The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR inBacillus

Abstract

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens.

Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase

“…Accordingly, the amounts of PurM, PurQ, PyrB and PyrP proteins were increased in the stationary Rho + proteome ( S1 Table ). These differences might reflect the altered levels of (p)ppGpp and GTP in Rho + cells, as it has been shown that (p)ppGpp stimulates activity of PurR, the repressor of the pur operon [ 114 ], while GTP antagonizes the activity of PyrR, the repressor of the pyr operon [reviewed in 115 ].…”

Bacillus subtilis produces (p)ppGpp in response to the bacteriostatic antibiotic chloramphenicol to prevent its potential bactericidal effect

“…Here, we found that regulation of GTP biosynthesis enzymes by (p)ppGpp is a key to the prevention of chloramphenicol lethality in B. subtilis (Figure 6). Given the conservation of GTP biosynthesis regulations in Firmicutes 32,36,38,43,44,46 , it is possible that similar protection mechanisms also apply to Firmicute pathogens such as E. faecalis, in which we also observed (p)ppGpp-dependent lethality by chloramphenicol (Figure 1D). On the other hand, Gram-negative bacteria such as E. coli do not appear to require (p)ppGpp to survive chloramphenicol 26 , indicating that Gram-negative bacteria have evolved different protection mechanisms against this antibiotic.…”

“…In B. subtilis, (p)ppGpp regulates many cellular processes, including DNA replication 34,35 , transcription 36 , ribosome biogenesis 37,38 and protein translation and secretion [39][40][41] . However, its most well-characterized effect in Gram-positive bacteria is the regulation of purine metabolism 31,36,37,[42][43][44][45][46][47] . In B. subtilis, (p)ppGpp inhibits purine biosynthesis enzymes GuaB, Gmk, HprT, and XprT, with a strong impact on GTP levels and viability 32 .…”

“…How does (p)ppGpp prevent chloramphenicol from killing B. subtilis? In B. subtilis, (p)ppGpp regulates many cellular processes, including DNA replication 34,35 , transcription 36 , ribosome biogenesis 37,38 and protein translation and secretion [39][40][41] . However, its most well-characterized effect in Gram-positive bacteria is the regulation of purine metabolism 31,36,37,[42][43][44][45][46][47] .…”

From magic spot ppGpp to MESH1: Stringent response from bacteria to metazoa

“…While there are common and conserved themes, the specific protein targets and regulatory mechanisms of (p)ppGpp may differ between different bacterial species. For example, while (p)ppGpp affects the global and regional transcription of Escherichia coli through the direct binding to the DksA and the RNA polymerase (RNAP) [ 6 ], it affects transcription by affecting the GTP level and binding to specific transcription factors, such as PurR [ 7 ] and MglA/SspA complex [ 8 ], in other bacteria. In addition, (p)ppGpp can reduce DNA proliferation by binding to DnaG (synthetase of the priming RNA required for DNA replication) [ 9 ], affecting the expression and stability of DnaA (replication initiation ATPase) [ 10 ] and modulating the supercoiling state of oriC [ 11 ].…”