Long-term persistence of bacterial DNA

Abstract

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).