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Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity
ACS Synthetic Biology  (IF5.11),  Pub Date : 2021-08-31, DOI: 10.1021/acssynbio.1c00250
Christine R. Zheng, Abhyudai Singh, Alexandra Libby, Pamela A. Silver, Elizabeth A. Libby

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 peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.