Biology subjects

Milanov, M.

Publications and source records attributed to Milanov, M..

2 recordsLinked to original sources

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2025

The small membrane protein YohP induces membrane depolarization and ppGpp accumulation in Escherichia coli.

Small membrane proteins represent an abundant and ubiquitous class of proteins that are often up-regulated when cells encounter unfavorable conditions, yet details about their exact function are largely missing. In bacteria, these proteins consist of typically less than 50 amino acids and contain a single transmembrane domain, but lack any detectable catalytic activity. Thus, the benefit of producing these proteins during stress conditions is unknown. In the current study we used a multidisciplinary approach to determine the function of the 27 amino acid long protein YohP in E. coli. Our proteomics approach revealed that YohP production leads to an up-regulation of proteins involved in membrane protection and to a down-regulation of many enzymes involved in key metabolic processes, such as nucleotide biosynthesis. Further biochemical characterizations revealed increased cardiolipin content in the membrane, a partial dissipation of the membrane potential and reduced membrane fluidity in YohP-containing membranes. Finally, our data show that YohP production induces the stringent response and leads to elevated levels of (p)ppGpp. Overall, our data indicate that the YohP-induced proteome and membrane changes initiate a state of metabolic silencing that protects E. coli against stress and helps to conserve cellular resources.

microbiology↗