Controlled Delivery and Light-Induced Release of Magic Spot Nucleotides in Escherichia coli

The "magic spot nucleotides" (MSNs) ppGpp and pppGpp (also: (p)ppGpp) are bacterial alarmones central to the conserved stringent response, a stress adaptation mechanism that helps bacteria adapt to stress conditions and hostile environments. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope-labeled MSN analogues and their delivery into Escherichia coli comparing different approaches. A cyclodextrin-based synthetic nucleotide transporter provides particular advantages. Upon 400 nm irradiation, these probes were photo-released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometry and studied their ability to alter growth in a (p)ppGpp0 mutant. This work provides the first demonstration that highly charged, photocaged MSNs can traverse the bacterial envelope, be photo-released intracellularly, and be metabolically tracked in real time. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in bacteria.