Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 322 (5909), 1843-5

CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Affiliations

CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Luciano A Marraffini et al. Science.

Abstract

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

Figures

Figure 1
Figure 1
A CRISPR locus provides immunity against plasmid conjugation in S. epidermidis. (A) Organization of the RP62a CRISPR locus. Repeats and spacers (colored boxes) are followed by CRISPR associated genes (cas1, cas2, cas6) and cas subtype M. tuberculosis genes (csm1 to csm6) (5). An AT-rich “leader” sequence precedes the repeat-spacer region (black box). LAM104 is an isogenic Δcrispr strain lacking only the repeat and spacer sequences. (B) The staphylococcal conjugative plasmid pG0400 spc1 target sequence [pG0(wt), highlighted in yellow] is shown on the top. This sequence was altered to introduce synonymous mutations to generate pG0(mut), with changes shown in red. (C) To restore interference in strain LAM104, two plasmids were introduced: pCRISPR and pCRISPR-L. (D) Conjugation was carried out by filter mating in triplicate; the cfu/ml values (mean +/− SD) obtained for recipients and transconjugants are shown. Recipient strains, complementing plasmids, and donor conjugative plasmids are indicated. Conjugation efficiency (Conj. Eff.) was calculated as the recipients/transconjugants ratio.
Figure 2
Figure 2
CRISPR interference requires an intact target sequence in plasmid DNA but not mRNA. (A) Disruption of the pG0400 nes target sequence with the orf142-I2 self-splicing intron, generating the conjugative plasmid pG0(I2). (B) Conjugation efficiency was measured as in Fig. 1D, using RP62a and the Δcrispr mutant LAM104 as recipients for pG0(wt) and pG0(I2). To test for interference with spliced nes mRNA, RP62a and LAM104 transconjugants were also used as donors of pG0(I2) to S. epidermidis ATCC 12228.
Figure 3
Figure 3
Plasmid transformation is subject to CRISPR interference. (A) Introduction of the wild-type and mutant nes target sequences (mutations highlighted in grey) into the plasmid pC194 (d, direct; i, inverted). The origin of replication (ori) as well as protein-coding genes are indicated. Stem-loops denote the rep and cat transcriptional terminators. (B) RP62a and the Δcrispr mutant LAM104 were transformed in triplicate with the plasmids described in (A). Transformation efficiency was calculated as cfu/µg DNA (mean +/− SD).

Similar articles

See all similar articles

Cited by 541 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback