Dynamics of Escherichia coli type I-E CRISPR spacers over 42 000 years

Abstract

CRISPR-Cas are nucleic acid-based prokaryotic immune systems. CRISPR arrays accumulate spacers from foreign DNA and provide resistance to mobile genetic elements containing identical or similar sequences. Thus, the set of spacers present in a given bacterium can be regarded as a record of encounters of its ancestors with genetic invaders. Such records should be specific for different lineages and change with time, as earlier acquired spacers get obsolete and are lost. Here, we studied type I-E CRISPR spacers of Escherichia coli from extinct pachyderm. We find that many spacers recovered from intestines of a 42 000-year-old mammoth match spacers of present-day E. coli. Present-day CRISPR arrays can be reconstructed from palaeo sequences, indicating that the order of spacers has also been preserved. The results suggest that E. coli CRISPR arrays were not subject to intensive change through adaptive acquisition during this time.

Keywords: Escherichia coli; CRISPR arrays; CRISPR spacers; palaeo DNA.

Fig. 1

Escherichia coli type I-E CRISPR-Cas system spacer retrieval from K12 strain and a palaeo DNA sample. (a) A Logo of the E. coli type I-E CRISPR repeat is shown at the top. The arrows above and below the Logo indicate primers used in PCR amplification. A scheme showing expected products of PCR amplification from an E. coli type I-E CRISPR array using repeat-specific primers is presented below. Repeats are dark grey, and spacers are light grey. Expected amplification products are shown below as black lines with their sizes indicated. (b) The procedure outlined in (a) was applied to E. coli K12 strain containing two CRISPR arrays (CRISPR1 and CRISPR2, schematically shown at the bottom, with repeats indicated in grey, and spacers are in colour). Rightward horizontal arrows indicate promoters in the leader of each array. Leader-proximal spacers are coloured with lighter shades of blue, while leader-distant spacers are shown in progressively darker colours. The number of Illumina reads corresponding to each spacer is shown on the histograms above. (c, d) Results of E. coli type I-E CRISPR spacer amplification from K12 strain (c) and mammoth intestinal (‘Int’) and stomach (‘St’) content samples (d). Lanes marked as ‘−’ show results obtained with mock-purified DNA.

Fig. 2

Comparison of ancient and present-day Escherichia coli type I-E CRISPR spacers. (a) Comparison of spacer cluster sets. Numbers within circles correspond to unique and overlapping spacer clusters. Blue circle represents clusters obtained from the mammoth sample; red circle represents known E. coli type I-E spacer cluster set. (b) An ancestral CRISPR array is schematically shown at the top. Repeats are light grey, and spacers are coloured. The leader (light grey rectangle with arrow) is shown on the left. With the passage of time, additional spacers (coloured with lighter shades of blue) are acquired at the leader-proximal end, while internal spacers (dark-coloured) are lost. A resulting contemporary array is shown at the bottom. Expected ratios of recently acquired (spacer-proximal) and ancient (spacer-distal) spacers in the ancestral and contemporary arrays are shown at the right. (c) The overall frequency of ‘ancient’ and ‘recent’ E. coli type I-E CRISPR spacer clusters from known CRISPR arrays present in public databases (DB) and in the mammoth sample is shown. Data for CRISPR1 and CRISPR2 arrays are shown separately.

Fig. 3

Reconstruction of contemporary CRISPR arrays from reads containing two or three spacers from the mammoth sample. Mapping results of neighbouring spacer pairs and triplets on five selected CRISPR arrays from contemporary Escherichia coli are shown. Repeats are grey, and spacers are coloured. The leader regions are marked by grey triangles on the left of each array. Leader-proximal spacers are coloured with lighter shades of blue, while leader-distant spacers are dark-coloured. Detected reads containing neighbouring spacer pairs or triplets are shown by thin grey lines above each array.