Expanded view of the CRISPR/Cas locus of pKLM/pLN, pF8E, pG6, and pG6::pRS4R.

<p>View of <i>cas</i> genes and CRISPR arrays correlated to phage resistance phenotype. pKLM/pLN contains the full spacer array which gives resistance to all 4 listed phages. pF8E contains an insertion within the array which disrupts spacers s1-s9 as well as resistance to phage encoded by those spacers. pG6 contains a deletion of spacers s2-s9, and resistance to phage encoded by those spacers is lost. pG6::pRS4R contains an engineered s4 which provides resistance to all 3 phages it has identity to.</p

Plasmid profiles.

<p>Plasmid profile of donor <i>L. lactis</i> DGCC7167 + pGh9::IS<i>S1</i>, phage resistant transconjugants K (derived from recipient <i>L. lactis</i> 1403S) and KLM (derived from recipient LM2345).</p

Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in <em>Lactococcus lactis</em>

<div><p><em>Lactococcus lactis</em> is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, <em>L. lactis</em> has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial <em>L. lactis</em> strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in <em>L. lactis</em>. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.</p> </div

List and nucleotide sequence of pKLM/pLN CRISPR spacers.

<p>nt = nucleotides.</p><p>Underlined nucleotides indicate mismatches against respective proto-spacer target.</p>*<p>A single nucleotide gap denoted by an underscore space in sequence.</p

List of bacterial strains, plasmids, and bacteriophages.

<p>(+) = positive phenotype/(−) = negative phenotype/(R) = resistant.</p><p>Lac = lactose fermentation/Sm = streptomycin/Em = erythromycin.</p><p>Cm = chloramphenicol/Sp = spectinomycin/Rf = rifampicin.</p><p>Km = kanamycin (<i>E. coli</i> only)/Ap = ampicillin (<i>E. coli</i> only).</p><p>TS ori = temperature sensitive origin of replication.</p><p>Tra = conjugative transfer.</p><p>Repeat = lactococcal CRISPR repeat sequence.</p

pKLM map and plasmid comparison.

<p>Rings from inside out: pKLM ORFs; pKLM GC content; nucleotide identity with pLN; nucleotide identity with pMRC01.</p

Escape phage analysis.

<p>Alignment of pKLM/pLN spacer s3 with the corresponding proto-spacer region in 73 M5952 escape phage isolates grouped A - I. (Bold denotes single nucleotide mutation).</p