Pathogens encounter extreme changes in pH during the course of infection. For example acidic pH in the “Salmonella-containing vacuole” or SCV is considered to be a major inducing signal for stimulating expression of Salmonella pathogenicity island 2 (SPI-2) genes. We set out to determine the consequences to Salmonella during growth in the SCV. A recently developed DNA nanomachine (I-switch) used fluorescence resonance energy transfer to map spatiotemporal pH changes during endosomal maturation in eukaryotes (1). This high performance pH sensor (pH range 5.5 – 7) is equally efficient in vitro and in the cytoplasm. We employed the I–switch first with E. coli in order to measure intracellular pH changes in response to osmotic signalling. The donor/acceptor ratio (D/A) of the in vitro FRET curve showed perfect agreement with the intracellular standard curve. The intracellular pH decreased ~ 1 unit (from 7.08 to 6.05) upon 400 mM salt stress, whereas the osmotic regulator mutant strain ∆ompR showed < 0.4 pH unit decrease, indicating that OmpR is a major regulator of osmolality in E. coli. In Salmonella enterica serovarTyphimurium, a similar pH decrease ~ 1 unit (from 6.78 to 5.75) was observed upon salt stress, but the ∆ompR strain exhibited an even lower response to osmotic stress than in E. coli. We then used the I-switch to measure the internal pH of Salmonella in the macrophage vacuole during systemic infection. Salmonella intracellular pH decreased ~ 1 unit (from 6.78 to 5.80) that was apparent twenty minutes after infection. Our study represents a novel application of an autonomous DNA nanomachine and indicates that the trigger of virulence genes in Salmonella upon entry inside macrophages likely results from changes in intracellular pH. Supported by Mechanobiology Institute, NUS and VA 5I01BX000372 to LJK.1