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

DNA and protein precursors were incorporated into trichloroaceticacid-precipitated material by bacterial cell suspensions duringincubation for 50 to 100 days at -15^oC. Incorporation did notoccur at -70^oC and was inhibited by antibiotics. The resultsdemonstrate that bacteria can perform macromolecular synthesisunder conditions that mimic entrapment in glacial ice.

INTRODUCTION

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Introduction

Bacteria can remain viable for hundreds of thousands of yearswhen 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 accumulatethrough amino acid racemization, DNA depurination, and exposureto natural ionizing radiation (12, 13). However, it is knownthat temperature and the presence of water strongly influencemacromolecular decay and that protein and nucleic acid decayrates are drastically reduced within materials with low wateractivity, such as amber (4) and perhaps ice as well. It alsoseems possible that such entrapped microbes might carry outslow rates of metabolism to repair the incurred macromoleculardamage. Thin veins of liquid exist between ice crystals thatcould provide a habitat for microorganisms within apparentlysolid ice (15), and studies of permafrost (17) and South Polesnow (6) have detected low levels of metabolic activity at subzerotemperatures. Therefore, to explore the concept that microorganismstrapped in glacial ice might also be metabolically active, reconstructionexperiments were undertaken to determine if macromolecular synthesisoccurs at -15°C when bacteria isolated from glacial icecore samples were refrozen.

Bacterial strains and culture conditions.

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Bacterial strains and culture...

A ${gamma}$ -proteobacterial Psychrobacter species (8) (isolate Trans1;16S ribosomal DNA [rDNA] GenBank accession no. AF479327) andEscherichia coli (Ohio State reference no. 422) were grown inLuria-Bertani medium (18), and an actinobacterial Arthrobacterspecies (7) (isolate G200-C1; 16S rDNA GenBank accession no.AF479341) was cultured in R2 medium (16). Cultures (25 ml) wereincubated aerobically with shaking (200 rpm) at 22°C in125-ml Erlenmeyer flasks to the late exponential growth phaseand were diluted to an initial A₆₀₀ of 0.2 and allowed to growto an A₆₀₀ of 0.6. The cells present were then harvested bycentrifugation at 17,000 x g for 5 min, washed twice with distilledwater, and resuspended at an A₆₀₀ of 0.2 in distilled water(equivalent to 1.3 x 10⁸ and 2 x 10⁸ CFU ml^-1 for Trans1 andG200-C1, respectively). Aliquots (500 µl) of these cellsuspensions were placed in 1.5-ml Eppendorf tubes and chilledto 4°C.

Precursor incorporation assays.

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Precursor incorporation assays.

Cell suspensions were maintained on ice, and 100 µl ofa chilled working solution (10 µCi ml^-1; diluted 1:100from stock) of either [³H]thymidine (ICN Biomedicals, catalogno. 24060; 60 to 90 Ci/mmol in sterile water) or [³H]leucine(ICN Biomedicals, catalog no. 20036E; 40 to 60 Ci/mmol in asterile 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°Cfreezer, except for control cell suspensions that were maintainedat -70°C. At designated experimental time points, 100 µlof 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 centrifugationat 18,000 x g for 15 min. The pellet was washed with 500 µlof 5% TCA, recentrifuged for 10 min at 18,000 x g, washed with500 µl of 70% ethanol, and suspended in 1 ml of EcoscintH scintillation fluid (Life Sciences, Inc., catalog no. LS-275).The Eppendorf tube was placed into a scintillation vial, andthe radioactivity present was quantitated by liquid scintillationcounting (Beckman model LS-7500 scintillation counter).

Incorporation into TCA-precipitable material at -15°C.

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Incorporation into tca...

