aureus Newman (accession number NC_009641) was performed using pKOR1 [23] yielding single mutants CQ33, CQ65 and CQ66, respectively. Correct deletion was confirmed by PCR and by sequencing. Furthermore, strain stability was confirmed by pulsed field gel electrophoresis of total genome

SmaI digests [55]. To complement the secDF mutant, secDF with its LY3039478 endogenous promoter was amplified from S. aureus strain Newman with primers listed in additional file 2 table S1. The amplified region was ligated into the SalI/BamHI restriction sites of pCN34, a low copy (20-25 copies/cell) E. coli-S. aureus shuttle vector [56]. The junction region was sequenced as a control. The resulting Salubrinal nmr plasmid pCQ27 was electroporated into RN4220 with subsequent transduction into the strains of interest. To construct MRSA PRN1371 cell line strains, the plasmid pME2, containing the mecA promoter and gene from strain COLn [28], was either electroporated or transduced into the strains selected. Promoter predictions were performed by BPROM http://​linux1.​softberry.​com/​berry.​phtml. Rho-independent transcriptional terminators were retrieved from the CMR terminator list http://​cmr.​jcvi.​org/​tigr-scripts/​CMR/​CmrHomePage.​cgi. Transmission electron microscopy (TEM) Cells were grown to exponential phase, harvested at OD600 0.5 and fixed for one hour in 2.5% glutaraldehyde in phosphate buffered saline

(PBS) pH 7.4. Electron microscopy was performed by the Center for Microscopy and Image Analysis, University of Zurich. Resistance profiles For qualitative susceptibility comparisons, bacterial suspensions of McFarland 0.5 were swapped across LB agar plates containing antibiotic gradients and incubated at 35°C for 20-24 h. Glycopeptides were tested on Brain Heart Infusion (BHI) (Difco) agar with a bacterial suspension of McFarland 2 [57]. Spontaneous and Triton X-100 induced autolysis Cells were grown to an OD600

of 0.7, pelleted by centrifugation and washed with 0.85% NaCl. The cells were then resuspended in 0.01 M Na-phosphate buffer pH 7 and the OD600 was adjusted to 0.7. After splitting the cultures, 0.01% Triton X-100 (Fluka) or an equal Neratinib clinical trial volume of PBS pH 7 was added. Cultures were incubated at 37°C and the decrease of OD600 was measured. Zymographic analyses Cultures were grown to an OD600 = 0.7, centrifuged and the filtered supernatants (pore size 0.45 μm, TPP) stored at – 20°C until further use. The cell wall peptidoglycan was digested in SMM buffer (0.5 M sucrose, 0.02 M maleate, 0.02 MgCl2 pH 6.5) supplemented with 72 μg/ml lysostaphin and 2 mM phenylmethylsulfonyl fluoride (PMSF) [38]. Cell wall containing supernatant was separated from the protoplasts and stored at – 20°C until further use. Protein concentrations were measured by Bradford assay (BioRad).