The cultures were then removed and the plates were washed twice with distilled water. The number of the adherent cells in an area of 1 mm2 was counted under an optic microscope (Olympus CKX41) to estimate the primary attachment ability of the bacteria in different mediums. The culture of S. aureus NCTC8325 was started
by diluting the overnight culture to an OD600 nm=0.05 and was then allowed to grow at 37 °C (200 r.p.m.) to exponential phase (OD600 nm=0.6). The cultures were then treated with 5 mM dithiothreitol, 10 mM cysteine or 20 mM BME, respectively, for 30 min. Staphylococcus aureus cells were predigested with digestion buffer containing 40 U mL−1 lysostaphin, 10 mg mL−1 lysozyme and 10% (v/v) glycerol. RNA extraction was performed using the SV Total RNA Isolation System (Promega). Residue DNA in extracted RNA was removed by treatment with 10 U of DNaseI (Takara) at 37 °C for an hour. Purified total RNA were qualified and quantified Selleckchem ERK inhibitor by a DU730 Nucleic LGK974 Acid/Protein Analyzer (Beckman Coulter). Reverse transcription of ica was carried out following the technical manual of ImProm-II Reverse Transcription System (Promega) with primer PicaD-R (TCACGATTCTCTTCCTCTC). Q-PCR was performed using StepOne Real-time System (Applied Biosystems) with primers PicaD-F (ATGGTCAAGCCCAGACAG) and PicaD-R. The crude extracellular matrix was prepared as described previously (Sadovskaya et al., 2005). Briefly, S. aureus
cells were grown in the respective medium at 37 °C with moderate shaking (50 r.p.m.) to reach an OD600 nm of 2.0. Bacterial cells were collected by centrifugation (3000 g), resuspended in phosphate-buffered saline (pH 7.4) and sonicated immediately on ice to extract the cell-associated extracellular material. Bacterial cells and insoluble material were removed by centrifugation (10 000 g). Then, the Elson–Morgan assay was performed to measure the amount of PIA in the supernatant after acidolysis as described previously
(Morgan & Elson, 1934). The cultures of S. aureus NCTC8325 were started by diluting the overnight culture to an OD600 nm=0.05 in TSB or TSB supplemented with 5 mM dithiothreitol and incubating at 37 °C (200 r.p.m.) for an additional 6 h. Total protein was purified Methane monooxygenase by trichloroacetic acid/ice-cold acetone precipitation as described previously (Resch et al., 2006). Protein sample (500 μg) was loaded in each 17-cm gradient gel strip in the pH range of 4–7 and separated by isoelectrofocusing (IEF) using a Protean IEF cell (Bio-Rad). Second-dimension electrophoresis was carried out on a sodium dodecyl sulfate polyacrylamide gel. The gels were stained with Coomassie G-250 and then scanned on a flatbed scanner. Images were analyzed with imagemaster 2d-platium 5.0 (Amersham), setting the threshold at 3.0-fold. Differential protein dots of interest were marked and 21 of them were excised manually and digested with trypsin (Worthington). The digests were analyzed by HPLC-ES-MS (MDLC-LTQ, Amersham).