[³H]thymidine and [³H]leucine were incorporated into TCA-precipitatedmaterial by cells frozen in distilled water during incubationat -15°C (Fig. 1). The majority of the incorporation occurredwithin the first 50 days after freezing. Incorporation did notoccur in identical samples incubated at -70°C, a temperaturebelow that predicted for liquid water to exist in ice (14),or by controls pretreated with 5% TCA before freezing. The radioactivecounts observed in TCA-treated controls were similar in samplesincubated at -15 and 22°C (1,000 to 5,000 dpm), indicatingthat $~$ 0.2% of the radiolabel persisted following $~$ 1:1,000 dilutionof the mixture during precipitation and washing. Since thereis not a significant difference between the radioactive countsobtained in samples prefixed in TCA and those incubated at -70°Cin distilled water, this suggests that the background in theseexperiments results from persistence of residual radiolabeland is not due to acid conditions effecting chemical binding.

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FIG. 1. Incorporation of [³H]thymidine and [³H]leucine into TCA-precipitable material over 172 days by frozen suspensions of Psychrobacter sp. strain Trans1 and Arthrobacter sp. strain G200-C1 at -15 and -70°C. Duplicate samples were measured at each time point. The y-axis error bars denote standard deviations from the mean.

No precursors were incorporated after >5 months at -15°Cby cells frozen in their respective growth media (data not shown).The presence of substrate in these undefined media may havediluted the radiolabel and prevented detection of a low levelof incorporation over this period of time. Another explanationis that nutrient-fed cultures were not metabolically stressedprior to freezing, as would occur when cells in logarithmicgrowth were washed and reconstituted in distilled water. Duringmetabolic arrest, the imbalance between decreased anabolismand continuing catabolism causes a burst of free radical production,resulting in damage to DNA and other cellular components (3).It is possible that incorporation by cells in ice made fromdistilled water represents activity directed toward repair ofdamage incurred prior to freezing.

In experiments in which the viable count was also determined,the recovery of G200-C1 decreased fourfold during the freezingprocess and then an additional fourfold during the first 12days of incubation at -15°C (Fig. 2). Due to a technicalerror, a day 0 time point is not available for the Trans1 isolate,but the viable count was 12-fold lower after 2 days at -15°Cthan before freezing. In both cases, the numbers of CFU permilliliter recovered stabilized by 150 days postfreezing. Interestingly,70 to 80% of the thymidine incorporation occurred during thefirst 12 days of incubation, and this coincided with the periodof time when the numbers of CFU per milliliter became stable,after the initial 17-fold decrease that occurred during freezingand initial storage for 12 days at -15°C. The presence ofciprofloxacin (ICN Biomedicals, catalog no. 199020) reducedthymidine incorporation by 30 to 40%, and the presence of chloramphenicol(Sigma, catalog no. 100K9113) reduced leucine incorporation50 to 60% (Table 1). Curiously, incubations in the presenceof nalidixic acid (Sigma, catalog no. N-3143), resulted in atwofold increase in thymidine incorporation in both species,even though growth of G200-C1 is inhibited in the presence of15 µg of this antibiotic per ml. Precursor incorporationwas similarly affected in aqueous cell suspensions incubatedat 22°C for 20 h in the presence of chloramphenicol andnalidixic acid (data not shown). Although the amount of incorporationunder liquid conditions after 20 h at 22°C was substantiallylarger (10 to 100x) than after 23 days at -15°C, leucineincorporation was 25 to 85% lower in the presence of chloramphenicol,and thymidine incorporation increased twofold in the presenceof nalidixic acid.

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FIG. 2. Incorporation of [³H]thymidine and numbers of CFU per milliliter for Psychrobacter sp. strain Trans1 and Arthrobacter sp. strain G200-C1 over 274 days of incubation at -15°C. Before freezing, the cell suspensions of Trans1 and G200-C1 contained 1.3 x 10⁸ CFU ml^-1 and 2 x 10⁸ CFU ml^-1, respectively. The 2-day number of CFU per milliliter represents the first data point for Trans1. Each point shown was the result of triplicate measurements. The y-axis error bars denote standard deviations from the mean.

