Recent functional insights into the role of (p)ppGpp in bacterial physiology

Abstract

The alarmones guanosine tetraphosphate and guanosine pentaphosphate (collectively referred to as (p)ppGpp) are involved in regulating growth and several different stress responses in bacteria. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of (p)ppGpp metabolism and (p)ppGpp-mediated regulation. In this Review, we summarize these recent insights, with a focus on the molecular mechanisms governing the activity of the RelA/SpoT homologue (RSH) proteins, which are key players that regulate the cellular levels of (p)ppGpp. We also discuss the structural basis of transcriptional regulation by (p)ppGpp and the role of (p)ppGpp in GTP metabolism and in the emergence of bacterial persisters.

Figure 1. (p)ppGpp metabolism by RelA , SpoT and Rel

(a) (p)ppGpp synthesis by RSH enzymes. The products of the RSH-catalyzed reaction guanosine 3′-diphosphate 5-′diphosphate (ppGpp) and guanosine 3′-diphosphate 5-'triphosphate (pppGpp) are commonly referred to as (p)ppGpp. The γ-phosphate moiety of GTP and pppGpp are highlighted in red. (b) (p)ppGpp metabolism in Escherichia coli. The ‘long’ RSHs RelA and SpoT synthesize (p)ppGpp from GTP and GDP, generating AMP as a by-product. Interconversion of pppGpp to ppGpp is catalyzed by guanosine pentaphosphate phosphatase (GppA) and translational GTPases, such as the translocase EF-G. SpoT catalyzes the degradation of pppGpp and ppGpp to form GTP and GDP, respectively. Interconversion of GDP to GTP is catalyzed by nucleoside diphosphate kinase (Ndk). Adapted from ref Cashel, M., et al., The stringent response, in Escherichia coli and Salmonella: cellular and molecular biology. 1996, ASM Press: Washington DC. p. 1458–1496. (c) The distribution of ‘long’ RSHs and RSH domain structure. The domains of the long RSHs SpoT, RelA and Rel are shown, along with the distribution of these proteins in bacteria. The coloured boxes representing each domain show their approximate location along the length of the proteins, with dashed borders indicating domains with reduced or absent functional activity. In the case of the SpoT (p)ppGpp synthesis (SYNTH) domain, its synthetic activity is weak, whereas hydrolytic activity is absent in the RelA (p)ppGpp hydrolysis (HD) domain. The HD and SYNTH domains comprise the N-terminal domain (NTD), whereas the ThrRS, GTPase and SpoT (TGS), helical, Conserved Cysteines (CC) and Aspartokinase, Chorismate mutase and TyrA (ACT) domains together comprise the C-terminal domain (CTD). The phylogenetic tree summarizes the evolutionary relationships among bacteria that contain or lack long RSHs. The red arrow indicates the duplication event that led to the emergence of RelA and SpoT from an ancestral Rel protein in the lineage of the Gamma- and Beta-proteobacteria. The Planctomycete, Verrucomicrobia and Chlamidiale superphylum (PVC) of bacteria do not encode any long RSHs. In the absence of a reliable root of the bacterial tree of life, it is not known whether long RSHs evolved after the divergence of PVC bacteria, or whether they were lost in this lineage.

Figure 2. Mechanism of action of RelA

(a) Amino acid starvation leads to the accumulation of deacylated tRNAs in the cytosol that bind to the ribosomal A-site. This ribosomal state is recognized by RelA, which binds to the 50S subunit and adopts an active conformation. Activation of RelA and consequent synthesis of (p)ppGpp leads to its dissociation from the ribosome, followed by numerous rounds of (p)ppGpp synthesis in the dissociated state^, . Increased (p)ppGpp levels direct cellular metabolic resources to amino acid synthesis, which restores normal levels of tRNA aminoacylation. Aminoacylated tRNA is delivered to the ribosome by elongation factor Tu (EF-Tu) in direct competition with binding of RelA and deacylated tRNA. (b-c) Cryo-EM structure of RelA in complex with the ribosome. On binding to the ribosomal A-site, RelA interacts with the Sarcin-Ricin Loop (SRL) and ribosomal protein L11, as well as deacylated tRNA in the A-site, which adopts a highly distorted conformation (referred to as A/T-like tRNA). The RelA cryo-EM structure (3D-EM database e accession code EMD-2373) is reproduced from, with permission.

