The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

I was lucky to start my research career as the molecular biology revolution was taking hold, providing a constantly increasing set of tools and questions to investigate. Starting from a fascination with bacteria and their ability to adapt to different conditions, I've investigated post-translational mechanisms and their role in the ability of E. coli to respond to stress. My research career has been primarily at the National Institutes of Health, where I run a group within the Laboratory of Molecular Biology, NCI and hold the title of NIH Distinguished Investigator. Our lab has been interested in both energy-dependent proteolysis, discussed very briefly here, and small regulatory RNAs (sRNAs). The major group of such sRNAs act by pairing with target mRNAs with the aid of the RNA chaperone Hfq, mediating both positive and negative regulation of translation and mRNA stability. Both in our own lab and in a continuing and highly productive collaboration with the laboratory of Gisela (Gigi) Storz, we have used global approaches to identify novel sRNAs, identified how many of them are regulated, both at the level of transcription and stability, and worked on understanding the role of these sRNAs in regulatory networks. Our continued work explores regulators of sRNA and Hfq function. Here, Gigi and I have split summaries of our findings, and hope that our two chapters will be read together.

Most bacterial small regulatory RNAs (sRNAs) modulate gene expression by forming complementary base pairs with target messenger RNAs (mRNAs), dependent upon the RNA chaperone Hfq. Hfq has three RNA-binding faces (proximal, rim, and distal), facilitating simultaneous binding of sRNAs and target mRNAs. Here, we systematically examined the functional impact of amino acid substitutions in the RNA-binding faces on in vivo pairing, using the RNA interaction by ligation and sequencing (RIL-seq) approach. The distal and proximal Hfq-binding face mutants retained substantial numbers of RNA-RNA interactions (significant chimera counts or S-chimeras). However, the rim face mutant R16A showed a near-complete loss of S-chimeras, although Hfq R16A retained partial RNA-binding activity as well as partial regulatory activity. Intracellular RIL-seq, a method with fewer in vitro processing steps, led to more S-chimeras in R16A, but there were still fewer than for wild-type Hfq and somewhat different sets of prevalent RNA-RNA pairs. Our analysis provides insights into how the RNA-binding faces of Hfq contribute to pairing in vivo, documents the key role for the rim face in stabilizing RNA pairs on Hfq, and highlights intriguing differences captured by different RNA-RNA interactome approaches.

This study assessed the influence of general stress-response alternative sigma factors RpoS (σS) and SigB (σB) on tolerance of Escherichia coli (E. coli MG1655 and its isogenic mutant E. coli MG1655 ΔrpoS) and Listeria monocytogenes (L. monocytogenes EGD-e and its isogenic mutant L. monocytogenes EGD-e ΔsigB) to the essential oils (EOs) from Origanum vulgare L.—oregano (OVEO) and Rosmarinus officinalis L.—rosemary (ROEO), as well as the changes in tolerance of parental and ΔrpoS and ΔsigB mutant strains to OVEO, ROEO and pulsed electric fields (PEF) following overnight exposure to subinhibitory concentrations (1/2×minimum inhibitory concentration—MIC) of each tested EO. MIC values of OVEO and ROEO against the mutant cells were usually lower than those found against the parental cells. Survivor curves showed that mutant cells were more sensitive to these EOs than parental cells. The recovery of survivors in selective media showed a greater proportion of cells sublethally injured at their cell envelopes in the mutant strains compared with the parental strains. Induction of increased direct-tolerance to OVEO and ROEO or cross-tolerance to PEF was not observed after pre-exposure of parental and mutant cells to EOs. Otherwise, parental and mutant cells of E. coli and L. monocytogenes pre-exposed to OVEO or ROEO showed decreased tolerance when further treated with the homologous stressing agent at 2×MIC. Still, mutant cells pre-exposed to OVEO or ROEO showed lower tolerance to PEF than parental strains. These results showed the influence of σS and σB in tolerance of single strains of E. coli and L. monocytogenes, respectively, to OVEO and ROEO. Moreover, the deletion of σS and σB resulted in decreased tolerance to OVEO, ROEO or PEF in tested strains following exposure to OVEO or ROEO at a subinhibitory concentration.

During cold shock, bacteria shut down translation of all but a set of cold-shock proteins critical for recovery; in this issue of Molecular Cell, Delaleau et al.1 show that Rho-dependent transcription termination plays an important role in cold adaptation, via temperature-regulated termination of the cold-shock protein mRNAs.

