Core CWSS genes include: murZ (MurA isozyme), involved in the early steps of cell wall biosynthesis [10]; pbp2 and sgtB, involved in transglycosylation; and fmtA, a penicillin binding protein with low affinity to β-lactams [3, 11, 12]. Therefore activation of the CWSS is predicted to enhance cell wall synthesis [2]. This is substantiated by the identification of clinical isolates with

point mutations in the vraSR operon that lead to increased basal expression of the CWSS in the absence of inducing agents, with PD-1/PD-L1 Inhibitor 3 clinical trial the resulting phenotypes including thickened cell walls and increased levels of glycopeptide and ß-lactam resistance [13, 14]. The VraSR system of S. aureus has been found to be induced by a much wider range of cell wall active antibiotics than the homologous LiaRS systems of Bacillus subtilis and Streptococcus mutans, which are only induced by lipid II-interacting antibiotics and not by those that inhibit the earlier or later stages of cell wall synthesis [15–18]. However,

the sizes and compositions of VraSR regulons reported so far vary quite extensively and appear to be heavily dependent upon the strains and experimental procedures used. Huge variations in levels of CWSS gene induction were found not only to be dependent upon the types of antibiotic used but also on the antibiotic concentrations [2, 19, 20]. In this study we created a highly sensitive reporter gene construct CA4P datasheet to indirectly measure the kinetics of VraSR-dependent signal transduction in the presence of antibiotic concentrations ranging from sub- to supra- minimum inhibitory concentrations (MIC), for a selection of antibiotics with 4SC-202 supplier different cell envelope targets (Figure 1). This allowed us to compare maximal induction capacities and BCKDHA determine optimal conditions, including concentrations and exposure times, for measuring CWSS induction by different

antibiotics. Methods Bacterial strains and growth conditions The strains and plasmids used in this study are listed in Table 1. Bacteria were grown at 37°C in Luria Bertani (LB) broth (Difco Laboratories), shaking at 180 rpm with a 1:5 culture to air ratio, or on LB agar plates. All optical density (OD) measurements given were taken at OD600 nm. Media were supplemented with the following antibiotics when appropriate: 10 μg/ml tetracycline (Sigma), 10 μg/ml chloramphenicol (Sigma), 100 μg/ml ampicillin (Sigma) or 200 ng/ml anhydrotetracycline (Vetranal). Strains were stored at -80°C in skim milk. Table 1 Strains and plasmids Strain/plasmid Relevant genotype a Reference/source S. aureus RN4220 Restriction-negative derivative of NCTC8325-4 [48] BB255 NCTC8325 derivative, cured of plasmid pI524 [49] BB255ΔVraR BB255 containing vraR mutation, truncating VraR after the 2nd amino acid This study E.