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1 NASA/NSSTC, XD12, 320 Sparkman Drive, Room 4247, Huntsville, AL 35805, USA
2 Laboratory for Structural Biology, The University of Alabama in Huntsville, MSB, Huntsville, AL 35899, USA
3 Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
4 American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110, USA
Correspondence
Elena V. Pikuta
elena.pikuta{at}msfc.nasa.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FTR1T is AF450136.
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). Some of these species are known as fish pathogens (Hiu et al., 1984). The species Carnobacterium piscicola (Collins et al., 1987) and Lactobacillus maltaromicus (Miller et al., 1974) were recently identified as synonyms and the two species were transferred to C. maltaromaticum (Mora et al., 2003). All species of the genus are facultative anaerobes and are capable of reducing resazurin in aerobic media during growth. In nature, they may act as primary agents responsible for the reduction of oxygen levels in ecosystems and for the creation of advantageous conditions for the development of obligately anaerobic micro-organisms (Franzmann et al., 1991).
In this article we describe a novel psychrotolerant, facultatively anaerobic bacterium isolated from the permafrost tunnel in Fox, Alaska. In previous work, we described in detail the isolation of the novel bacterium and characterized the ecosystem in which it was found (Hoover et al., 2002; Pikuta & Hoover, 2003). Since the geological age of the sample is dated as the Pleistocene epoch, we suggest the name Carnobacterium pleistocenium sp. nov. for the novel species.
Strain FTR1T was isolated from an ice core sample collected from the lower level of a frozen lenticular ice lens (Fig. 1) associated with a Pleistocene thermokarst pond (age 
32 000 years) in the CRREL (Cold Regions Research and Engineering Laboratory) Fox Permafrost Tunnel, which is approximately 15 km north of Fairbanks, Alaska (Hoover & Gilichinsky, 2001).
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); and 1 ml trace mineral solution (Whitman et al., 1982). The final pH was 7·14–7·2. High-purity nitrogen was used as the gas phase. A pure culture was obtained by the dilution method in Hungate tubes and supported on medium with a modified NaCl concentration of 0·5 % (w/v). The ninth dilution of the morphologically monotypic culture was chosen for growth of colonies on agar medium. Isolation of colonies was performed by the roll-tube method on 3 % (w/v) agar medium. After 10–14 days incubation at 2 °C, colonies of strain FTR1T were white to cream in colour and round, convex lens-shaped (in deep agar) with a diameter of around 1–2 mm. Colonies grown on the agar surface were conical in shape and the centre had a denser consistency and darker colour than the perimeter. The surface of colonies was granulated and rough with thinner, irregular, torn edges. Pure cultures in all experiments were incubated at 4–22 °C. The purity of the culture was checked by microscopic control during this study.
The morphology of the novel isolate was examined under a phase-contrast microscope (Fisher Micromaster) and a Hitachi S-4000 field-emission scanning electron microscope was used to examine the ultramicrostructure of the cell surface. An epifluorescence microscope Leitz Diaplan was used with DAPI and BacLite live/dead cell stains. Cells of strain FTR1T were small, short rods with rounded ends and were 0·7–0·8 µm wide and 1·0–1·5 µm long (Fig. 2). Cells were motile and stained Gram-positive. Spores were never observed. Cells occurred singly, in pairs or in short, irregular curved chains.
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). All substrates were added to the medium, containing 0·1 g yeast extract l–1, at concentrations of 3 g l–1. End products of peptone fermentation in the liquid phase were determined by HPLC. Separation was done on an Aminex HPX-87H (Bio-Rad) column with 5 mM H2SO4 as the mobile phase. Gases were measured using a gas chromatograph 3700 (Varian) equipped with a Porapak Q column and TCD detector. Nitrogen was used as the gas carrier. The novel isolate was a facultative anaerobe and grew well under aerobic and anaerobic conditions. During growth under aerobic conditions resazurin was reduced (i.e. became colourless). Strain FTR1T had a negative catalase reaction. The novel isolate could grow without NaCl with a longer lag phase (three passages on a medium in which all sodium salts were replaced by potassium salts and chloride salts were replaced by sulfates). The NaCl range for growth was 0–5 % (w/v), with optimum growth at 0·5 % (w/v). At 7 % NaCl, growth was absent. Strain FTR1T grew in pH range 6·5–9·5 and had optimum growth at pH 7·3–7·5. The temperature range for growth of the novel isolate was 0–28 °C with optimal growth at 24 °C.
