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

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Protein kinase activity is crucial for bacterial signaling but challenging to measure directly at the single-cell level.
• Existing methods using transcriptional reporters are often confounded by pathway crosstalk, hindering specific kinase activity deconvolution.

Purpose of the Study:

• To develop and validate modular, direct, and specific sensors for bacterial kinase activity.
• To investigate the activity of the conserved bacterial Ser/Thr kinase, PrkC, under various conditions.

Main Methods:

• Engineered FRET-based sensors and a synthetic transcription factor (based on lactose repressor, LacI) responsive to phosphorylation.
• Utilized sensors to measure PrkC activity in single bacterial cells and colonies.
• Applied sensors to assess PrkC response to cell-wall active antibiotics.

Main Results:

• Demonstrated the utility of novel sensors for real-time measurement of bacterial kinase activity.
• Showed that PrkC activity increases upon exposure to cefotaxime, an antibiotic blocking late-stage peptidoglycan synthesis.
• Confirmed that PrkC activity does not increase with fosfomycin, an antibiotic affecting early-stage peptidoglycan synthesis.

Conclusions:

• Developed versatile and specific bacterial kinase sensors applicable from single cells to colonies.
• Established a link between PrkC kinase activity and the inhibition of late-stage bacterial cell wall synthesis.
• The modular sensor design offers potential for broader application in studying diverse bacterial signaling pathways.