Methylobacterium extorquens RSH Enzyme Synthesizes (p)ppGpp and pppApp in vitro and in vivo, and Leads to Discovery of pppApp Synthesis in Escherichia coli

Abstract

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 γ - proteobacteria, such as Escherichia coli . In Firmicutes and α - , δ - , and 𝜀 - proteobacteria, 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 in vitro and in vivo properties of an RSH enzyme from Methylobacterium extorquens (RSH Mex ). 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 2+ . In addition, it can synthesize pppApp, which has not been demonstrated in vitro for any Rel/SpoT/RSH enzyme so far. In vivo , our studies also show that RSH Mex is active in Escherichia coli cells, as it can complement E. coli ppGpp 0 growth defects and affects rrnB P1- lacZ fusion activity in a way expected for an RSH enzyme. These studies also led us to discover pppApp synthesis in wild type E. coli cells (not carrying the RSH Mex enzyme), which to our knowledge has not been demonstrated ever before. In the light of our recent discovery that pppApp directly regulates E. coli RNAP transcription in vitro 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.

Estimates of RelSeq, Mesh1, and SAHMex Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays

“…Methylobacterium extorquens (a SAH-bearing bacterium) and E. coli have been chosen as the model organisms. As we reported elsewhere, we found pppApp to be synthesized by M. extorquens and E. coli cells in vivo, and RSH Mex enzyme (a long bifunctional RSH protein) is the source of both, (p)ppGpp and pppApp in M. extorquens (Sobala et al, 2019). At the same time, we confirmed and dissected the role of (p)ppApp in regulating E. coli RNAP activity at the ribosomal rrnB P1 promoter (Bruhn-Olszewska et al, 2018).…”

“…SAH Mex was purified under similar conditions, except that the His8-SUMO tags were cleaved with in-house purified his-tagged-Ulp1 SUMO protease. To this end, after initial fractionation and purification, the pooled fractions were dialyzed against the following buffer: 20 mM Tris-HCl pH 8.0, 250 mM NaCl, 10% glycerol, and processed as described for the His8-SUMO tagged proteins in Sobala et al (2019). The storage buffer was the same as for the Mesh1 and MESH1 proteins mentioned above.…”

Possible Roles for Basal Levels of (p)ppGpp: Growth Efficiency Vs. Surviving Stress

“…Synthetases of (p)ppGpp transfer the intact 5'-βγ pyrophosphate group from ATP to the ribose 3' hydroxyl group of GTP or GDP, while (p)ppGpp hydrolases regenerate the GDP and GTP substrates by removal of the same pyrophosphate ( Cashel and Kalbacher, 1970 ; Que et al, 1973 ; Sy and Lipmann, 1973 ; Kari et al, 1977 ). Many combinations of synthetase and hydrolase proteins are found throughout bacterial and plant kingdoms, along with uncertainty of functional implications or even the identities of the alarmone produced ( Atkinson et al, 2011 ; Avilan et al, 2019 ; Ronneau and Hallez, 2019 ; Sobala et al, 2019 ; Jimmy et al, 2020 ).…”

“…This conclusion rests on the assumption that Mesh1 activity is limited solely to (p)ppGpp hydrolysis. Traces of (p)ppApp have been detected by TLC in E. coli ( Sobala et al, 2019 ), therefore, until the full range of Mesh1 substrates is known within E. coli , conclusions as to the mechanism of growth inhibition by Mesh1 are qualified.…”

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

“…For example, another small alarmone synthetase SasB in Enterococcus faecalis can produce pGpp in vitro ( Gaca et al, 2015 ). On the other hand, Rel from Methylobacterium extorquens can synthesize pppApp both in vitro and when expressed in E. coli ( Sobala et al, 2019 ), and the secreted toxin Tas1 from Pseudomonas aeruginosa can produce pppApp, ppApp, and pApp in target E. coli cells to mediate contact dependent inhibition ( Ahmad et al, 2019 ). These findings suggest that stress responses mediated by (p)ppGpp synthetases may not be strictly limited to the alarmones pppGpp and ppGpp.…”