RpoS is an alternative sigma factor needed for the induction of the general stress response in many gammaproteobacteria. Tight regulation of RpoS levels and activity is required for bacterial growth and survival under stress. In Escherichia coli, various stresses lead to higher levels of RpoS due to increased translation and decreased degradation. During non-stress conditions, RpoS is unstable, because the adaptor protein RssB delivers RpoS to the ClpXP protease. RpoS degradation is prevented during stress by the sequestration of RssB by anti-adaptors, each of which is induced in response to specific stresses. Here, we examined how the stabilization of RpoS is reversed during recovery of the cell from stress. We found that RpoS degradation quickly resumes after recovery from phosphate starvation, carbon starvation, and when transitioning from stationary phase back to exponential phase. This process is in part mediated by the anti-adaptor IraP, known to promote RpoS stabilization during phosphate starvation via the sequestration of adaptor RssB. The rapid recovery from phosphate starvation is dependent upon a feedback loop in which RpoS transcription of rssB, encoding the adaptor protein, plays a critical role. Crl, an activator of RpoS that specifically binds to and stabilizes the complex between the RNA polymerase and RpoS, is also required for the feedback loop to function efficiently, highlighting a critical role for Crl in restoring RpoS basal levels.

Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.

The Rcs (regulator of capsule synthesis) phosphorelay is a conserved cell envelope stress response mechanism in enterobacteria. It responds to perturbations at the cell surface and the peptidoglycan layer from a variety of sources, including antimicrobial peptides, beta-lactams, and changes in osmolarity. RcsF, an outer membrane lipoprotein, is the sensor for this pathway and activates the phosphorelay by interacting with an inner membrane protein IgaA. IgaA is essential; it negatively regulates the signaling by interacting with the phosphotransferase RcsD. We previously showed that RcsF-dependent signaling does not require the periplasmic domain of the histidine kinase RcsC and identified a dominant negative mutant of RcsD that can block signaling via increased interactions with IgaA. However, how the inducing signals are sensed and how signal is transduced to activate the transcription of the Rcs regulon remains unclear. In this study, we investigated how the Rcs cascade functions without its only known sensor, RcsF, and characterized the underlying mechanisms for three distinct RcsF-independent inducers. Previous reports showed that Rcs activity can be induced in the absence of RcsF by a loss of function mutation in the periplasmic oxidoreductase DsbA or by overexpression of the DnaK cochaperone DjlA. We identified an inner membrane protein, DrpB, as a multicopy RcsF-independent Rcs activator in E. coli. The loss of the periplasmic oxidoreductase DsbA and the overexpression of the DnaK cochaperone DjlA each trigger the Rcs cascade in the absence of RcsF by weakening IgaA-RcsD interactions in different ways. In contrast, the cell-division associated protein DrpB uniquely requires the RcsC periplasmic domain for activation; this domain is not needed for RcsF-dependent signaling. This suggests the possibility that the RcsC periplasmic domain acts as a sensor for some Rcs signals. Overall, the results add new understanding to how this complex phosphorelay can be activated by diverse mechanisms.

Bacterial small RNAs (sRNAs) regulate gene expression by base- pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5- related N- acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activ-ity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq- dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction inter-fered with HqbA suppression of Hfq. However, an acetylation- deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low -affinity RNAs but not high -affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWVand proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low -affinity RNA binders.

The specialized sigma factor RpoS mediates a general stress response in Escherichia coli and related bacteria, activating promoters that allow cells to survive stationary phase and many stresses. RpoS synthesis and stability are regulated at multiple levels. Translation of RpoS is positively regulated by multiple small RNAs in response to stress. Degradation of RpoS, dependent upon the adaptor protein RssB, is rapid during exponential growth and ceases upon starvation or other stresses, increasing accumulation of RpoS. E. coli carrying mutations that block the synthesis of polyamines were previously found to have low levels of RpoS, while levels increased rapidly when polyamines were added. We have used a series of reporters to examine the basis for the lack of RpoS in polyamine-deficient cells. The polyamine requirement was independent of small RNA-mediated positive regulation of RpoS translation. Mutations in rssB stabilize RpoS and significantly bypassed the polyamine deficit, suggesting that lack of polyamines might lead to rapid RpoS degradation. However, rates of degradation of mature RpoS were unaffected by polyamine availability. Codon optimization in rpoS partially relieved the polyamine dependence, suggesting a defect in RpoS translation in the absence of polyamines. Consistent with this, a hyperproofreading allele of ribosomal protein S12, encoded by rpsL, showed a decrease in RpoS levels, and this decrease was also suppressed by either codon optimization or blocking RpoS degradation. We suggest that rpoS codon usage leads it to be particularly sensitive to slowed translation, due to either lack of polyamines or hyperproofreading, leading to cotranslational degradation. We dedicate this study to Herb Tabor and his foundational work on polyamines, including the basis for this study.

