Bacterial strains and plasmids
Media and antibiotics
SILAC labeling
Cell lysis and protein extraction
Protein digestion in solution
Phosphopeptide enrichment
High-pH reversed-phase peptide fractionation on commercial spin columns
Offline high-pH reversed-phase peptide fractionation
SDS–polyacrylamide gel electrophoresis and in-gel digestion
Incorporation and mixing check
Protein purification by StageTips
LC-MS/MS measurement
MS data processing and analysis
Protein production for His-tag affinity purification
His-tag affinity purification
In vitro kinase assay measured by MS
Dynamics of HipA and HipA7 in vitro (auto)phosphorylation measured by autoradiography
Determination of cell viability on SMG plates
Cells were grown in 20 ml of M9 medium with glycerol containing chloramphenicol (25 μg/ml) and ampicillin (25 μg/ml) to the exponential phase. At OD600nm of around 0.4, hipA was induced with 0.2% arabinose for 95 min. Cultures were then treated with ciprofloxacin (2 μg/ml; Sigma-Aldrich) for 5 hours. For determination of colony-forming units (CFU), 1 ml of aliquots was taken before arabinose addition, 95 min after hipA induction, and 5 hours of ciprofloxacin treatment. Cells were washed with phosphate-buffered saline, serially diluted, plated on LB agar plates containing 0.4% glucose, and grown for 24 to 40 hours at 37°C. Persistence was calculated by dividing the number of CFU/ml of ciprofloxacin-treated culture with the CFU/ml of the culture before antibiotic addition and presented as a frequency of surviving (persister) cells in log10 scale.