Small Alarmone Synthetase SasA Expression Leads to Concomitant Accumulation of pGpp, ppApp, and AppppA in Bacillus subtilis

Abstract

(p)ppGpp is a highly conserved bacterial alarmone which regulates many aspects of cellular physiology and metabolism. In Gram-positive bacteria such as B. subtilis , cellular (p)ppGpp level is determined by the bifunctional (p)ppGpp synthetase/hydrolase RelA and two small alarmone synthetases (SASs) YjbM (SasB) and YwaC (SasA). However, it is less clear whether these enzymes are also involved in regulation of alarmones outside of (p)ppGpp. Here we developed an improved LC-MS-based method to detect a broad spectrum of metabolites and alarmones from bacterial cultures with high efficiency. By characterizing the metabolomic signatures of SasA expressing B. subtilis , we identified strong accumulation of the (p)ppGpp analog pGpp, as well as accumulation of ppApp and AppppA. The induced accumulation of these alarmones is abolished in the catalytically dead sasA mutant, suggesting that it is a consequence of SasA synthetase activity. In addition, we also identified depletion of specific purine nucleotides and their precursors including IMP precursors FGAR, SAICAR and AICAR (ZMP), as well as GTP and GDP. Furthermore, we also revealed depletion of multiple pyrimidine precursors such as orotate and orotidine 5′-phosphate. Taken together, our work shows that induction of a single (p)ppGpp synthetase can cause concomitant accumulation and potential regulatory interplay of multiple alarmones.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis

“…In addition, and in contrast to (p)ppGpp, (p)ppApp is inefficiently degraded by P. aeruginosa SpoT, which results in (p)ppApp accumulation and likely contributes to its function as an energy depleting toxin [12]. Currently, the long RSH homologue from Methylobacterium extorquens and the small alarmone synthetase, SasA, from B. subtilis are the only other known enzymes that endogenously produce pyrophosphorylated derivatives of adenosine; however, these enzymes differ from Tas1 in that they also synthesize (p)ppGpp in response to stress [114,115]. (p)ppApp has also been reported in cell lysates of E. coli, but the identity of the enzyme responsible for its production is unknown [114].…”

“…Currently, the long RSH homologue from Methylobacterium extorquens and the small alarmone synthetase, SasA, from B . subtilis are the only other known enzymes that endogenously produce pyrophosphorylated derivatives of adenosine; however, these enzymes differ from Tas1 in that they also synthesize (p)ppGpp in response to stress [ 114 , 115 ]. (p)ppApp has also been reported in cell lysates of E .…”

Unique features of magic spot metabolism in Clostridioides difficile

“…35 To date, every organism that synthesizes pGpp directly or indirectly also synthesizes ppGpp and pppGpp. 31,40,47,[62][63] Utilization of GDP and GTP to exclusively synthesize pGpp by both a RSH and a SAS enzyme has not previously been reported. While it is still possible that RelC synthesizes the longer forms of magic spot despite its non-conserved guanosine binding motif, the dependence of CdRSH and CdRelQ on guanosine βphosphate bond hydrolysis indicates that pGpp is a very important, and possibly the only, clostridial alarmone.…”

Re-evaluation of Diadenosine Tetraphosphate (Ap4A) From a Stress Metabolite to Bona Fide Secondary Messenger

“…This has potentially limited our understanding and appreciation of individual Ap 4 N in mammalian cells. Advances in detection via liquid chromatography/tandem mass spectroscopy have aided the quantitation of different Ap n N and have led to the identification of Ap 4 U in extracellular environments ( Schulz et al, 2014 ; Götz et al, 2019 ; Ji et al, 2019 ; Fung et al, 2020 ). Establishing new LC-MS/MS methods for the analysis of individual intracellular Ap n N and other Np n N will clarify the biological relevance of these dinucleotides in stress responses in mammalian systems.…”