In enterobacteria such as Escherichia coli, the general stress response is mediated by as, the stationary phase dissociable promoter specificity subunit of RNA polymerase. as is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of fulllength RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD antiadaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the a4-115-a5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify as-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full as engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Hfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), the Escherichia coli mutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression of mutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination.

As key players of gene regulation in many bacteria, small regulatory RNAs (sRNAs) associated with the RNA chaperone Hfq shape numerous phenotypic traits, including metabolism, stress response and adaptation, as well as virulence. sRNAs can alter target messenger RNA (mRNA) translation and stability via base pairing. sRNA synthesis is generally under tight transcriptional regulation, but other levels of regulation of sRNA signaling are less well understood. Here we used a fluorescence-based functional screen to identify regulators that can quench sRNA signaling of the iron-responsive sRNA RyhB in Escherichia coli. The identified regulators fell into two classes, general regulators (affecting signaling by many sRNAs) and RyhB-specific regulators; we focused on the specific ones here. General regulators include three Hfq-interacting sRNAs, CyaR, ChiX, and McaS, previously found to act through Hfq competition, RNase T, a 3 ' to 5 ' exonuclease not previously implicated in sRNA degradation, and YhbS, a putative GCN5-related N-acetyltransferase (GNAT). Two specific regulators were identified. AspX, a 3 ' end-derived small RNA, specifically represses RyhB signaling via an RNA sponging mechanism. YicC, a previously uncharacterized but widely conserved protein, triggers rapid RyhB degradation via collaboration with the exoribonuclease PNPase. These findings greatly expand our knowledge of regulation of bacterial sRNA signaling and suggest complex regulatory networks for controlling iron homeostasis in bacteria. The fluorescence-based genetic screen system described here is a powerful tool expected to accelerate the discovery of novel regulators of sRNA signaling in many bacteria.

ABSTRACTHfq, a bacterial RNA chaperone, stabilizes small regulatory RNAs (sRNAs) and facilitates sRNA base-pairing with target mRNAs. Hfq has a conserved N-terminal domain and a poorly conserved disordered C-terminal domain (CTD). In a transcriptome-wide examination of the effects of a chromosomal CTD deletion (Hfq1-65), theEscherichia colimutant was most defective for the accumulation of sRNAs that bind the proximal and distal faces of Hfq (Class II sRNAs), but other sRNAs also were affected. There were only modest effects on the levels of mRNAs, suggesting little disruption of sRNA-dependent regulation. However, cells expressing Hfq lacking the CTD deletion in combination with a weak distal face mutation were defective for the function of the Class II sRNA ChiX and repression ofmutS, both dependent upon distal face RNA binding. Loss of the region between amino acids 66-72 was critical for this defect. The CTD region beyond amino acid 72 was not necessary for distal face-dependent regulation, but was needed for functions associated with the Hfq rim, seen most clearly in combination with a rim mutant. Our results suggest that the C-terminus collaborates in various ways with different binding faces of Hfq, leading to distinct outcomes for individual sRNAs.

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.

