Bacterial strains, plasmids, and primers are listed in Supplementary Tables S1–S3 . For cloning and genetic manipulation, E. coli was cultivated in LB medium. S. aureus strains with the pRB473-XylR-Brx-roGFP2 plasmids were cultivated in LB medium with 1% xylose to ensure constitutive expression of the biosensor. For stress experiments, S. aureus cells were grown in LB until an optical density at 540 nm (OD540) of 1.0 and were transferred to Belitsky minimal medium (BMM) with 1% xylose. The fully reduced control cells were treated with 10 mM DTT and the fully oxidized control was treated with 5 mM diamide for 20 min each, harvested with 10 mM N-ethylmaleimide (NEM) to block the biosensor redox state, and transferred to the microplate wells. The samples for stress exposure were transferred to the microplates, and different oxidants were injected into the wells of microplates. The Brx-roGFP2 biosensor fluorescence emission was measured at 510 nm after excitation at 405 and 488 nm using the CLARIOstar microplate reader (BMG Labtech) as described next for the in vitro measurements. Three biological tests were performed for each stress experiment.
For the survival assays, S. aureus COL wild type and the bshA mutant were treated with NaOCl and H2O2 at an OD500 of 1.0 in BMM and serial dilutions were plated on LB agar plates and counted for colony-forming units. The survival assays were performed in three biological replicates for each strain.
For the determination of the growth-inhibitory and sub-lethal antibiotics concentrations, S. aureus was cultivated in RPMI medium and the antibiotics erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, or oxacillin were added at an OD500 of 0.5 to monitor the reduction in growth as previously described (8 (link)). The measurements of the biosensor responses after antibiotics treatment were performed in S. aureus Brx-roGFP2 cells that were grown in RPMI medium and treated with sub-lethal antibiotics doses that reduced the growth rate. Cells were harvested after different times of antibiotics treatment, washed with phosphate-buffered saline (PBS), and blocked with NEM before the microplate reader measurements. Four biological replicates were performed for each antibiotics stress experiment. Sodium hypochlorite, diamide, DTT, H2O2 (35% w/v), and antibiotics (erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, and oxacillin) were purchased from Sigma-Aldrich.
For the survival assays, S. aureus COL wild type and the bshA mutant were treated with NaOCl and H2O2 at an OD500 of 1.0 in BMM and serial dilutions were plated on LB agar plates and counted for colony-forming units. The survival assays were performed in three biological replicates for each strain.
For the determination of the growth-inhibitory and sub-lethal antibiotics concentrations, S. aureus was cultivated in RPMI medium and the antibiotics erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, or oxacillin were added at an OD500 of 0.5 to monitor the reduction in growth as previously described (8 (link)). The measurements of the biosensor responses after antibiotics treatment were performed in S. aureus Brx-roGFP2 cells that were grown in RPMI medium and treated with sub-lethal antibiotics doses that reduced the growth rate. Cells were harvested after different times of antibiotics treatment, washed with phosphate-buffered saline (PBS), and blocked with NEM before the microplate reader measurements. Four biological replicates were performed for each antibiotics stress experiment. Sodium hypochlorite, diamide, DTT, H2O2 (35% w/v), and antibiotics (erythromycin, rifampicin, vancomycin, ciprofloxacin, gentamicin, ampicillin, fosfomycin, lincomycin, linezolid, and oxacillin) were purchased from Sigma-Aldrich.
