Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity

Abstract

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.

Experimental measurement and computational prediction of bacterial Hanks‐type Ser/Thr signaling system regulatory targets

“…Upon phosphorylation of the substrate sequence, a conformational change occurs, resulting in a change in the FRET signal. These sensors were successfully used for eukaryotic Ser/Thr kinases such as PKC (Violin et al, 2003) and Aurora B (Fuller et al, 2008) among more than 20 others (Gonzalez-Vera & Morris, 2015), and their modular nature proved adaptable to the bacterial Hanks-type Ser/Thr kinase PrkC from B. subtilis (Zheng et al, 2021). This modular design was used to swap the substrate peptide among four variants and observe sequence-specific changes in phosphorylation activity.…”

“…Therefore, creating a transcriptional reporter for this family of kinases required the design of a synthetic transcription factor. Using the design principles of the bacterial FRET sensor and protein engineering, a modular synthetic transcription factor that specifically responds to PrkC activity in B. subtilis was created (Zheng et al, 2021). The design of this transcription factor relies on the ability of Hanks-type kinases to phosphorylate short substrate peptides.…”

“…As an additional consideration, the modular phosphopeptide binding domain (FHA2) used in the B. subtilis study has been characterized to be partially sensitive to the choice of amino acid in the +3 position relative to the phosphosite (Durocher et al, 2000). For example, better sensitivity was achieved using an I in the +3 position as a biosensor in both the PrkC study in B. subtilis (Zheng et al, 2021) and was used for a FRET biosensor for Aurora B activity in eukaryotic cells (Fuller et al, 2008). After optimization, the modular nature of this sensor and its single-cell sensitivity could allow quantitative measurements of the specificity of a large substrate library, with the high throughput and accuracy required for training machine learning models.…”