Activation of the transcriptional regulator SlyA by the PhoP/PhoQ two-component system controls intracellular expression of numerous factors influencing Salmonella virulence. By dissecting the SlyA regulon using stable isotope labeling with amino acids in cell culture analysis, we found that SlyA enhances overall transcription of PhoP-activated loci. This amplification of cellular responses to Mg2+ occurs when SlyA binds to the phoPQ promoter thereby activating phoP autoregulation via a positive feedback mechanism. SlyA footprints a DNA region located one helical turn upstream of the PhoP box, which overlaps the H-NS-binding motif required for signal-dependent phoP repression in high Mg2+ conditions. Therefore, binding of SlyA likely antagonizes H-NS and facilitates the interaction of PhoP to its own promoter, subsequently activating the phoPQ operon. Establishment of this regulatory circuit allows SlyA to exert its effect on the PhoP/PhoQ system specifically in Salmonella, which may confer an additional transcriptional regulation. Thus, our results provide a molecular mechanism that determines SlyA-dependent activation of PhoP-regulated genes in modulating Salmonella virulence. Evidence from this study also suggests a function of SlyA as a mediator in signal transduction from the PhoP/PhoQ system to other bacterial two-component systems in Salmonella. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.