Salmonella enterica serovar Typhimurium must adapt to rapid environmental shifts, including those encountered upon entry and during replication to survive within macrophages during pathogenesis. Despite extensive RNA-seq-based investigations, questions remain regarding the range, timing and magnitude of response dynamics. Here we constructed a comprehensive GFP-reporter strain library representing 2,901 computationally identified Salmonella promoter regions to study time-resolved Salmonella transcriptional responses. Promoter activity was measured during in vitro growth and during intracellular infection of RAW 264.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in SPI2-related promoter activity. We also discovered previously unidentified transcriptional activity from 234 promoters. These analyses revealed metabolic shifts including requirements for mntS expression to support manganese homeostasis and expression of Entner-Doudoroff pathway-associated genes to support growth within macrophages. Our library and datasets, made available through the online tool SalComKinetics, provide resources for systems-level interrogation of Salmonella transcriptional dynamics.
© 2025. The Author(s), under exclusive licence to Springer Nature Limited.