0, 10 mM 2-mercaptoethanol, 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% v/v Nonidet NP-40) and 3 μl 1 M CaCl2. The resulting solution was applied to a column containing 200 μl of Calmodulin-Sepharose beads (Stratagene) that had been washed with 10 ml of Calmodulin Binding Buffer. The column was then rotated for 1 h at 4°C. Elution was performed by gravity flow and the beads washed three times with 10 ml Calmodulin Binding Buffer. The bound proteins were eluted

with Calmodulin Elution Buffer (10 mM Tris-HCl pH 8.0, 10 mM 2-mercaptoethanol, 150 mM NaCl, 1 mM magnesium acetate, 1 mM imidazole, 2 mM EGTA, 0.1% v/v Nonidet NP-40) in 10×200 μl fractions. Proteins purifed as described from the equivalent of 15 l of original culture were

TCA precipitated and separated using a gradient of 4-12% (w/v) SDS-PAGE and silver stained. Two independent and equivalent experiments CHIR-99021 research buy were undertaken Distinctive bands were in-gel tryptic digested, this website and prepared for positive-ion MALDI mass spectra (Applied Byosystems 4700 Proteomics Analyzer), MS spectra were acquired, and the strongest peaks with a signal to noise greater than 40 were selected for CID-MS/MS analysis (Technology Facility, University of York). Mass spectral data were submitted to database searching using the MASCOT program (Matrix Science Ltd.) The Mowse scoring algorithm uses empirically determined factors to assign a statistical weight to each individual peptide match. The threshold level indicates that a match is significant if it would be expected to else occur at random

with a frequency of less than 5%. Therefore, individual ions with scores greater than the calculated threshold level indicate identity or extensive homology. Subcellular localisation S. aureus SH1000 (2 l culture) was grown to an OD600~3 and immediately transferred to an ice slurry for 10 min. Cells were harvested (6,000 rpm, 10 min, 4°C), broken using a Braun homogeniser, and the membrane/ribosome fraction purified by ultracentrifugation at 50,000 rpm for 2.5 h in a 70.1 Ti rotor (Beckman). The resulting pellet was resuspended in 7 ml of 0.01 M Tris-HCl pH 8.2, 14 mM magnesium acetate, 60 mM potassium acetate, 1 mM DTT containing 0.5% (v/v) Triton X-100 to solubilise membranes. The ultracentrifugation step was repeated and the pellet resuspended in 6 ml of the above buffer containing 1 M NH4Cl. The sample was ultracentrifuged again and the resulting pellet was resuspended in 5 ml of the above buffer. YsxC overexpression, purification and production of antisera A His(6)tagged version of YsxC was constructed by cloning the ysxC gene PCR-amplified from S. aureus SH1000 (using primers 5′elc4 and 3′elc4, and ReadyMix, ABgene) into the 3′-dA overhang site of the overexpression vector pETBlue-1 AccepTor vector (Novagen). The resulting plasmid (pELC4) was subsequently electroporated into E. coli TunerTM (DE3) pLacI (Novagen).