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.

Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction-modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.

CRISPR-Cas systems provide immunity in bacteria and archaea against nucleic acids in the form of viral genomes and plasmids, and influence their coevolution. The first main step of CRISPR-Cas activity is the immune adaptation through spacer(s) acquisition into an active CRISPR locus. This step is also mandatory for the final stage of CRISPR-Cas activity, namely interference. This chapter describes general procedures for studying the CRISPR adaptation step, accomplished by producing bacteriophage-insensitive mutants (BIMs) or plasmid-interfering mutants (PIMs) using various spacer acquisition analyses and experiments. Since each bacterial or archaeal species (and even strain) needs specific conditions to optimize the acquisition process, the protocols described below should be thought of as general guidelines and may not be applicable universally, without modification. Because Streptococcus thermophilus was used as the model system in the first published study on novel spacer acquisition and in many studies ever since, the protocols in this chapter describe specific conditions, media, and buffers that have been used with this microorganism. Details for other species will be given when possible, but readers should first evaluate the best growth and storage conditions for each bacterium-foreign element pair (named the procedure settings) and bear in mind the specificity and variability of CRISPR-Cas types and subtypes. Also, we suggest to be mindful of the fact that some CRISPR-Cas systems are not "naturally" active in terms of the ability to acquire novel CRISPR spacers, and that some systems may require specific conditions to induce the CRISPR-Cas activity for spacer acquisition.