The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence. IMPORTANCE Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues. These amino acid residues were crucial for PhoP phosphorylation or dimerization, DNA-binding ability of PhoP, and Salmonella virulence. Interestingly, methylation negatively regulated the activity of PhoP. A bacterial chemotaxis methyltransferase CheR was involved in PhoP methylation. Methylation of PhoP could stabilize it in an inactive conformation. Our work provides a more informative depiction of PhoP PTM and markedly improves our understanding of the coordinate regulation of bacterial chemotaxis and virulence.
