Bacterial strains and plasmids
Media and antibiotics
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
Measurement of persistence
For quantitative (phospho)proteomic experiments, E. coli cells were differentially labeled using the following stable isotope–labeled lysine derivatives: 4,4,5,5-D4 l-lysine (Lys4, medium-heavy lysine, K4, Cambridge Isotope Laboratories), 13C615N2 l-lysine (Lys8, heavy lysine, K8, Cambridge Isotope Laboratories), or l-lysine (Lys0, light lysine, K0, Sigma-Aldrich) (52). Both precultures and main cultures were grown in M9 minimal medium containing 0.0025% (w/v) Lys0, Lys4, or Lys8. The experimental design of all experiments is summarized in table S3.