The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design.IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

Wang et al., 2020Wang B. Grant R.A. Laub M.T. ppGpp coordinates nucleotide and amino-acid synthesis in E. coli during starvation.Mol. Cell. 2020; 80 (this issue): 29-42Abstract Full Text Full Text PDF Scopus (7) Google Scholar show that binding of the second messenger ppGpp to inosine-guanosine kinase (Gsk) in E. coli modulates the levels of the key metabolite phosphoribosyl pyrophosphate (pRpp), decreasing purine synthesis to favor amino acid synthesis during stress adaptation. Wang et al., 2020Wang B. Grant R.A. Laub M.T. ppGpp coordinates nucleotide and amino-acid synthesis in E. coli during starvation.Mol. Cell. 2020; 80 (this issue): 29-42Abstract Full Text Full Text PDF Scopus (7) Google Scholar show that binding of the second messenger ppGpp to inosine-guanosine kinase (Gsk) in E. coli modulates the levels of the key metabolite phosphoribosyl pyrophosphate (pRpp), decreasing purine synthesis to favor amino acid synthesis during stress adaptation. All cells use small molecule effectors to signal changing conditions and adjust metabolism to best ensure survival and growth. For bacteria like E. coli, capable of growing in a large variety of environments, adapting to stress/starvation and then recovering from it is critical. Many bacteria, including E. coli, synthesize guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from GTP in response to starvation (Figure 1); these small molecules can rapidly and reversibly remodel transcription and metabolism (Potrykus and Cashel, 2008Potrykus K. Cashel M. (p)ppGpp: still magical?.Annu. Rev. Microbiol. 2008; 62: 35-51Crossref PubMed Scopus (786) Google Scholar). How do they do this, and what is the best way to unravel the myriad effects of these signals? Recent studies, including the study by Wang et al. discussed here (Wang et al., 2020Wang B. Grant R.A. Laub M.T. ppGpp coordinates nucleotide and amino-acid synthesis in E. coli during starvation.Mol. Cell. 2020; 80 (this issue): 29-42Abstract Full Text Full Text PDF Scopus (7) Google Scholar), highlight how (p)ppGpp binds to and regulates key enzymes of purine metabolism, clarifying just how complex and integrated the response is. (p)ppGpp was first identified as a molecule critical for the dramatic changes in metabolism in response to amino acid starvation, called the stringent response (Cashel, 1975Cashel M. Regulation of bacterial ppGpp and pppGpp.Annu. Rev. Microbiol. 1975; 29: 301-318Crossref PubMed Scopus (205) Google Scholar). The subsequent 50+ years of study have made it clear how profound its effects can be. Cells stop translation and growth when ppGpp is overproduced; total absence of ppGpp, via deletion of the two enzymes that synthesize it, RelA and SpoT (Figure 1), results in significant growth defects and multi-amino acid auxotrophy (Potrykus and Cashel, 2008Potrykus K. Cashel M. (p)ppGpp: still magical?.Annu. Rev. Microbiol. 2008; 62: 35-51Crossref PubMed Scopus (786) Google Scholar). New results from a number of groups, including the Wang et al. paper in this issue of Molecular Cell, are starting to sort out the range of physiological roles of ppGpp. Most effective have been studies allowing global identification of the proteins with which ppGpp interacts, followed by investigation of what happens when a specific target becomes blind to ppGpp. In E. coli, a primary target for ppGpp is RNA polymerase (RNAP). Direct binding of ppGpp to sites on RNAP, aided by the protein cofactor DksA, downregulates ribosomal RNA and ribosomal protein synthesis and upregulates transcription of genes for amino acid biogenesis. ppGpp synthesis is stimulated by amino acid starvation; therefore, these adjustments to decrease translation and improve amino acid synthesis make sense. Comparing cells carrying wild-type RNAP to RNAP mutated for the two sites of ppGpp binding (RNAP 1−2−), Gourse and coworkers found that the multiple transcriptional changes seen immediately after ppGpp induction virtually all disappear in the RNAP 1−2−strain (Sanchez-Vazquez et al., 2019Sanchez-Vazquez P. Dewey C.N. Kitten N. Ross W. Gourse R.L. Genome-wide effects on Escherichia coli transcription from ppGpp binding to its two sites on RNA polymerase.Proc. Natl. Acad. Sci. USA. 2019; 116: 8310-8319Crossref PubMed Scopus (62) Google Scholar). However, high levels of ppGpp still inhibit growth of cells carrying the RNAP 1−2− mutations (Wang et al., 2019Wang B. Dai P. Ding D. Del Rosario A. Grant R.A. Pentelute B.L. Laub M.T. