RSH Enzyme Synthesizes (p)ppGpp and pppApp and , and Leads to Discovery of pppApp Synthesis in .

In bacteria, the so-called stringent response is responsible for adaptation to changing environmental conditions. This response is mediated by guanosine derivatives [(p)ppGpp], synthesized by either large mono-functional RelA or bi-functional SpoT (synthesis and hydrolysis) enzymes in β and γproteobacteria, such as . In Firmicutes and α, δ, and 𝜀proteobacteria, large bifunctional Rel-SpoT-homologs (RSH), often accompanied by small (p)ppGpp synthetases and/or hydrolases devoid of regulatory domains, are responsible for (p)ppGpp turnover. Here, we report on surprising and properties of an RSH enzyme from (RSH). We find that this enzyme possesses some unique features, e.g., it requires cobalt cations for the most efficient (p)ppGpp synthesis, in contrast to all other known specific (p)ppGpp synthetases that require Mg. In addition, it can synthesize pppApp, which has not been demonstrated for any Rel/SpoT/RSH enzyme so far. , our studies also show that RSH is active in cells, as it can complement ppGpp growth defects and affects P1- fusion activity in a way expected for an RSH enzyme. These studies also led us to discover pppApp synthesis in wild type cells (not carrying the RSH enzyme), which to our knowledge has not been demonstrated ever before. In the light of our recent discovery that pppApp directly regulates RNAP transcription in a manner opposite to (p)ppGpp, this leads to a possibility that pppApp is a new member of the nucleotide second-messenger family that is widely present in bacterial species.