0 was suspended in 0.8 ml of 50 mM Tris-HCl (pH 6.8). A sample of 15 μl of the protein extracts was analysed

on NuPAGE® 4-12% Bis-Tris gels (Invitrogen) check details using the X Cell SureLock® Mini-Cell system (Invitrogen) as recommended by the supplier. The gels were Coomassie stained using GelCode® Blue Stain Reagent (Pierce). DNA-binding analysis Gel retardation analysis were performed as described by Nan et al by mixing 100 ng of plasmid DNA (pBluescript II SK+(Stratagene)) with increasing amounts of peptide in 20 μl binding buffer (5% glycerol, 10 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 20 mM KCL and 50 μg ml-1 bovine serum albumin) [28]. Reaction mixtures were incubated 1 h at room temperature and subjected Selleck AZD8931 to 1% agarose gel electrophoresis and visualised using ethidium bromide. Transposon library in L. monocytogenes and S. aureus Transposon mutagenesis of L. monocytogenes 4446 was performed with the temperature-sensitive plasmid pLTV1 as described, but with modifications [29]. L. monocytogenes 4446 harbouring pLTV1 was grown overnight

at 30°C in BHI containing 5 μg/ml erythromycin. The bacterial culture was then diluted 1:200 in BHI containing 5 μg/ml erythromycin and grown for 6 h at 42°C. Aliquots were plated onto BHI containing 5 μg/ml erythromycin GW3965 clinical trial plates and incubated at 42°C. Colonies were harvested from the plates in BHI and stored in 30% glycerol at -80°C. To determine the transposition frequency, the transposon library was plated onto BHI containing 5 μg/ml erythromycin. One hundred colonies were picked and streaked

onto BHI plates containing 5 μg/ml erythromycin, 10 μg/ml chloramphenicol, and 12.5 μg/ml tetracycline, respectively, and mafosfamide incubated at 30°C for 48 h. The transposition frequency was calculated as the percentage of colonies growing only on BHI + 5 μg/ml erythromycin and BHI+10 μg/ml chloramphenicol (harbouring only the transposon) but not on BHI+12.5 μg/ml tetracycline (still harbouring the plasmid). Transposon mutagenesis of S. aureus 8325-4 with bursa aurealis was performed as described [30]. Screening of transposon library for plectasin resistant mutants The transposon mutant libraries were screened on agar plates for increased resistance to plectasin as compared to wild-type sensitivity. Wild-type sensitivity was determined by plating approx. 1.0 × 107 CFU/ml on TSB agar containing plectasin (S. aureus) and approx. 1.0 × 105 CFU/ml on Muller Hinton Broth agar plates (MHB, 212322 Becton Dickinson) with plectasin (L. monocytogenes). Plates were incubated at 37°C for 3 days and inspected for growth. The transposon libraries were screened on TSB agar with 300 μg/ml, 500 or 750 μg/ml plectasin (S. aureus) or MHB plates with 250 μg/ml or 500 μg/ml plectasin (L. monocytogenes) at 37°C for up to 7 days. Identification of transposon mutant Chromosomal DNA was purified from resistant mutants using FAST DNA kit, Bio101, Qiagen, Germany).