transferring the membranes into fresh PBS, they were scanned using the Odyssey Infrared Imaging System (LiCor Biosciences). As a cell lysis control, antibodies against cytoplasmic protein TrxA were used in the same concentration as PknB antibodies. Cloning, Expression and Purification of S. aureus TrxA All procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of competent E. coli DH5a cells were carried out as previously described [81]. Genomic DNA of S. aureus RN4220 [82] was isolated using the Genelute Bacterial Genomic DNA kit (Sigma). The trxA gene on this genomic DNA was PCR-amplified using the primers GGGGGCATATGGCAATCGTAAAAGTAA and GGGGGC- TCGAGTAAATGTTTATCTAAAACTTC. The PCR product was cloned into HincII (HindII) restricted pUC18 [83]. After verification of the sequence, the trxA gene was excised from this plasmid with NdeI and XhoI, and ligated into the same restriction sites of pET26b(+) (Novagen, Inc.), downstream of the T7 promoter and upstream of an in-frame His(6)tag sequence. The resulting pET26-SatrxA plasmid was checked by sequencing and used to transform E. coli BL21(DE3) (Invitrogen) for high-level trxA expression and purification. 10 ml of an overnight culture of this strain was used to inoculate 1 liter fresh LB medium and grown until an OD600 of 0.7 was reached. Then, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM to induce TrxA production. After 3 hrs of induction, cells were harvested by centrifugation and resuspended in binding buffer (20 mM NaPi, 300 mM NaCl, 10% (v/v) glycerol, 5 mM imidazole, 3 mM DTT, pH 7.4). Next, cells were disrupted by two passages through a French Press (2500 PSI). Cellular debris was removed by centrifugation (30 min at 30000 g, 4uC), and the clarified supernatant fraction was applied to a nickel-charged IMAC column (5 ml HisTrap HP, GE Healthcare). Unbound sample was washed from the column with binding buffer using an A¨KTA explorer (GE Healthcare). Next, the His-tagged TrxA protein was eluted from the column using binding buffer with a gradient of increasing imidazole concentrations (up to 500 mM imidazole). The eluted fractions were checked for the presence of pure TrxA protein using SDS-PAGE and subsequent silver-staining. Further purification was achieved by concentrating the proteins with Vivaspin columns (Vivascience) and loading them on a Superdex 75 gel filtration column (Amersham) pre-equilibrated with 20 mM NaPi, 150 mM NaCl, 10% glycerol and 3.5 mM DTT, pH 7.4. Fractions containing the purified TrxA proteins were pooled and dialyzed 3 times against 20 mM Tris-HCl, pH 7.6, with 150 mM NaCl. Specific polyclonal antibodies against the purified TrxA protein were raised in rabbits (Eurogentec). Kinome Array Analysis PepChipTM Kinomics slides containing 976 fully annotated, disease-related kinase phosphorylation sites in triplicate (Pepscan, Lelystad, The Netherlands, http://www.pepscan.com/) were incu- bated with 50 ml of the ser/thr kinase PknB incubation mix (0.8 mg/ ml PknB kinase domain, 60 mM HEPES, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 1 mM DTT, 50% glycerol, 50 mM ATP supple- mented with 1Mbq [c-33P] ATP, 0.03% Brij-35, 50 mg/ml bovine serum albumin, 3 mM Na3VO4, 50 mg/ml PEG 8000) for 90 min in a humidified incubator. As a negative control we used the ‘‘empty’’ incubation mix without PknB. After incubation the peptide arrays were washed twice in 2 M NaCl (1% Tween-20) and PBS-T. Next, the arrays were rinsed twice in demineralised water and air-dried. The dried slides were transferred to a phosphor imager plate (Fuji Storm 860, Stanford, GE, USA) and exposed for 72 hours. The density of the spots was measured and analyzed with array software. PknB Phosphorylation Profile Peptide Array Data Analysis To analyze the intensity of spots and to correct for background phosphorylation, the ScanAnalyze software and grid tools were used, and the resulting data were exported to an excel sheet. Three replicate data sets were taken for further statistical analysis. To this end, the Spearman correlation coefficient was calculated for each combination of the three sets. The average and standard deviation for each peptide were determined and plotted in an amplitude- based hierarchical fashion. If only background phosphorylation is present, this amplitude-based distribution can be described by a single exponent. Thus, determining the exponent describing amplitude behavior of the 500 least phosphorylated peptides should give an adequate description of array background phosphorylation and, in practice, this was indeed the case. 125 Peptides which exhibited the incorporation of c-33P in the absence of added kinase were excluded from further analysis. Peptides of which the average phosphorylation minus 1.96 times the standard deviation was higher as the value expected from describing the background distribution were considered to represent true phosphorylation events. Two-sided heteroscedastic t-tests were also performed on each set of values to determine significance (p,0.05). Sequence Logos Sequence logos were created with the weblogo server at http:// weblogo.berkeley.edu/logo.cgi using either the preferred sub- strates of PknB, or known phosphorylation sequences for human kinases as available at http://www.phosphosite.org/homeAction. do In Vitro Phosphorylation of ATF-2 by Staphylococcal PknB In order to confirm the phosphorylation of ATF-2 by PknB, the in vitro assay was performed. Purified staphylococcal kinase PknB (26 mg) was incubated with 50 mg of Activating Transcription Factor fusion protein (Cell Signaling) in kinase incubation mix (50 mM HEPES, 1 mM DTT, 0.01 Brij35, 3 mM MnCl2, 3 mM MgCl2, 50 mM ATP) for 30 minutes at 37uC in waterbath. As a positive control, 0.1 mg of p38-alpha MAP kinase (Cell Signaling) was incubated with 50 mg of ATF-2. As negative controls, the following reaction mixtures were used: PknB with ATF-2 but without ATP; PknB with ATP but without ATF-2; and ATF-2 with ATP but without PknB. Phosphorylation Site Identification and Protein Identification by Mass Spectrometry Trypsin (Promega) was activated by 15 min incubation at 30uC in activation buffer and then added 1:200 to the samples, digestion was allowed to proceed over night at 37uC. The resulting peptides were separated by liquid chromatography and measured online by ESI-mass spectrometry using a nanoACQUITY UPLCTM system (Waters, Milford, MA) coupled to an LTQ OrbitrapTM mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Peptides were desalted onto a trap column (SymmetryH C18, Waters). Elution was performed onto an analytical column (BEH130 C18, Waters) by a binary gradient of buffer A (0.1% (v/v) acetic acid) and B (100% (v/v) acetonitrile, 0.1% (v/v) acetic acid) over a period of 50 min with a flow rate of 400 nl/min. The LTQ Orbitrap was operated in data-dependent MS/MS mode using MSA for phospho-relevant masses. Proteins were identified by searching all MS/MS spectra in .dta format against all S. aureus NCTC 8325 proteins and added ATF-2 protein (extracted from the NCBI database) using SorcererTM-SEQUESTH (ThermoFin- PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9057