Simultaneous Capillary Electrophoresis - Mass Spectrometry profiling of (p)ppGpp and dinucleoside polyphosphates reveals oscillatory (p)ppGpp dynamics and a potential synchronisation between alarmones

Abstract To survive stressful conditions, bacteria enter the stringent response mediated by the magic spot nucleotides (MSN) guanosine 3’5’-bispyrophosphate (ppGpp) and guanosine 3’-diphosphate 5’-triphosphate (pppGpp). Another group of so-called alarmones elevated under stress are dinucleoside polyphosphates (Np n Ns), such as diadenosine triphosphate (Ap 3 A). How these two groups of alarmones intersect with one another is poorly understood. Here, we present a sensitive method using capillary electrophoresis coupled to mass spectrometry (CE-MS) with heavy isotope labeled internal references to separate and quantify magic spot nucleotides in parallel with Np n Ns. This approach uncovered oscillatory fluctuations in MSN and some Np n N during exponential growth, suggesting more dynamic regulation of the stringent response than previously thought. In addition, up to eleven Np n Ns were detected after prolonged growth. Np n N levels were also quantified under various amino acid starvation conditions, revealing a pronounced accumulation in late stationary phase. In summary, our parallel assignment and quantification of highly charged signaling molecules in bacteria under stress conditions paves the way to better study and understand the cross-talk between different alarmones.