Strain FTR1T had a chemoorganoheterotrophic metabolism and was capable of growth on the following substrates: D-glucose, D-fructose, D-mannose, D-maltose, sucrose, lactose, starch, D-mannitol, peptone, Bacto tryptone, Casamino acids and yeast extract. The best growth was observed on D-trehalose and the weakest growth was on D-arabinose. No growth was observed with formate, acetate, lactate, pyruvate, propionate, butyrate, citrate, ethanol, methanol, glycerol, acetone, betaine, trimethylamine or triethylamine.
Metabolic end products of culture on glucose were acetate (1·2 mM) and ethanol (6·3 mM) in the liquid phase and traces of CO2 in the gas phase.
Concentrations of antibiotics used for testing were 250 µg ml–1 for ampicillin, kanamycin, gentamicin, tetracycline and rifampicin and 125 µg chloramphenicol ml–1. Strain FTR1T was sensitive to all antibiotics tested.
For extraction of fatty acid methyl esters, the culture was incubated for 4 days at 22 °C on the medium described above. The extraction procedure and instruments were described previously (Pikuta et al., 2003). The major fatty acids for strain FTR1T were C14 : 0, C16 : 1cis7, C16 : 0, C18 : 1cis9 and C18 : 0 (Table 1).
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). The 16S rRNA gene of strain FTR1T was amplified and sequenced as described previously (Hoover et al., 2003), except for a slight change in the thermal cycling profile (time of initial denaturation at 95 °C was 2 min) and the use of a modified set of 16S rRNA-specific sequencing primers: 5'-GCCAGCAGCCGCGGTAATAC (Escherichia coli positions 516–535), 5'-TCGTCAGCTCGTGTCGTGAG (1061–1080), 5'-GGGTTGCGCTCGTTGCGGG (1113–1095) and 5'-GCGCTCGCTTTACGCCCAAT (581–562).
The consensus sequence was aligned with eight closely related sequences using CLUSTAL W (Thompson et al., 1994). Pairwise distances were computed with MEGA version 2.1 (Kumar et al., 2001) using the Jukes–Cantor model (Jukes & Cantor, 1969). An unrooted phylogenetic tree was constructed with the same program using the neighbour-joining method (Saitou & Nei, 1987).
A sequence covering 1486 nucleotides of the 16S rRNA gene was obtained, corresponding to positions 28–1492 of the E. coli 16S rRNA sequence. No difference was observed between the sequences of the three selected clones. The G+C content of this sequence was 53·5 mol%. The sequence was compared with all sequences presently available in the GenBank database and appeared to be highly similar to sequences from Carnobacterium species. A phylogenetic dendrogram was constructed based on 1379 common nucleotides sites, and the dendrogram shows the position of strain FTR1T among all currently known species of the genus Carnobacterium (Fig. 3). According to the pairwise distance table (not shown), based on the same 1379 common nucleotide sites, strain FTR1T appears to belong to a cluster including C. alterfunditum, C. inhibens and C. viridans, with 99·78, 98·54 and 98·83 % similarity, respectively. Since the similarity of the novel isolate was determined to be highest to C. alterfunditum pf4T, it was suggested that an additional genetic comparison be performed for the two strains.
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; Gillis et al., 1970). Purified genomic DNA (200 µg) from strain FTR1T and C. alterfunditum pf4T was sonicated to generate DNA fragments of 500–700 bp. Any residual RNA and single-stranded DNA was removed by treatment with RNase A and S1 nuclease, respectively (Ausubel et al., 1987). The concentration and purity of the DNA were determined from the A260 and the A260/A280 ratio using a Shimadzu UV-160 spectrophotometer. DNA (80 µg) from each of these micro-organisms was then denatured in 1x SSC buffer (pH 7·0) by increasing the temperature of the sample from 26 to 100 °C (at a rate of 1 °C min–1) and the A260 was recorded. The experiment was conducted in triplicate. The Tm was determined by calculating the temperature at which the hyperchromicity reached half of the value obtained after complete melting. The Tm of the genomic DNA of strain FTR1T was 62±2 °C (mean±SD, n=4), whereas it was 53±3 °C (n=4) for C. alterfunditum pf4T.
