Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.
At a glance
- Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433 (1987) , , , &
- Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002) , , &
- Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol. Microbiol. 36, 244–246 (2000) , , &
- Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005) , , &
- Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005) , , &
- CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005) , &
- Virus population dynamics and acquired virus resistance in natural microbial communities. Science 320, 1047–1050 (2008) &
- CRISPR/Cas, the immune system of Bacteria and Archaea. Science 327, 167–170 (2010) &
- CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007) et al.
- CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008) &
- Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus . J. Bacteriol. 190, 1401–1412 (2008) et al.
- CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Rev. Genet. 11, 181–190 (2010) &
- RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009) et al.
- CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol. Microbiol. 72, 259–272 (2009) et al.
- Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol. 75, 1495–1512 (2010) et al.
- Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus . Proc. Natl Acad. Sci. USA 99, 7536–7541 (2002) et al.
- Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008) , , , &
- Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus . RNA 14, 2572–2579 (2008) , , &
- Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008) et al.
- Phage response to CRISPR-encoded resistance in Streptococcus thermophilus . J. Bacteriol. 190, 1390–1400 (2008) et al.
- Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family. Plasmid 45, 171–183 (2001) &
- Distribution and analysis of plasmids in Streptococcus thermophilus . J. Ind. Microbiol. 1, 157–163 (1986) &
- Role of galK and galM in galactose metabolism by Streptococcus thermophilus . Appl. Environ. Microbiol. 74, 1264–1267 (2008) et al.
- Analysis of two theta-replicating plasmids of Streptococcus thermophilus . Plasmid 58, 174–181 (2007) &
- The mode of replication is a major factor in segregational plasmid instability in Lactococcus lactis . Appl. Environ. Microbiol. 59, 358–364 (1993) , , , &
- Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009) , , &
- Genomic organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing Streptococcus thermophilus strain. Appl. Environ. Microbiol. 71, 4057–4068 (2005) et al.
- Global gene expression analysis of two Streptococcus thermophilus bacteriophages using DNA microarray. Virology 340, 192–208 (2005) , , &
- 2001) & Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
- Morphological and genetic diversity of temperate phages in Clostridium difficile . Appl. Environ. Microbiol. 73, 7358–7366 (2007) &
- An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence. Appl. Environ. Microbiol. 65, 3800–3804 (1999) , , , &
- Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages. Appl. Environ. Microbiol. 68, 588–596 (2002) &
- Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein. Plasmid 43, 205–213 (2000) , , &
- Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623 (1988) , &
- Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686 (1988)
- Characterization of lactococcal bacteriophages from Québec cheese plants. Can. J. Microbiol. 38, 875–882 (1992) et al.
- Supplementary Information (506K)
This file contains Supplementary Figures 1-2 with legends and Supplementary Tables 1-3.