David A. Gilichinsky

Metabolic activity of permafrost bacteria below the freezing point

ABSTRACT Metabolic activity was measured in the laboratory at temperatures between 5 and −20°C on the basis of incorporation of14C-labeled acetate into lipids by samples of a natural population of bacteria from Siberian permafrost (permanently frozen soil). Incorporation followed a sigmoidal pattern similar to growth curves. At all temperatures, the log phase was followed, within 200 to 350 days, by a stationary phase, which was monitored until the 550th day of activity. The minimum doubling times ranged from 1 day (5°C) to 20 days (−10°C) to ca. 160 days (−20°C). The curves reached the stationary phase at different levels, depending on the incubation temperature. We suggest that the stationary phase, which is generally considered to be reached when the availability of nutrients becomes limiting, was brought on under our conditions by the formation of diffusion barriers in the thin layers of unfrozen water known to be present in permafrost soils, the thickness of which depends on temperature.

Phylogenetic diversity of a bacterial community determined from Siberian tundra soil DNA.

Genomic DNA was isolated from the active layer of tundra soil collected from the Kolyma lowland, Northeast Eurasia, near the Arctic Ocean coast. The SSU (small subunit) rRNA genes were amplified with eubacterial primers from the bulk genomic community DNA and cloned into plasmid vectors. Forty-three SSU rDNA clones were obtained, and all of them had different RFLP patterns. Phylogenetic analysis based on partial sequences (about 300 bp) established with the maximum likelihood method revealed the presence of three major and several minor groups that fell into 11 of the established lines of bacteria, and one sequence that could not be assigned to any of the described groups. Most of the clones belonged to the alpha (20.9%) and delta (25.6%) subdivisions of the Proteobacteria, with lesser proportions in the beta (9.3%) and gamma (4.7%) subdivisions, groups typically isolated from soil by culture methods. Fewer than 12% of the clones belonged to Gram-positive bacteria, and 16% of the clones were related to Fibrobacter. The majority of the clones (70%) had sequences that were 5-15% different from those in the current databases, and 7% of the clones had sequences that differed by more than 20% from those in the database. The results suggest that these tundra-derived clones are very diverse in phylogeny, and that many probably reflect new genera or families. Hence, most of the tundra soil bacterial community has never been isolated and thus the physiology and function of its dominant members appears to be unknown.

Characterization of Viable Bacteria from Siberian Permafrost by 16S rDNA Sequencing

A bstractViable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is −10°C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30°C. The majority of the bacterial isolates were rod shaped and grew well at 30°C; but two of them did not grow at or above 28°C, and had optimum growth temperatures around 20°C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, β-proteobacteria, γ-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and β-proteobacteria, and all γ-proteobacteria, came from samples with an estimated age of 1.8–3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000–8,000 years (Alas suite).

Ancient bacteria show evidence of DNA repair

Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability.

Long-term persistence of bacterial DNA

The persistence of bacterial DNA over geological timespans remains a contentious issue. In direct contrast to in vitro based predictions, bacterial DNA and even culturable cells have been reported from various ancient specimens many million years (Ma) old [1–8]. As both ancient DNA studies and the revival of microorganisms are known to be susceptible to contamination [8–10], it is concerning that these results have not been independently replicated to confirm their authenticity. Furthermore, they show no obvious relationship between sample age, and either bacterial composition or DNA persistence, although bacteria are known to differ markedly in hardiness and resistance to DNA degradation [11]. We present the first study of DNA durability and degradation of a broad variety of bacteria preserved under optimal frozen conditions, using rigorous ancient DNA methods [8–10]. The results demonstrate that nonspore-forming gram-positive (GP) Actinobacteria are by far the most durable, out-surviving endosporeformers such as Bacillaceae and Clostridiaceae. The observed DNA degradation rates are close to theoretical calculations [9], indicating a limit of ca. 400 thousand years (kyr) beyond which PCR amplifications are prevented by the formation of DNA interstrand crosslinks (ICLs). The twelve permafrost samples (0-8.1 Ma) investigated were obtained from northeast Siberia and Beacon Valley, Antarctica. DNA preservation at these sites is exceptional due to constant subzero temperatures, largely neutral pH, and anaerobic conditions. Epifluorescence microscopy revealed ~107cells/gram wetweight in the bacterial size range. The cell counts are in agreement with previous results obtained on permafrost [2,3]. 16S rDNA sequences of 120 bp and 600 bp could be reproducibly amplified from samples up to 400–600 kyr, and show an inverse relationship between PCR amplification efficiency and fragment length that is typical of ancient DNA [8–10,12]. Controls for surface contamination during sampling were negative. Chimeric sequences were excluded from analysis, along with sequences that failed a bootstrap test for independent reproducibility [13]. DNA concentrations and taxonomic diversity were found to decrease with age until 400–600 kyr, at which point the percentage of templates with ICLs reached 100% (Figure 1A–C). Sequences from the older samples appear to be a subset of those from younger material, and all identified bacterial taxa are known soil inhabitants, indicating that permafrost is a nonextremophile environment. There were clear age-related patterns in taxon survival across geographically widespread samples (separated up to 1400 km). Sequences of non-sporeforming GP Actinobacteria, affiliated largely to the genus Arthrobacter (99–100% similarity), consistently persisted for the longest time, followed by GP endospore-forming Bacillaceae and Clostridiaceae and finally gram-negative (GN) bacteria, mostly Proteobacteria (Figure 1D).

