Materials and Methods

Characterization of a Type II-A CRISPR-Cas System inStreptococcus mutans

MATERIALS AND METHODSStrain, phage, and culture conditions. S. mutans strain P42S and the lytic phage M102AD were obtained from the Félix d’Hérelle Reference Center for Bacterial Viruses (www.phage.ulaval.ca). The bacterial strain was grown in brain heart infusion (BHI) medium at 37°C with 5% CO2. For growth on plates, 1.25% agar was added to BHI medium. Phage M102AD was amplified using an exponentially growing culture of P42S. Phage-infected cultures were incubated at 37°C until lysis. The resulting lysate was then filtered (0.45 μm) and stored at 4°C until use. Phage titration was performed using the double-layer plaque assay, and the top agar consisted of BHI medium supplemented with 0.75% agar.Identification and analysis of the CRISPR-Cas system in S. mutans P42S. The cas genes and the CRISPR array sequences were obtained from the whole-genome sequence analysis of S. mutans P42S. The genomic DNA was first extracted as described elsewhere (52), with the following modifications. Briefly, the proteinase K and SDS steps were separated into a 15-min proteinase K (0.4 mg/ml) step, followed by a 2-h SDS (1%) step. After the potassium acetate step and subsequent centrifugation, the supernatant was treated with RNase A (2 μg/ml) for 1 h at 37°C. The protocol then was resumed with the isopropanol step as described previously (52). The genomic DNA of S. mutans P42S was prepared for sequencing using the Nextera XT DNA library preparation kit according to the manufacturer’s instructions. The library was sequenced on a MiSeq apparatus using a MiSeq reagent kit v2 (Illumina). Sequences were assembled into 18 contigs using Ray Assembler 2.3.0 (53) and fused using Mauve Assembly Metrics (54). CRISPR loci were identified by searching for repeat sequences as listed in the CRISPR database (https://crispr.i2bc.paris-saclay.fr/). The cas gene sequences from other S. mutans strains were obtained from NCBI and compared to the genome of S. mutans P42S. Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) was used to determine the percent identity between the DNA sequences and between the translated amino acid sequences.BIM assay. An overnight culture of S. mutans P42S was transferred (1%) to fresh BHI medium and grown to an optical density at 600 nm (OD600) of 0.3 to 0.5. Mixtures of 100 μl of the S. mutans P42S culture and 100 μl of phage M102AD lysate (titer between 107 and 109 PFU/ml) then were mixed in BHI top agar and poured directly onto solid medium. Plates were incubated at 37°C for 48 to 72 h, and surviving cells were analyzed for spacer acquisition by amplifying the CRISPR locus as described elsewhere (16). The primers CR-F (5′-AATGTCGTGACGAAAATTGG-3′) and CR-R (5′-GAAGTCATCGGAACGGTCAT-3′) were used to amplify the CRISPR locus found in S. mutans P42S. PCR products were sequenced with an ABI 3730xl analyzer at the Plateforme de Séquençage et de Génotypage des Génomes at the CHU of Québec City. BIM assays were also performed with UV-damaged phage lysates as described previously (19).Phage adsorption assay. An overnight culture of S. mutans P42S was transferred (1%) to fresh BHI medium and grown until an OD600 of 0.7. Phage M102AD (103 PFU) was added to 900 μl of this culture and allowed to adsorb for 15 min at 37°C. Cultures then were centrifuged for 1 min at maximum speed in a microcentrifuge, and the titer of the supernatant was determined to estimate the phage fraction that did not adsorb to the host cells.Plasmid interference assay. An approach similar to the one described by Serbanescu et al. was performed (30), except that we used plasmid pNZ123 (55). pNZ123 contains 2,497 bp, provides chloramphenicol resistance to the host cells, and is readily transformable in S. mutans. The 24 bp between the XhoI and EcoRI restriction sites (positions 149 to 173) were removed, and the resulting linearized plasmid (2,473 bp) was purified from an agarose gel using the QIAquick gel purification kit as described by the manufacturer. Various DNA inserts were ligated between the XhoI and EcoRI sites of the gel-purified plasmid (see below).A 30-bp protospacer sequence, targeted by one of the spacers already present in the CRISPR locus of the wild-type (WT) strain S. mutans P42S and flanked by the nucleotide sequence TAAAT (see below) at the 3′ end, was first cloned between the XhoI and EcoRI sites to generate pNZ123-sp1. The recombinant plasmid was confirmed by sequencing. pNZ123 and pNZ123-sp1 then were independently transformed (see below) into S. mutans P42S.Another 30-bp protospacer targeted in strain BIM1 was cloned between the XhoI and EcoRI sites of pNZ123. The cloned protospacer was flanked by 5 bp found downstream in the phage genome (positions 24306 to 24335) to generate pNZ123-sp2. pNZ123 and pNZ123-sp2 