MATERIALS AND METHODSBacterial strains and growth conditionsCultivation of S. aureus RN4220 (42) and derivatives JW263 and JW418 (29), and S. aureus Newman (43) was carried out in brain-heart infusion (BHI) medium at 37°C. Media was supplemented with chloramphenicol at 10 μg/ml, erythromycin at 10 μg/ml, kanamycin at 25 μg/ml or spectinomycin at 250 μg/ml for plasmid maintenance. Media was supplemented with 1 mM IPTG to induce expression of genes under the control of the IPTG-inducible Pspank-hy promoter. See Supplementary Table S1 for a full list of bacterial strains and plasmids used in this study.Plasmid constructionThe plasmids used in this study are listed in Supplementary Table S1. The sequences of oligonucleotides used in this study are listed in Supplementary Table S2. The plasmid cloning strategies are listed in Supplementary Table S3.Inducible adaptation assay and amplification of newly acquired spacers by DR-PCR for high-throughput sequencingOvernight culture of the indicated S. aureus strains harboring pCas1-2 and pCRISPR/pDR were diluted 1:100 in 50 ml BHI and grown 1 h shaking at 37°C. An uninduced sample was obtained by pelleting 10 ml of the culture and removing supernatant. Pellets were kept at −80°C until all time points were collected. Then, freshly made IPTG was added to the remaining culture before it was placed back shaking at 37°C. Every hour, for 3–5 h post-induction, 10 ml of the culture was pelleted and frozen, OD600 was measured and calibrated back to ∼0.3 by adding the appropriate volume of BHI supplemented with 1 mM IPTG. The remaining of the culture was left shaking at 37°C overnight. The next day, 1.5 ml of the overnight culture was pelleted and frozen. Plasmids were isolated from S. aureus pellets with a modified QIAprep Spin Miniprep Kit (Qiagen) protocol: bacterial cell pellets were resuspended in 250 μl P1 buffer supplemented with lysostaphin at a final concentration of 107 μg/ml (AMBI Products) and incubated at 37°C for 15 min followed by the standard QIAprep protocol. 250 ng of each sample were used as input for DR-PCR, using the Phusion DNA Polymerase (Thermo) with primers NA101/NA102 or NA169/170 for type III-A or type II-A, respectively (see Supplementary Table S2 for a full list of primers used in this study). Primer annealing temperature was set to 64 and 54°C for type III-A and II-A, respectively, and extension time was 10 s. Both plasmid isolation and DR-PCR preparation were performed in a PCR-free room to avoid contaminations. After amplification, 5 ul of DR-PCR products were analyzed by agarose gel electrophoresis to verify successful amplification. The remaining sample underwent cleanup with the MinElute PCR Purification Kit (Qiagen) and size selection using PippnHT 3% cassette with a timed protocol set at extraction between 26 and 35 min. Size selected products were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina). For maintaining the small sized product, 2.2× Sample Purification Beads (Ilumina) were used after end repair. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.RNA sequencingOvernight culture of RN4220 was diluted 1:200 in 10 ml BHI and grown 1:10 h shaking at 37°C. Culture was then pelleted, and supernatant was removed. Pellets were resuspended in 100ul RNase free PBS supplemented with 100 μg/ml lysostaphin (AMBI Products), incubated for 5 min at 37°C, and sarkosyl was added at 1%. RNA was purified from lysed pellets using the Zymo Direct-Zol RNA miniprep plus kit, and genomic DNA was removed by Ambion Turbo DNA-free kit. For the rRNA-depleted sample, Illumina Ribo-Zero rRNA removal (Bacteria) kit was used to remove rRNA. Untreated and rRNA-depleted RNA samples were prepared for sequencing using the TruSeq Stranded mRNA Library prep kit, beginning at the RNA fragmentation step. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.High throughput sequencing data analysisSpacers were extracted from MiSeq FASTQ files using a Python code that finds all sequences flanked by two DR sequences. The sequences, location and abundance of all spacers detected in this study are provided in the Supplementary Data File spreadsheet. For generating Weblogos, a python code searching for fully aligned spacers gave an output of spacer sequence, strand, start and end positions, spacer length, 20 bp upstream and downstream of spacer, and number of reads for that unique spacer sequence. Then, spacers were filtered to length of 35 or 30 bp in type III-A or type II-A, respectively, and motif search was performed by WebLogo (version 3.7.4.)For generating genome alignment maps, all spacers were aligned to the indicated bacterial chromosome using bowtie2. Genome positions covered by aligned spacers were counted and aggregated using the Python pysam package (version 0.15.3). For spacers that aligned to more than one position (n > 1) counts were divided by number of aligned position (1/n). Genome was either divided to 10 or 1 kb bins, or analyzed at a single nucleotide resolution, as indicated in each figure legend. RPM values were calculated as chromosomal reads within a bin per million total aligned reads. RNA-seq sequences underwent the same pipeline of analysis.
Article TitleDifferent modes of spacer acquisition by theStaphylococcus epidermidistype III-A CRISPR-Cas system
Datasets generated from Illumina sequencing has been made publicly available at NCBI, BioProject accession number PRJNA769698. The authors declare that all other data supporting the findings of this study are available within the article and itsSupplementary Information files, or are available from the authors upon request.