Stringent response plays a major role in modulating gene transcription in bacterial cells. It is signaled by a bacterial alarmone, guanosine tetra(penta)-phosphate, (p)ppGpp. Direct detection of (p)ppGpp in living cells is crucial to our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. We also incorporated the (p)ppGpp sensors with other existing sensors, such as c-di-GMP sensor and tetracycline sensors to further investigate the biological correlation among these molecules in cells. These RNA-based sensors can be broadly adapted to study the bacterial stringent response and other biological phenomena such as antibiotic resistance and biofilm formation.

Live-cell imaging of stringent response-related small molecules with RNA-based fluorescent sensors

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ACS Fall 2022

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