Ribo-seq
Preinocula cultures of S. aureus 15981 strain were grown in 10 ml MH at 37C and 180 rpm overnight. Preinocula cultures were diluted to a final OD600 of 0.05 in 2L-Erlenmeyer flasks containing 400 ml of MH broth, which were incubated with agitation (200 rpm) at 22 and 37C until cells reached an early exponential phase (OD600nm = 0.4). For total RNA sequencing, we recovered 20 ml of cultures to extract total RNAs as previously described (Toledo-Arana et al., 2009). Total RNAs were sequenced at the Gene Expression Analysis Platform from IBPM-CNRS and fastq files were aligned to the S. aureus NCTC 8325 reference genome (NC_007795.1) using SeqMan NGen software from DNASTAR package. For ribosome profiling, two minutes before harvesting, chloramphenicol (1mM) was added to the cultures. Cell cultures were harvested by centrifugation for 10 min at 4,400 x g and 4C. Bacterial pellets were resuspended in 500 l of lysis buffer R (20 mM Tris-HCl pH 8.0, 100 mM NH4Cl, 10 mM MgCl2, 0.1% Nonidet p-40, 0.4% Triton X-100, 1mM Chloramphenicol, 100 U/ml DNase I) and lysed twice in Lysing Matrix B tubes for 40s at 6.0 m/s in a FastPrep-24 instrument (MP Biomedicals). Tubes were centrifuged for 15 min at 21,000 x g and 4C. Supernatants were recovered in 1.5 ml Eppendorf tubes and RNA concentrations were determined. To obtain ribosome footprints, 1000 U of Micrococcal Nuclease S7 (Roche) was added per 1 mg of RNA. Samples were incubated for 1 h at room temperature with rotation at 190 rpm. Reaction was quenched by the addition of EDTA to a final concentration 6 mM. A fraction of RNA samples were loaded to the linear sucrose gradients 5-50% prepared on the Gradient Master IP (Biocomp). Tubes were centrifuged at 260,000 x g (39000 rpm, SW41 Ti rotor, Beckman Coulter) for 3 h at 4C using a piston gradient fractionator (Biocomp) combined with a fraction collector (Gilson). Fractions containing 70S and 70S with protected mRNA were stored at -20C until need. Hot acid phenol-chloroform RNA extraction from monosomes and library preparations for RNA sequencing were carried out as previously described (Kohl et al., 2024). Raw fastq sequencing data were trimmed using cutadapt to remove Illumina adapters (Martin, 2011). To remove rRNAs sequences, trimmed fastq files were aligned to S. aureus ribosomal sequences using Bowtie v.1.2.2 (Langmead et al., 2009). The rRNA-filtered fastq files were then aligned to the S. aureus NCTC 8325 reference genome (NC_007795.1) using Bowtie v.1.2.2. The obtained SAM files were sequentially converted to BAM, bedgraph and BigWig files using samtools v1.9, stranded-coverage and bedGraphToBigWig programs, respectively (Danecek et al., 2021).