During the final period, the project achieved several milestones that advanced our understanding of how small signaling molecules called magic spot nucleotides (MSN) function in living systems. We identified and validated proteins that interact with and regulate ppGpp, a central MSN, and showed how these partners influence cellular processes such as membrane depolarization. In parallel, we completed the development of photoactivatable MSN - light-sensitive molecular versions that can be modified, for example by introducing stable isotopes, sugars, or polycations to improve cellular uptake. These molecules were successfully tested in bacteria and other models, demonstrating broad biological applicability. A major breakthrough was the first delivery of MSN into living bacteria and their release on demand by irradiation. This enabled real-time tracking of MSN metabolism via capillary electrophoresis–mass spectrometry (CE–MS), which we refined into the most sensitive platform currently available for such studies. We also developed selective chemical sensors that detect MSN or related inorganic polyphosphates, enabling measurements in biological samples and high-throughput screening.
Detailed analyses of MSN levels in Escherichia coli during growth and nutrient stress revealed an unexpected coupling with another class of molecules, dinucleoside polyphosphates, suggesting stress messengers are more tightly linked than previously thought. We also began establishing a method to monitor MSN turnover using ¹⁸O-labeled water, useful for following metabolic fluxes in living cells.
Our work on chemical inhibitors of the bacterial enzyme RelA progressed substantially; we synthesized a diverse library of compounds to help clarify how MSN production is regulated in different bacterial species.
Throughout the project, we created an extensive toolbox of MSN derivatives, including authentic standards, isotope-labeled references, photoactivatable compounds, and affinity probes. We developed efficient new synthetic routes, such as chemoenzymatic approaches that now enable access to structures previously extremely difficult to obtain. Using CE–MS, we discovered that bacterial infection triggers MSN production in plants and addressed their molecular origin. We also observed oscillations in MSN levels correlating with other stress signals, revealing new directions for studying bacterial adaptation. The results were widely disseminated through 11 peer-reviewed publications, with three more in preparation. Team members presented findings at international conferences and workshops, earning poster prizes and winning science slams for the public. Importantly, the new analytical tools, datasets, and molecular probes are freely available to the scientific community, ensuring others can build upon this research.