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TABLE 1. Incorporation of [³H]thymidine and [³H]leucine by frozen cell suspensions during 23 days of incubation at -15°C^a

Biochemical fractionation.

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Biochemical fractionation.

The washed, TCA-precipitated material from cells incubated at-15°C for 280 days with [³H]thymidine or [³H]leucine wasfractionated according to the method of Kelley (11). This materialwas suspended in 30 µl of a 1:1 ethanol-ether mixture,incubated at 37°C for 30 min, and then centrifuged for 10min at 18,000 x g. The resulting supernatant was collected,and the pellet was resuspended in 30 µl of 5% TCA andincubated at 95°C for 30 min. After centrifugation at 18,000x g for 10 min, the supernatant was collected, and the radioactivityin all fractions was quantitated by liquid scintillation counting.

In both cases, $~$ 50% of the [³H]thymidine was incorporated intothe hot TCA extract containing nucleic acids, and $~$ 50% was incorporatedinto the insoluble residue that would contain proteins (Table2). Although less [³H]leucine was incorporated, the majorityof this precursor was incorporated into the protein fraction.Given that 7,400 to 8,400 dpm of thymidine and $~$ 2,100 dpm ofleucine were incorporated per sample into nucleic acid and protein,respectively, it can be calculated from the specific activitiesof the precursors (60 to 90 Ci of [³H]thymidine and 40 to 60Ci of [³H]leucine per mmol) and from the assumption that allcells present (1.1 x 10⁷ CFU ml^-1) (Fig. 2) participated inthe DNA and protein syntheses that 4,100 to 6,900 moleculesof thymidine and $~$ 2,000 molecules of leucine were incorporatedper cell after 280 days of incubation. Based on 3-Mbp genomesand an average protein size of 36 kDa, this incorporation correspondsto replication of <1% of the genome and synthesis of $~$ 100protein molecules. This incorporation is therefore insufficientfor reproduction.

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TABLE 2. Fractionation of TCA-insoluble material from cell suspensions incubated at -15°C in the presence of [³H]thymidine and [³H]leucine for 280 days^a

Implications for metabolism in glacial ice.

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Implications for metabolism in...

The results are consistent with macromolecular synthesis at-15°C by isolates recovered from polar and nonpolar glacialice cores under conditions comparable to those within glacialice. The 16S rDNA sequences of these psychrotrophic isolatesare >99% identical to those of species recovered from brinechannels in sea ice (5, 10), and therefore it was hypothesizedthat a biological activity at below-freezing temperatures mightbe exclusive to closely related strains. However, [³H]thymidineand [³H]leucine were also incorporated into TCA-precipitablematerial during 102 days of incubation at -15°C by a laboratorystrain of E. coli, indicating that this ability is not limitedto species specialized for survival in ice. For cells in 5%TCA, 3,800 ± 1,100 dpm (mean ± standard deviation)of [³H]thymidine and 4,100 ± 16 dpm of [³H]leucine wereincorporated (n = 2). For cells in distilled water, 15,000 ±470 dpm of [³H]thymidine and 17,000 ± 9,900 dpm of [³H]leucinewere incorporated (n = 2).

The data reported here add to the growing body of evidence formetabolism under frozen conditions (6, 17). They support theargument that bacteria trapped in glacial ice might repair macromoleculardamage that occurs while immured for extended periods. Possibly,cells remain metabolically active in solute-enriched water filmson the surface of entrapped particulates and air bubbles orin liquid veins between adjacent ice crystals (15).

ACKNOWLEDGMENTS

I am indebted to John Reeve for experimental guidance and valuableeditorial comments and thank Wade Jeffrey for providing proceduraladvice.

This research was supported at Ohio State University by NationalScience Foundation (NSF) grant OPP-9714206, awarded throughthe Life in Extreme Environments Initiative. I am presentlysupported at Montana State University by NSF grant OPP-0085400.

FOOTNOTES

* 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{at}montana.edu.

REFERENCES

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