RNase-free sucrose-10 mM phosphate buffer (pH 7.4) and held at 1°C until processing for SEM.

DNA sequencing. Melted snow (10 liters) from each snow sample was filtered through a 0.2-µm-pore-size Nalgene filtration unit on a clean bench. The sample retained on the membrane was then subjected to DNA purification using cetyltrimethylammonium bromide and chloroform according to a procedure as modified in reference 8. Briefly, the membrane was incubated with 1 ml of Tris-EDTA buffer containing lysozyme (1 mg/ml) at 37°C for 1 h with constant shaking (250 rpm). Subsequently, NaCl and sodium dodecyl sulfate were added at final concentrations of 0.7 M and 1%, respectively, and the mixture was incubated at 70°C for 15 min (gently hand shaken every 5 min). Next, the mixture was incubated with 1 mg of RNase per ml at 37°C for 30 min. Cetyltrimethylammonium bromide dissolved in 0.7 M NaCl was added at a final concentration of 1% (wt/vol), and the mixture was incubated at 56°C for 10 min. After a wash with an equal volume of chloroform, DNA in the aqueous phase was precipitated with an equal volume of isopropanol for 2 h or overnight at 20°C. To prevent contamination in any step of the procedure, a negative control was made by filtering 2 liters of the autoclaved distilled and deionized water used in the above-described experiments with the same filtration unit, and the membrane was subjected to the same procedure as the sample. After centrifugation and washing of the DNA pellet with 75% ethanol, the DNA was dissolved in autoclaved deionized and distilled water. Two microliters of the DNA preparation from both the sample and the negative control was used in a PCR to amplify the gene for the small subunit of ribosomal RNAs of prokaryotes (16S ribosomal DNAs (rDNAs). Primers were initially Bac1 (forward, 5'-AGA GTT TGA TCM TGG CTC AG-3' [M is A or C]) and Bac2 (reverse, 5'-ACC TTG TTA CGA CTT CAC-3') (15). In some cases, a secondary PCR was required and a nested primer was used, Bac5 (forward, 5'-ATG TGG TTT AAT TCG A-3') (15). One microliter of the first PCR product from both the sample and the negative control was used as the template for the secondary PCR. On the 16S rDNA sequences from the south pole samples, we found regions that were universally conserved among all bacteria, including the Deinococcus-Thermus group. A universal primer was designed based on one of these regions and used in a PCR for samples from the second sampling season, Bac14 (forward; CGG GAG GCA GCA GTT AGG AAT). The PCR products were extracted from agarose gel using Spin X ultrafiltration units and cloned into a TA cloning kit (19). Ten to 12 clones were randomly selected for sequencing with an ABI Prizm automatic sequencer. The nucleotide sequences obtained were compared against those in the GenBank database using FASTA and aligned with representatives of major bacterial divisions using CLUSTAL W. Phylogenetic analysis was performed using PHYLIP (10).

Fluorescent in situ hybridization. A protocol previously reported (2) was modified. First, 250 ml of snowmelt was fixed with formaldehyde (final concentration, 3.7% [vol/vol]) at 4°C for 2 h. The fixed water sample was then filtered onto a 0.2-µm-pore-size Nucleopore membrane at a vacuum pressure of 5 lb/in². Ethanol (50% [vol/vol] in sterilized double-distilled water) was applied to the membrane and incubated for 15 min. After the liquid was filtered, the cells retained on the membrane were transferred onto a slide by placing the membrane upside down on a poly-L-lysine-coated slide. The slide had been briefly rinsed with 70% ethanol to remove potentially contaminating bacteria before use. When the slide and the membrane became dry, the membrane was removed gently and a hydrophobic boundary was drawn with a PAP pen on the covered area on the slide (Energy Beam Sciences, Inc., Agawam, Mass.). The cells encircled within the boundary were then incubated with 50 µl of hybridization buffer (0.9 M HCl, 20 mM Tris [pH 7.5], 5 mM EDTA, 35% formamide, 0.01% sodium dodecyl sulfate) containing 5 ng of the oligonucleotide probe designed for eubacteria (Rhodamin-CGCGGTAATACGGAGGG) per µl. Incubation at 46°C lasted overnight. Next, the slide was washed in Oligo wash buffer (70 mM NaCl, 20 mM Tris · HCl [pH 7.4], 5 mM EDTA) at 48°C for 15 min. After a brief rinse in double-distilled water, the slide was mounted with Gel Mount. A negative control was made with a slide without samples being applied, and the slide was subjected to the same procedures as those described above.

DNA and protein synthesis. Assays using about 3.8 µCi of either [methyl-³H]thymidine or [³H]leucine (6, 17) were conducted on minicores of snow in sterile 7-ml polystyrene round-bottom tubes (12 by 75 mm) with snap-top closures. Incubations were done in a walk-in cold room at temperatures between 12 and 17°C, and in an attempt to duplicate irradiance at the south pole, some tubes were exposed to 250 µmol of photons m² s¹ (photosynthetically active radiation [PAR]) of radiant energy from incandescent lamps (typical December and January PAR at the snow surface at the south pole ranges from 750 to 1,200 µmol of photons m² s¹). Incubation temperature was measured within a 7-ml tube that contained snow and was placed under the lighting. Two-hundred-microliter volumes of high-specific-activity thymidine (73 Ci/mmol) or leucine (64 Ci/mmol) was injected with a Hamilton microliter syringe in several line injections into each tube removed from the cold room on ice in the Radiation Laboratory. Injected samples were immediately returned to the cold room. Controls included (i) zero time-harvested samples (i.e., samples injected with radioisotope and immediately fixed with trichloroacetic acid [TCA]); (ii) samples incubated at 80°C; and (iii) samples amended with TCA at initiation and incubated over several hours. After termination of assays by addition of TCA to a final concentration of 5% and rapid melting of snow, assays were conducted according to standard procedures and included both filtration and centrifugation (29) protocols. Both a procedure to determine flux of isotopes into bulk macromolecules (TCA-ethanol rinse) and that to determine flux into DNA (NaOH digestion, phenol-chloroform rinse) were used (6).

