Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance

Abstract

Summary Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a post-transcriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone

“…This idea also is supported by the observation that the stringent response seems to be linked to the cellular energy state, because decreased GTP levels render B. subtilis more capable of surviving amino acid starvation, albeit at lower growth rates (35,36). Both events are closely connected to ppGpp and pppGpp, which also inhibit multiple enzymes for GTP biosynthesis (35,37,38). Furthermore, these data strongly indicate that pppGpp and ppGpp execute different biological functions.…”

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

“…Gmk is the enzyme responsible for the conversion of GMP to GDP during de novo synthesis of GTP, whereas HprT is involved in the salvage pathway, converting both hypoxanthine to IMP and guanine to GMP. The activities of these enzymes from both B. subtilis and E. faecalis, as well as Gmk from S. aureus (Gmk SA ), have been shown to be inhibited in the presence of (p)ppGpp, thus lowering intracellular GTP levels to a range that supports survival during starvation (28)(29)(30).…”

“…To examine whether (p)ppGpp can directly inhibit the function of the staphylococcal HprT enzyme, and to confirm that Gmk SA can be inhibited, the enzymatic activities of both proteins were monitored in the presence of both ppGpp and pppGpp. Enzymatic assays monitoring the conversion of guanine to GMP by HprT or GMP to GDP by Gmk were set up as previously described (28). This analysis revealed that the HprT and Gmk enzymes from S. aureus are inhibited by both ppGpp and pppGpp ( Fig.…”

“…2 E and F). During stringent response activation, the levels of (p)ppGpp in the bacterial cell rise to 1-2 mM (28,52), levels that are more than sufficient to inhibit the functions of these enzymes. Altogether, these data reveal that RsgA, RbgA, Era, and HflX function as GTPases, the activities of which increase upon association with the ribosome and are inhibited upon interaction with (p)ppGpp.…”

“…Decreased GTP levels lead to a decrease in the transcription of mRNAs with a GTP-initiating nucleotide, which in Gram-positive bacteria includes most rRNA promoters (26). Aside from substrate depletion, (p)ppGpp also actively inhibit GTP synthesis in Bacillus subtilis and Enterococcus faecalis by blocking the functions of the hypoxanthine-guanine phosphoribosyltransferase HprT and the guanylate kinase Gmk, two enzymes involved in the GTP synthesis pathway (28)(29)(30). GTP levels are also important in some species for the activation of CodY, a global transcriptional regulator.…”