To determine the relatedness of genomic DNA between strain FTR1T and C. alterfunditum pf4T, DNA–DNA hybridization was performed by DNA reassociation kinetics as described previously (De Ley et al., 1970; Johnson, 1985). Purified, sonicated genomic DNA (80 µg) from each strain was added to 4x SSC buffer (pH 7·0) and 25 % deionized formamide. The DNA was denatured by raising the temperature to 100 °C and cooled to 5 °C above the respective melting temperatures. The temperature was then rapidly (1·5 min) lowered to the reassociation temperature and the A270 was recorded at 5 s intervals for a total of 20 min. The initial reassociation kinetics were determined by linear regression analysis. The experiment was conducted in triplicate. The percentage relatedness of the DNA was calculated using the equation described by De Ley et al. (1970). All statistical analyses were performed using Microsoft Excel. DNA–DNA hybridization established 39±1·5 % relatedness (mean±SD, n=3) between the genomes of strain FTR1T and C. alterfunditum pf4T.
The genome sizes of strain FTR1T and C. alterfunditum pf4T were determined from the DNA reassociation kinetics, following the equation described by Gillis et al. (1970). The genome sizes were 2·1x109 Da for strain FTR1T and 1·9x109 Da for C. alterfunditum pf4T.
The G+C content in genomic DNA of strain FTR1T was determined by following the procedure described by Starr & Mandel (1968) and Franzmann et al. (1991). The total G+C content of the purified genomic DNA for strain FTRT was 42±1·5 mol% (mean±SD, n=3) compared with 33–34 mol% for C. alterfunditum (Franzmann et al., 1991).
The novel isolate is the first representative of this genus to be found alive in ice entrained in Pleistocene permafrost. Detailed characterization of this ecosystem and the importance of its study to paleomicrobiology, biostratigraphy, geocryology, microbial evolution, conservation of the modern gene pool and the long-term viability of micro-organisms in deep anabiosis were described previously (Hoover et al., 2002). Indeed, there is still much less known about the microbiota preserved in Pleistocene permafrost and ice than is known about fossil Pleistocene mammals. In Table 2, distinguishing features of the novel isolate and C. alterfunditum pf4T are shown. Notwithstanding the different sources of isolation for the two strains, a very close phylogeny and physiology is demonstrated, while some metabolic features have significant differences. For example, the sugars that could be used as substrates are different, and lactic acid is absent from the end products of strain FTR1T. Also, the composition of fatty acid methyl esters in the novel isolate is different: 16 : 0 DMA is absent from strain FTR1T, but it represents around 0·8 % of the total in C. alterfunditum pf4T; 12 : 0 FAME and 18 : 2cis9,12 FAME are present in strain FTR1T, but they are absent from C. alterfunditum pf4T (Table 1
). DNA–DNA hybridization of genomic DNA between strain FTR1T and C. alterfunditum pf4T exhibited only 39 % relatedness. Also, the melting temperature, the G+C content of the genomic DNA and the genome sizes of the two micro-organisms are different (Table 2).
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Description of Carnobacterium pleistocenium sp. nov.
Carnobacterium pleistocenium (plei.sto.ce'ni.um. N.L. neut. adj. pleistocenium belonging to the Pleistocene, a geological epoch).
Cells are motile, small rods with rounded ends, 0·7–0·8x1·0–1·5 µm. Gram-positive. Growth occurs between 0 and 28 °C (optimum 24 °C) and at pH22 °C 6·5–9·5 (optimum pH 7·3–7·5). Range of NaCl for growth is 0–5 % (w/v); optimum growth at 0·5 % (w/v) NaCl. Facultative anaerobe. Catalase-negative. Heterotrophic growth occurs with D-glucose, D-fructose, maltose, D-mannitol, D-mannose, D-trehalose, lactose, D-ribose, D-arabinose, sucrose, starch, peptone, Bacto tryptone, Casamino acids and yeast extract. End products of growth are acetate, ethanol and traces of carbon dioxide (in gas phase). Sensitive to ampicillin, kanamycin, gentamicin, tetracycline, rifampicin and chloramphenicol.
The type strain, FTR1T (=ATCC BAA-754T=JCM 12174T=CIP 108033T), was obtained from a sample of permafrost from Fox Tunnel, Alaska.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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