Abstract
A lack of direct single-cell readouts for bacterial kinase activity remains a major barrier to our understanding of most signaling systems. At the single-cell-level, protein kinase activity is typically inferred by the activity of downstream transcriptional reporters. Complicating this approach in vivo, promoters are often co-regulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we have designed and constructed new, direct and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor that responds to phosphorylation. We demonstrate the utility of these reporters in measuring kinase activity in population-based and single-cell assays during various growth phases and antibiotic treatments. These sensors respond to a highly conserved bacterial Ser/Thr kinase, PrkC that has no known dedicated transcription factor and whose regulon is known to be convolved with an essential signaling system. We used these new sensors to measure PrkC activity in colonies, bulk culture, and single cells. Together these new sensors provide evidence for considerable heterogeneity in PrkC activity in actively growing populations. We further demonstrate 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). This is consistent with a model where PrkC senses and responds to blocks in the extracellular steps in cell wall synthesis. As the design of these phosphorylation sensors is modular, we anticipate that this work may have broad applications to other bacterial signaling systems in the future.
Competing Interest Statement
The authors have declared no competing interest.
