Luria broth lb

The wild type strain used in this study was L. monocytogenes serotype 1/2c strain LO28 (27 (link)). Isogenic mutant derivatives of this strain were constructed as described previously (24 (link)). Primers used for in-frame deletions are listed in Supplementary Table S1. All strains used in the study are listed in Supplementary Table S2. In order to grow L. monocytogenes strains, overnight cultures were diluted 100-fold into Brain Heart Infusion medium (BHI, Oxoid) and vigorously shaken at 37°C; when appropriate, kanamycin (50 μg/ml) was added. For northern blot experiments, the following concentrations were used to induce cell envelope stress: 4 μg/ml cefuroxime (Sigma-Aldrich), 0.03% bile salts (DIFCO, No. 3), 4% NaCl, pH5 adjusted with HCl and 2% ethanol. In stress tolerance assays overnight cultures were diluted 1000-fold into BHI adjusted with cefuroxime (4 μg/ml), bile salts (0.07%), NaCl (8%), pH5 and ethanol (5%); growth was monitored for 24 h.
For cloning purposes and green fluorescent protein (GFP) reporter experiments, Escherichia coli TOP10 and E. coli DH5α (Invitrogen) were used and grown in Luria Broth (LB, Oxoid) at 37°C. When appropriate, LB was supplemented with kanamycin (50 μg/ml), ampicillin (30 μg/ml), chloramphenicol (25 μg/ml) or erythromycin (150 μg/ml).

MRSA isolates relating to staphylococcal bacteremia cases were identified and collected from the University Hospitals of Leicester (UHL) archive and stored in tryptic soy broth (TSB; BD Diagnostics Systems) with 20% (vol/vol) glycerol at −80°C. Unless otherwise stated, isolates were cultured on Luria agar (LA; Oxoid) at 37°C in air followed by Luria broth (LB; Oxoid) and were incubated with shaking at 37°C. For nutrient-restrictive conditions, 6% horse blood agar (HBA; Oxoid) was used and strains were cultured at 37°C in 5% CO₂ followed by CRPMI medium (CRPMI medium is RPMI 1640 medium [Sigma-Aldrich] depleted of metal ions via treatment with 6% [wt/vol] Chelex 100 [Sigma-Aldrich] but with 10% [vol/vol] untreated RPMI 1640 medium reapplied to provide the minimum elements required for growth) (23 (link)) cultured statically at 37°C in 5% CO₂.

S. aureus strains, SH1000 and SH1000 Δfnb, were kindly provided by Simon Foster (University of Sheffield, Sheffield, United Kingdom). MRSA isolates were retrieved from skin infections of patients at Northern General Hospital, Sheffield, and kindly provided by Sue Whittaker. Liquid cultures were grown in Luria broth (LB; Oxoid, Ltd., Basingstoke, United Kingdom) microaerobically at 37°C in a humidified atmosphere with constant agitation. OD_600nm readings were taken using a Varioskan LUX microplate reader (ThermoFisher Scientific, Waltham, MA, USA). Solid cultures were grown on LB agar (Oxoid) overnight at 37°C. Freshly grown plates were used to inoculate all liquid cultures.