Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States.
ACS Synth Biol. 2021 Sep 17;10(9):2340-2350. doi: 10.1021/acssynbio.1c00250. Epub 2021 Aug 31.
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.
在单细胞水平上，蛋白激酶的活性通常可以通过下游的转录报告基因推断出来。然而，启动子通常受到几个途径的共同调控，使得特定激酶的活性难以分解。在这里，我们提出了细菌激酶活性的模块化、直接和特异性传感器，包括基于 FRET 的传感器，以及基于乳糖阻遏物（LacI）的合成转录因子，该因子经过工程改造以响应磷酸化。我们证明了这些传感器在测量不同生长条件下（从单细胞到菌落）的保守细菌丝氨酸/苏氨酸激酶 PrkC 的活性方面的实用性。我们还表明，PrkC 的活性会增加对细胞壁活性抗生素的反应，该抗生素会阻止肽聚糖合成的后期步骤（头孢噻肟），但不会阻止早期步骤（磷霉素）。这些传感器具有模块化设计，将来应该可以推广到其他细菌信号系统。
