Simultaneous Capillary Electrophoresis - Mass Spectrometry profiling of (p)ppGpp and dinucleoside polyphosphates reveals oscillatory (p)ppGpp dynamics and a potential synchronisation between alarmones
Prucker, I.; Milanov, M.; Moser, P.; Popp, C.; Haas, T. M.; Qiu, D.; Koch, H.-G.; Jessen, H. J.
Show abstract
To survive stressful conditions, bacteria enter the stringent response mediated by the magic spot nucleotides (MSN) guanosine 35-bispyrophosphate (ppGpp) and guanosine 3-diphosphate 5-triphosphate (pppGpp). Another group of so-called alarmones elevated under stress are dinucleoside polyphosphates (NpnNs), such as diadenosine triphosphate (Ap3A). 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 NpnNs. This approach uncovered oscillatory fluctuations in MSN and some NpnN during exponential growth, suggesting more dynamic regulation of the stringent response than previously thought. In addition, up to eleven NpnNs were detected after prolonged growth. NpnN 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.
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