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
Measurement of persistence
It is known that ΔrelA mutant exhibits relaxed phenotype when grown in the presence of single carbon amino acids (SMG) (31). E. coli MG1555, rplK S102A, rplK S102D, and ΔrelA deletion mutant (table S1) were grown in M9 medium with glucose for 24 hours to stationary phase. Aliquots of cells were serially diluted in M9 medium, plated on M9 agar plates supplemented with or without 1 mM amino acids SMG, and grown for 40 hours at 37°C.