Diversity in (p)ppGpp metabolism and effectors

Abstract

Bacteria produce guanosine tetraphosphate and pentaphosphate, collectively named (p)ppGpp, in response to a variety of environmental stimuli. These two remarkable molecules regulate many cellular processes, including the central dogma processes and metabolism, to ensure survival and adaptation. Work in Escherichia coli laid the foundation for understanding the molecular details of (p)ppGpp and its cellular functions. As recent studies expand to other species, it is apparent that there exists considerable variation, with respect to not only (p)ppGpp metabolism, but also to its mechanism of action. From an evolutionary standpoint, this diversification is an elegant example of how different species adapt a particular regulatory network to their diverse lifestyles.

Figure 1. Divergent mechanisms of transcription initiation regulation by (p)ppGpp in E. coli and B. subtilis

(A) In E. coli, this regulation is mediated by the tripartite interaction between RNAP, (p)ppGpp, and DksA. Cross-linking and crystallography suggest that ppGpp (blue spheres) binds to an interface between the β′ (magenta) and ω (green) subunits of RNA polymerase [21**,22*,23*]. This binding site is 28 Å away from the active site located between the β and β′ subunits (not visible under current view), which makes (p)ppGpp an allosteric regulator. DksA binds to the secondary channel of RNAP [24]. Classic examples of negatively and positively regulated promoters are those that direct ribosomal RNA and histidine biosynthesis, respectively. Structures of RNAP (PDB: 4JKR) and DksA (PDB: 1TJL) were used for figure preparation [22*,72]. (B) In B. subtilis, (p)ppGpp regulates GTP levels by directly inhibiting IMPDH, GMK, and HprT [3**,31], and by passively consuming GTP (GDP) during (p)ppGpp synthesis. pppGpp (blue spheres) binds the GMK active site and thus acts as a competitive inhibitor [37]. Lowering GTP levels (the downward red arrow) decreases transcription from ribosomal RNA promoters, which initiate with GTP [32], but activates transcription from amino acid biosynthesis promoters (e.g. the ilv-leu operon), in part through inactivating CodY [17,38]. The C-terminal domain of B. subtilis CodY (PDB: 2B0L) was used for figure preparation [73]. Solid lines indicate biosynthesis pathways, whereas dotted lines indicate regulation. Negatively and positively regulated promoters are colored in blue and red, respectively.

Figure 2. (p)ppGpp regulates various important cellular processes to allow survival and adaptation to stresses

Stresses, such as amino acid starvation and antibiotic treatment activate/upregulate (p)ppGpp synthetases (RSH and SAS) to produce (p)ppGpp (the wrench). In B. subtilis, by directly inhibiting several GTP biosynthesis enzymes, (p)ppGpp curtails production of GDP/GTP, the substrates for (p)ppGpp synthesis, thus constituting a negative feedback regulation and maintaining homeostasis of guanylate nucleotide pools [3**]. Importantly, through direct interaction with its targets, (p)ppGpp regulates replication, transcription, translation, and metabolism (differently colored bolts) to adjust the cellular physiology to survive and adapt to adverse conditions. The dotted lines indicate that (p)ppGpp also indirectly modulates replication, translation, and metabolism through its effects on transcription.