Figure 3. Direct regulation of RNAP activity by (p)ppGpp

(a) The E. coli RNAP holoenzyme (α_I: white, α_II, light gray, β, cyan; β’, pink; ω, dark gray; σ, orange) and promoter DNA (green) complex model showing how the (p)ppGpp and DksA interact with RNAP. The model shows the (p)ppGpp (blue) binding site on RNAP, which is located in the DPBB domain of the β’ subunit (magenta) and the N-termini of the ω subunit. Binding of (p)ppGpp to the DPBB domain may induce an allosteric signal to the catalytic Mg²⁺ (red sphere) for regulating the catalytic efficiency of RNAP. Alternatively, the (p)ppGpp binding at the shelf-core ratcheting axis may influence the shelf-core ratcheting and/or the DNA binding clamp swinging. The black dashed line indicates the shelf-core ratcheting axis and black arrows show directions of the ratcheting and the DNA binding clamp swinging. DksA is shown as a yellow cartoon model and a blue arrow shows its binding to the E. coli RNAP secondary channel, which may influence the orientation of the core and shelf modules for enhancing the potency of (p)ppGpp. This model was constructed by combining the X-ray crystal structures of E. coli RNAP-ppGpp complex (PDB: 4JK1/4JKR), T. thermophilus RNAP-promoter DNA complex (PDB: 4G7H) and E. coli DksA (PDB: 1TJL). (b) Taxonomic distribution of the MAR motif. Alignment of the N-terminal region of the ω subunit shows that the MAR motif (shaded box) that (p)ppGpp binds to is conserved only in the α-, β-, δ- and γ-proteobacteria. This suggests that the ancestor of Proteobacteria carried the MAR motif, and it was subsequently lost in the lineage that gave rise to the ε-proteobacteria. The phylogenetic tree on the left shows the evolutionary relationships among the groups of bacteria sampled, according to.

Figure 4. Role of (p)ppGpp in GTP homeostasis

In E. coli, the salvage pathway (red) utilizes either guanosine (GUO), guanine (GUA), inosine (INO) and hypoxanthine (HPX) as substrates. Guanosine kinase (Gsk) converts nucleosides GUO and INO to GMP and IMP, respectively. The de novo pathway uses phosphoribosyl pyrophosphate (PRPP) as a starting compound for the multi-step synthesis of inosine 5′-monophosphate (IMP), which is further converted to GTP. The transformation is achieved in four steps: IMP is first converted into xanthosine monophosphate (XMP) by the IMP dehydrogenase GuaB, and the GMP synthase GuaA then converts XMP into guanosine monophosphate (GMP). GMP is transformed into the final product, GTP, via sequential rounds of phosphorylation: the GMP kinase Gmk catalyzes the conversion of GMP into guanosine diphosphate (GDP), which is then converted to guanosine triphosphate (GTP) by the nucleoside diphosphate kinase Ndk. Cellular GTP concentrations have dual effect on bacterial physiology. Until a certain threshold concentration, increasing the GTP level increases the growth rate. However, further increase leads to cytotoxic effects, and at high concentrations GTP inhibits growth, negatively affecting bacterial survival upon amino acid starvation. The specific targets of (p)ppGpp-mediated control vary according to species and differ in E. coli^, _, B. subtilis and E. faecalis. In E. coli, (p)ppGpp inhibits the IMP dehydrogenase GuaB; in B. subtilis and E. faecalis (p)ppGpp inhibits hypoxanthine phosphoribosyltransferase (HprT, the enzyme that catalyzes both conversion of HPX to IMP and of GUA to GMP); in B. subtilis (p)ppGpp inhibits the GMP kinase Gmk.