The CRISPR-Cas immune system and genetic transfers : reaching an equilibrium

Horizontal gene transfer drives the evolution of bacterial genomes, including the adaptation to changing environmental conditions. Exogenous DNA can enter a bacterial cell through transformation (free DNA or plasmids) or through the transfer of mobile genetic elements by conjugation (plasmids) and transduction (bacteriophages). Favorable genes can be acquired, but undesirable traits can also be inadvertently acquired through these processes. Bacteria have systems, such as clustered regularly interspaced short palindromic repeat CRISPR–associated genes (CRISPR-Cas), that can cleave foreign nucleic acid molecules. In this review, we discuss recent advances in understanding CRISPR-Cas system activity against mobile genetic element transfer through transformation and conjugation. We also highlight how CRISPR-Cas systems influence bacterial evolution and how CRISPR-Cas components affect plasmid replication

CRISPR-Cas and restriction–modification systems are compatible and increase phage resistance

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

Phage-host interactions in Streptococcus thermophilus : genome analysis of phages isolated in Uruguay and ectopic spacer acquisition in CRISPR array

Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5′-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array

Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos- and pac-Type Streptococcus thermophilus Bacteriophages▿ †

The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described

Cleavage of Phage DNA by the <em>Streptococcus thermophilus</em> CRISPR3-Cas System

<div><p><em>Streptococcus thermophilus</em>, similar to other <em>Bacteria</em> and <em>Archaea</em>, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (<u>C</u>lustered <u>R</u>egularly <u>I</u>nterspaced <u>S</u>hort <u>P</u>alindromic <u>R</u>epeats loci coupled to <u>C</u>RISPR-<u>as</u>sociated genes). Two <em>S. thermophilus</em> CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the <em>S. thermophilus</em> CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.</p> </div

Determination of the cleavage site in 2972 phage genome by the BIM S79 CRISPR3-Cas system.

<p>Nucleotide positions of the phage genome sequence are given for each fragment and the CRISPR3 PAM is underlined. <b>A</b>. Phage 2972 genome sequence showing the protospacer PS79 and surrounding regions. The protospacer’s nucleotide positions (1^st, 27^th, and 30^th) are indicated by a vertical black line to highlight the distal dsDNA cleavage position occurring after the 27^th nucleotide, at the extremity near the PAM (5′-N<u>GG</u>N<u>G</u>-3′). <b>B</b>. Sequencing chromatograms of inverse PCR reactions following intramolecular ligation of the 3′-PS79 end, produced by CRISPR3 cleavage, and the blunt–end, produced by DraI restriction. <b>C</b>. Sequencing chromatograms of inverse PCR reactions following intramolecular ligation of the 5′-PS79 end, produced by CRISPR3 cleavage, and the blunt–end, produced by SspI restriction. In both B and C, the joining point corresponding to the ligation of both extremities is indicated.</p

Simultaneous cleavage of phage DNA by CRISPR1-Cas and CRISPR3-Cas systems of <i>S. thermophilus</i> BIMs.

<p><b>A</b>. Representation of the phage 2972 genome. Arrows symbolize the open reading frames (ORFs) as identified previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Delorme1" target="_blank">[18]</a> and colors indicate associated transcriptional modules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Khan1" target="_blank">[24]</a>. Three protospacers (PS61, PS78 and PS85) are positioned below the genome and the CRISPR-Cas systems that have acquired the respective spacers are specified (CRISPR1 or CRISPR3). <b>B</b>. The fragment sizes obtained from restriction and/or CRISPR digestion are specified. Probes used in the Southern blot assays are also indicated and details of the primers used are presented in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913.s001" target="_blank">Table S1</a>. <b>C</b>. Comparison by Southern blot assays of CRISPR-Cas cleavage occurring in the two selected BIMs S61/S78, and S61/S85, which share the S61 spacer (CRISPR3), but not a second spacer (CRISPR3 and CRISPR1, respectively). The Southern profiles show that independent cleavage of CRISPR1-Cas (PS85) and CRISPR3-Cas (PS61 and PS78) systems can occur simultaneously, when infected by phage 2972. Corresponding cleavage bands are identified with asterisks, and their sizes are specified. The two BIMs were infected with phage 2972 for 45 minutes before total DNA was extracted. Five micrograms of SpeI-restricted DNA was then loaded in each lane. NI, non-infected strain. C+, 10 ng of 2972 phage DNA digested with SpeI.</p

<i>S. thermophilus</i> BIMs used in the study and details on cleavage and EOP.

a<p>The other names are used in the text to help the reading.</p>b<p>BIM S4 was characterized elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Deveau2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Garneau1" target="_blank">[15]</a> and used here as a control.</p>c<p>Experimentally proved cleavage site are symbolized with plain arrows, previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Deveau2" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040913#pone.0040913-Garneau1" target="_blank">[15]</a> cleavage sites are represented by clear arrows, whereas not experimentally determined cleavage sites are represented by grey arrows.</p>d<p>PAMs are 5′-NN<u>AGAAW</u>-3′ and 5′-N<u>GG</u>N<u>G</u>-3′ for CRISPR1 and CRISPR3 protospacers, respectively. Consensus nucleotides are underlined.</p>e<p>EOPs are the mean of at least three independent assays.</p

Architecture of CRISPR1-Cas and CRISPR3-Cas systems of <i>S. thermophilus</i> DGCC7710.

<p><b>A</b>. CRISPR loci are represented by grey boxes. The percentages of amino acid identity between the Cas protein sequences are indicated in the grey shading. The percentages of identity were calculated by dividing the number of identical residues per the length of the alignment (i.e., the higher protein size). <b>B</b>. Csn2 amino acid alignment of CRISPR1-Cas and CRISPR3-Cas systems. Bars, dots, and hyphens emphase identical amino acids and gaps, respectively. Numbers at the right side are the positions on the related protein sequence. EMBOSS Needle (from the European Bioinformatics Institute) was used to align protein sequences (<a href="http://www.ebi.ac.uk/Tools/psa/emboss_needle/" target="_blank">http://www.ebi.ac.uk/Tools/psa/emboss_needle/</a>).</p

Characteristics of <i>S. thermophilus</i> DGCC7710 BIMs.

*<p>Only in CRISPR1.</p