Bacterial concentrations. Direct counts by epifluorescence microscopy revealed population densities from about 200 to 5,000 cells ml of snowmelt¹ (mean ± standard error, 3,140 ± 771 cells ml¹, n = 18) for both years sampled. This value compares to similar densities of bacteria in surface snows from the Ross Ice Shelf and typical concentrations of about 10⁶ bacteria ml of surface seawater¹ near McMurdo Station (12). In situ hybridization revealed the presence of eubacteria in the snowmelt from the south pole (Fig. 1A), while the negative control indicated no contamination. Examination of snowmelt using SEM indicated the presence of coccoid and rod-shaped bacteria, some of which appeared to be dividing (Fig. 1B).

View larger version (75K): [in this window] [in a new window]   FIG. 1.   Micrographs of fluorescent in situ hybridization (A) and SEM of bacteria from south pole snow. Bar = 10 µm (A) and 1.0 µm (B).

DNA. Amplification and sequencing of 16S rDNAs from DNAs isolated from bacteria in snow samples indicate the presence of bacteria that align most closely with the genus Deinococcus. The 16S rDNA sequences of these microbes were about 86 to 87% identical to those of Deinococcus. species (closest to D. grandis and D. geothermalis), the closest relatives found from GenBank databases. This genus has also been observed in soils of the Antarctic Dry Valleys that lie near the coast of Antarctica (28). We also cloned and sequenced one sample from Taylor Valley and found a sequence closest to Deinococcus sp. and D. grandis (84% identity). Phylogenetic analysis comparing our samples with representatives from the major 11 bacterial divisions, including some Antarctic and Arctic bacteria, showed that these south pole snow microbes are clustered with the Deinococcus-Thermus group (Fig. 2) and possibly make up a new genus. As reported before, Deinococcus and Thermus are closely related (3). The 16S rRNA cloned from south pole snow samples also contained bacteria from psychrophilic or unidentified taxa. FASTA comparison showed that these sequences (~1.2 kb cloned, >800 bp sequenced and matched with existing sequences) were most closely related to Alcaligenes sp. (clone Spd17), Cytophaga sp. (clones Spd1_8 and Spd2B_4), and Bacteroides sp. (Spd1_12). Based on the number of clones randomly selected for sequencing, Deinococcus-like organisms accounted for about 10 to 20% of the total snow bacteria.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Phylogenetic trees based on the 16S rDNA sequence. (A) Maximum-parsimonious analysis performed using PHYLIP with 100 replicates of bootstrap. Included in this analysis were two identical south pole sequences (SP1) that showed highest similarity to the Deinococcus group, representatives from the 11 major bacterial divisions, and some polar bacteria (denoted with asterisks). Bootstrap values are shown at the nodes. Scale bar = 50 steps. (B) Neighbor-joining analysis carried out with PHYLIP. The scale bar indicates a 10% substitution. GenBank accession numbers for these bacteria are given in parentheses following the species names in panel A.

DNA and protein synthesis. Remarkably, we also detected low levels of apparent DNA and protein synthesis in minicores of snow incubated with tritiated ([³H]thymidine and [³H]leucine, respectively) precursors of these macromolecules incubated at temperatures between 12° and 17°C (Fig. 3) (29). Over the course of 2 to 18 h, radioactive counts recovered in the macromolecular fraction generally increased by 1.5- to 5-fold, relative to counts in zero time-harvested and control samples incubated at 80°C or with TCA from the outset. Positive results were noted for 16 of 18 leucine experiments and 12 of 18 thymidine experiments.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Time courses of incorporation of [3H]leucine and [methyl-3H]thymidine into protein and DNA in south pole surface snows. First-order regressions are indicated. (A) The slope is equivalent to a rate of 0.31 ± 0.12 (n = 3) pmol of leucine liter of snowmelt1 h1 for the sample taken in January of 2000 and 0.09 ± 0.03 pmol liter of leucine of snowmelt1 h1 for the sample taken in January of 1999. (B) Results are for two thymidine-uptake experiments in January 2000 using samples from the very surface and snows 1-m deep. For the surface samples, the rate of thymidine incorporation was 0.13 pmol liter of snowmelt1 h1, and for the 1-m samples, the rate was 0.07 ± 0.02 pmol liter of snowmelt1 h1. Experimental samples from 1999 were incubated at 12°C, while those from 2000 were incubated at 15 to 17°C. Results from additional controls (incubated at 80°C or amended with TCA at zero time) from January 2000 are also indicated.

Nucleotide sequence accession number. The nucleotide sequences of the Taylor Valley sample were assigned GenBank accession numbers AF239213 and AF239800, respectively.