were independently transformed into S. mutans P42S BIM1.Finally, the activity of ectopically acquired spacers (see below) was assayed by a similar experiment. The 30-bp protospacer (positions 18743 to 18714) targeted in BIM2 and flanked by the 5 bp downstream was cloned between the XhoI and EcoRI sites of pNZ123 to generate pNZ123-sp3. pNZ123 and pNZ123-sp3 then were independently transformed into S. mutans P42S BIM2.Transformation of S. mutans. The plasmid constructs were transformed into S. mutans using natural competence (56) through the addition of the competence-stimulating peptide (CSP). The active form of this peptide has the following sequence: NH2-SGSLSTFFRLFNRSFTQA-COOH (57, 58). The first peptide batch was kindly provided by Céline Lévesque from the University of Toronto. All subsequent batches were ordered from Biomatik (www.biomatik.com). An overnight culture of S. mutans P42S was transferred to fresh BHI medium and grown at 37°C until the OD600 reached 0.1. Aliquots of 500 μl then were collected and 1 μg of plasmid DNA was added to them. Along with the plasmid construct, the CSP was added at a concentration of 1 μM to the growing culture (59). If no CSP was added, the quantity of plasmid DNA was increased to 10 μg. The cultures were incubated at 37°C and 5% CO2 for 2.5 h and spun down, and the cell pellets were resuspended in 100 μl of BHI. Samples were plated onto BHI agar plates supplemented with 10 μg/ml chloramphenicol. Plates were incubated at 37°C for 72 h.Determination of PAM sequence. Based on the CRISPR analysis of various S. mutans BIMs obtained after the challenge with the virulent phage M102AD, we identified several newly acquired spacers. The analysis of the sequences flanking the protospacers (26) in the genome of phage M102AD led to the identification of a preferred 5-bp PAM motif at the 3′ end of the protospacer. To determine the importance of each of these five base pairs, we designed a plasmid-based interference experiment as described above. Between the XhoI and EcoRI sites of pNZ123, one of the spacers already present in the CRISPR locus of S. mutans P42S was flanked by a 5-bp motif and several derivatives. The pNZ123-sp1 plasmid was used as a control, as it contains the protospacer flanked by the most commonly observed PAM (TAAAT). Other versions of the plasmid included one or two mismatches in the motif, as a nucleotide was replaced by one of the three other alternatives. All plasmids were transformed in duplicate into S. mutans P42S WT and derivatives, and their transformability was compared. The constructs and insert sequences are listed in Table S2 in the supplemental material.TABLE S2Construct insert sequences. Protospacer sequences with flanking PAM are in italics. Download Table S2, DOCX file, 0.02 MB.Copyright © 2020 Mosterd and Moineau.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Data availability. The complete genome sequence of phage M102AD was previously deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"DQ386162","term_id":"88698119","term_text":"DQ386162"}}DQ386162 (10). The sequences of the cas genes of S. mutans P42S are available in GenBank under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"MT008463","term_id":"1852011443","term_text":"MT008463"}}MT008463 (cas9), {"type":"entrez-nucleotide","attrs":{"text":"MT008464","term_id":"1852011464","term_text":"MT008464"}}MT008464 (cas1), {"type":"entrez-nucleotide","attrs":{"text":"MT008465","term_id":"1852011467","term_text":"MT008465"}}MT008465 (cas2), {"type":"entrez-nucleotide","attrs":{"text":"MT008466","term_id":"1852011469","term_text":"MT008466"}}MT008466 (csn2), and {"type":"entrez-nucleotide","attrs":{"text":"MT008467","term_id":"1852011471","term_text":"MT008467"}}MT008467 (tracrRNA).

Article TitleCharacterization of a Type II-A CRISPR-Cas System inStreptococcus mutans

Abstract

The complete genome sequence of phage M102AD was previously deposited in GenBank under accession number{"type":"entrez-nucleotide","attrs":{"text":"DQ386162","term_id":"88698119","term_text":"DQ386162"}}DQ386162(10). The sequences of thecasgenes ofS. mutansP42S are available in GenBank under accession numbers{"type":"entrez-nucleotide","attrs":{"text":"MT008463","term_id":"1852011443","term_text":"MT008463"}}MT008463(cas9),{"type":"entrez-nucleotide","attrs":{"text":"MT008464","term_id":"1852011464","term_text":"MT008464"}}MT008464(cas1),{"type":"entrez-nucleotide","attrs":{"text":"MT008465","term_id":"1852011467","term_text":"MT008465"}}MT008465(cas2),{"type":"entrez-nucleotide","attrs":{"text":"MT008466","term_id":"1852011469","term_text":"MT008466"}}MT008466(csn2), and{"type":"entrez-nucleotide","attrs":{"text":"MT008467","term_id":"1852011471","term_text":"MT008467"}}MT008467(tracrRNA).


Login or Signup to leave a comment
Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.