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.2012 Sep;85(6):1044-56.
doi: 10.1111/j.1365-2958.2012.08171.x. Epub 2012 Jul 27.

Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms

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Free PMC article

Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms

Susanne Erdmann et al. Mol Microbiol.2012 Sep.
Free PMC article

Abstract

Central to the disparate adaptive immune systems of archaea and bacteria are clustered regularly interspaced short palindromic repeats (CRISPR). The spacer regions derive from invading genetic elements and, via RNA intermediates and associated proteins, target and cleave nucleic acids of the invader. Here we demonstrate the hyperactive uptake of hundreds of unique spacers within CRISPR loci associated with type I and IIIB immune systems of a hyperthermophilic archaeon. Infection with an environmental virus mixture resulted in the exclusive uptake of protospacers from a co-infecting putative conjugative plasmid. Spacer uptake occurred by two distinct mechanisms in only one of two CRISPR loci subfamilies present. In two loci, insertions, often multiple, occurred adjacent to the leader while in a third locus single spacers were incorporated throughout the array. Protospacer DNAs were excised from the invading genetic element immediately after CCN motifs, on either strand, with the secondary cut apparently produced by a ruler mechanism. Over a 10-week period, there was a gradual decrease in the number of wild-type cells present in the culture but the virus and putative conjugative plasmid were still propagating. The results underline the complex dynamics of CRISPR-based immune systems within a population infected with genetic elements.

© 2012 Blackwell Publishing Ltd.

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Figures

Fig. 1
Fig. 1
CRISPR loci and viruses infectingS. solfataricus P2. A. Scheme of six CRISPR loci ofS. solfataricus P2, their associated leader regions (L), and genes encoding adaptation-associated Cas1, Cas2 and Cas4. Numbers of repeat-spacer units within CRISPR arrays are given. B and C. Electron micrographs of virus particles isolated from (B) supernatant of the enrichment culture, and (C)S. solfataricus P2 6 days post infection with the virus mixture in (B). Samples were negatively stained with 1% uranyl acetate and size bars are included.
Fig. 2
Fig. 2
Growth curves of virus-infectedS. solfataricus P2 and spacer uptake in CRISPR loci. A. Growth curves for uninfected (dark blue) and virus-infected (light blue) wild-type cells, and for uninfected (dark green) and infected (light green) CRISPR-minus strain. B. Growth curves for uninfected (dark blue) and virus-infected (light blue) wild-type cells at onset of spacer uptake (10–12 days).A600 measurements were made every 6 h. C. PCR products amplified from leader proximal regions of CRISPR loci A to F of wild-type strain and matched to growth curves (Dpi3 and Dpi12). Dpi, days post infection.
Fig. 3
Fig. 3
Assembly of newly inserted spacer sequences. Composition of one contig generated from overlapping spacer sequences on both DNA strands. Arrowheads indicate the direction away from the leader (corresponding to spacer crRNA sequence). Two spacers (in red) have opposite orientations. Spacers are preceded by a CCN protospacer-associated motif (PAM) (Lillestølet al., 2009) (in blue and bold type). CCN motifs with no detected spacers (green). Loci origins and spacer lengths are indicated.
Fig. 4
Fig. 4
Yields of virus and putative conjugative plasmid in single clones carrying new spacers. Reinfection with the purified virus mixture of isolated single clones containing new spacers in locus C (S11), loci C and D (S3) and in loci C, D and E (S10). PCR products oforf174 within an operon in viral contig 1 (Table 3) and of thetraG-like gene in contig 2 of the putative conjugative plasmid (Table 4) were PCR-amplified from DNA isolated from cultures 2 and 20 days post infection (Dpi).
Fig. 5
Fig. 5
Yields of virus and putative conjugative plasmid in the wild-type culture over a 74-day period. PCR products are shown fororf174 of the virus and thetraG-like gene of the putative conjugative plasmid isolated from cultures 2–74 days post infection (Dpi). Template DNA was isolated from 2 ml of infected cells and concentrations were adjusted to be the same for each sample prior to PCR analysis.
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