Fig 7. Phosphorylation of RbsR1 on residues T175, T209 and RsbS on residue S56 requires the kinase RsbT.

Western blots probed with (A) anti-RsbR1 and (B) anti-RsbS antibodies. Wild type and all mutant strains were grown to mid-log phase at 37°C in BHI and then untreated (-) or treated (+) at pH 5.0 for 15 min. Total protein extracted was separated in Phos-tag gels. The lower bands correspond to non-phosphorylated isoforms of RsbR1 or RsbS in the respective blots. The middle bands in anti-RsbR1 blots correspond to mono-phosphorylated RsbR1 at the T209 and the higher band corresponds to mono-phosphorylated RsbR1 at T175. The higher band on the RsbS blot corresponds to RsbS phosphorylated on S56. The ΔrsbR1 mutant extracts were included as negative controls since RsbR1 and RsbS are not expressed in this strain.