Live‐Cell Imaging of Guanosine Tetra‐ and Pentaphosphate (p)ppGpp with RNA‐based Fluorescent Sensors**

Abstract

Guanosine tetra-and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for 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 cellto-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.

Genetically Encoded RNA-based Bioluminescence Resonance Energy Transfer (BRET) Sensors

“…56-58 Naturally existing RNA riboswitches that can selectively recognize SAM 59 and ppGpp 60,61 have been identified and used previously for engineering FR-based fluorescent sensors. 13,62…”

“…[56][57][58] Naturally existing RNA riboswitches that can selectively recognize SAM 59 and ppGpp 60,61 have been identified and used previously for engineering FR-based fluorescent sensors. 13,62 To convert these riboswitches into BRET sensors, we again focused on the design of transducer sequences between the aptamer domain of riboswitch and the Pepper P2 stem region (Fig. 1a and Table S1).…”

Bacteriophage anti-defense genes that neutralize TIR and STING immune responses

“…Cells were briefly pelleted (8,000 x g, 45 s), then shifted to M9 medium containing 0.25% CAA, 0.5% glucose and 0.5mM IPTG, in the absence or presence of 2.5% α-MG to induce chemical starvation; or M9 containing 1% glycerol, 0.5mM IPTG and 0.75% arabinose to induce both nutrient deprivation and expression of TIR-STING from the pBAD vector. Cells were incubated with DFHBI-1T dye 1 h after shift to inducing media as described (Sun et al, 2021). Images were obtained using a Zeiss LSM 880 Airyscan confocal microscope and were quantified using ImageJ (1.53r) software.…”

“…We next utilized an RNA-based fluorescent (p)ppGpp sensor (Sun et al, 2021) to monitor the effects of TIR-STING expression on cellular alarmone pools. Live-cell imaging revealed significant fluorescence activation following induction of TIR-STING (Figure 4A), with a mean 2.7-fold increase in signal intensity relative to glucose-fed control cells, similar to the increase seen in cells exposed to a chemical inducer of starvation, α-methylglucose (α-MG) (Figures 4A and 4B).…”

“…Live cell imaging E. coli BL21(DE3) cells carrying the S2 fluorescent sensor plasmid (Sun et al, 2021) were grown in M9 medium supplemented with 0.25% CAA and 0.5% glucose to an A 600 of 0.2-0.4. Cells were briefly pelleted (8,000 x g, 45 s), then shifted to M9 medium containing 0.25% CAA, 0.5% glucose and 0.5mM IPTG, in the absence or presence of 2.5% α-MG to induce chemical starvation; or M9 containing 1% glycerol, 0.5mM IPTG and 0.75% arabinose to induce both nutrient deprivation and expression of TIR-STING from the pBAD vector.…”