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments

Abstract Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4°C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost.

Supercooled Water Brines Within Permafrost—An Unknown Ecological Niche for Microorganisms: A Model for Astrobiology

This study describes brine lenses (cryopegs) found in Siberian permafrost derived from ancient marine sediment layers of the Arctic Ocean. The cryopegs were formed and isolated from sediment ~100,000-120,000 years ago. They remain liquid at the in situ temperature of -10 degrees C as a result of their high salt content (170-300 g/L). [(14)C] Glucose is taken up by the cryopeg biomass at -15 degrees C, indicating microbial metabolism at low temperatures in this habitat. Furthermore, aerobic, anaerobic heterotrophs, sulfate reducers, acetogens, and methanogens were detected by most probable number analysis. Two psychrophilic microbes were isolated from the cryopegs, a Clostridium and a Psychrobacter. The closest relatives of each were previously isolated from Antarctica. The cryopeg econiche might serve as a model for extraterrestrial life, and hence is of particular interest to astrobiology.

Biogeochemical activity of anaerobic microorganisms from buried permafrost sediments

Permafrost sediment samples, ranging in age from 7 thousand to 2 million years, from the northeastern region of Russian Arctic were analyzed for evidence of reducing conditions, viable populations of anaerobic bacteria and their metabolic end products. Field analyses of samples showed that all sediments were reduced with a redox potential ranging from +40 to —256 mV. Ferrous iron, acid‐soluble sulfide, and methane were detected in the frozen sediments. Direct bacterial counts were 107 to 108 cells/g sediments as determined by epifluorescence microscopy using acridine orange. Denitrifiers and (H2 + CO2)‐utilizing methanogens were detected in all samples, and acetoclastic methanogens, sulfate reducers, and Fe(Ill) reducers were detected in some samples and at much lower numbers. [3SS]Sulfide production from [35S]sulfate was detected in soils incubated anaerobically at 4°C for 6 months. Thus anaerobic metabolic activity was present at temperatures near freezing. These results suggest that viable anaerobic ba...

Long-term preservation of microbial ecosystems in permafrost.

It has been established that significant numbers (up to 10 million cells per gram of sample) of living microorganisms of various ecological and morphological groups have been preserved under permafrost conditions, at temperatures ranging from -9 to -13 degrees C and depths of up to 100 m, for thousands and sometimes millions of years. Preserved since the formation of permafrost in sand-clay sediments of the Pliocene-Quaternary period and in paleosols and peats buried among them, these cells art the only living organisms that have survived for a geologically significant period of time. The complexity of the microbial community preserved varies with the age of the permafrost. Eukaryotes are found only in Holocene sediments; while prokaryotes are found to greater ages, i.e., Pliocene and Pleistocene. The diversity of microorganisms decreases with increasing age of sediments, and as a result cocci and corynebacteria are predominant. Enzyme activity (catalase and hydrolytic enzymes) and photosynthetic pigments (chlorophyll and pheophytin have also been detected in permafrost sediments. These results permit us to outline some approaches to the search for traces of life in the permafrost of Martian sediments by borehole core sampling. It is in the deep horizons (and not on the planet surface), isolated by permafrost from the external conditions, that results similar to those obtained on Earth can be expected.

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate ∼100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be ∼400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.