biostudies-literatureZheng CRIntelligence Advanced Research Projects ActivityArmy Research OfficeNational Institute of General Medical SciencesNIGMS NIH HHS2340-2350https://www.ebi.ac.uk/biostudies/studies/S-EPMC8498941biostudies-literatureUnknown10(9)At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidogACS synthetic biologyModular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.PMC8498941N660011824505W911NF1910243R01GM124446R01 GM124446Libby AZheng CRSingh ASilver PALibby EAfalseModular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidog2021-01-01T00:00:00Z2021 Sep2025-04-26T16:31:51.313Z2025-02-19T00:47:39.912ZS-EPMC84989413446348210.1021/acssynbio.1c00250