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.Nat. Chem. Biol. 2019; 15: 141-150Crossref PubMed Scopus (65) Google Scholar), and ppGpp interacts with and affects the activity of multiple enzymes (Link et al., 2015Link H. Fuhrer T. Gerosa L. Zamboni N. Sauer U. Real-time metabolome profiling of the metabolic switch between starvation and growth.Nat. Methods. 2015; 12: 1091-1097Crossref PubMed Scopus (132) Google Scholar). Laub and coworkers took a global approach to identifying ppGpp binders, creating cross-linkable ppGpp derivatives and using them to identify more than 50 protein targets in E. coli, including many of those previously identified (Wang et al., 2019Wang B. Dai P. Ding D. Del Rosario A. Grant R.A. Pentelute B.L. Laub M.T. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.Nat. Chem. Biol. 2019; 15: 141-150Crossref PubMed Scopus (65) Google Scholar). Many (but not all) GTPases were targets of ppGpp inhibition, consistent with the similarity between ppGpp and GTP substrates. In addition, a variety of enzymes of purine metabolism were identified as ppGpp targets. Using structure-guided mutagenesis coupled with metabolomics, the Laub lab has created mutant enzymes that have lost ppGpp binding ability. Expressing these ppGpp-blind alleles in vivo, tracking changes in metabolites in the mutant cells, and selecting suppressors of growth phenotypes have allowed them to begin to unravel this network of interactions. The results of studying Gsk, the salvage enzyme that feeds inosine and guanosine into the purine synthesis pathway, suggest intricate connections and feedback loops between the synthesis of amino acids and nucleotides, reinforcing the importance of balanced and regulated metabolic switches (Wang et al., 2020Wang B. Grant R.A. Laub M.T. ppGpp coordinates nucleotide and amino-acid synthesis in E. coli during starvation.Mol. Cell. 2020; 80 (this issue): 29-42Abstract Full Text Full Text PDF Scopus (7) Google Scholar). At the center of many of these metabolic switches is phosphoribosyl pyrophosphate (pRpp). pRpp is the starting point for synthesis of both purine and pyrimidine nucleotides as well as for some amino acids (histidine and tryptophan) and NAD+ (Hove-Jensen et al., 2016Hove-Jensen B. Andersen K.R. Kilstrup M. Martinussen J. Switzer R.L. Willemoës M. Phosphoribosyl Disphosphate (PRPP): biosynthesis, enzymology, utilization, and metabolic significance.Microbiol. Mol. Biol. Rev. 2016; 81 (e00040–e00016)Google Scholar). pRpp synthesis is dependent on PrsA (ribose-phosphate diphosphokinase), an enzyme sensitive to inhibition by ADP (Figure 1). Laub and coworkers determined the structure of Gsk bound to ppGpp and used the structure to design a Gsk mutant blind to ppGpp. When the mutant cells were fed inosine or guanosine in the presence of high ppGpp levels, ADP and ATP levels rose and pRpp levels decreased. Likely, the decrease in pRpp directly reflects ADP inhibition of PrsA. The consequences of this were dramatic, emphasizing the balance that the cells must usually exert on pRpp use. First, cells failed to make the critical pyrimidine triphosphate UTP. Addition of uridine allowed UTP synthesis without pRpp and bypassed that defect. However, even with uridine present, the Gsk mutant cells fared poorly when shifted to grow without exogenous amino acids. This defect was tracked to lack of histidine and tryptophan synthesis. Therefore, abrogating ppGpp feedback of just this one enzyme of purine metabolism was sufficient to disrupt pRpp levels and how it is used. PurF, the first enzyme of purine biosynthesis, also uses pRpp as the starting material and is itself inhibited by ppGpp (Wang et al., 2019Wang B. Dai P. Ding D. Del Rosario A. Grant R.A. Pentelute B.L. Laub M.T. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.Nat. Chem. Biol. 2019; 15: 141-150Crossref PubMed Scopus (65) Google Scholar); it may well be that PurF inhibition is also important to redirect the use of pRpp. This remains to be tested. Inhibition of purine biosynthesis during the stringent response is a common feature in bacteria. Guanylate kinase (Gmk) is a critical target of ppGpp inhibition in many bacteria but not in E. coli, leading to the suggestion that regulation of RNAP by ppGpp may have evolved relatively recently, later than Gmk-ppGpp (Liu et al., 2015Liu K. Myers A.R. Pisithkul T. Claas K.R. Satyshur K.A. Amador-Noguez D. Keck J.L. Wang J.D. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.Mol. Cell. 2015; 57: 735-749Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In B. subtilis, substituting the ppGpp-resistant Gmk from E. coli for the native enzyme interfered with adaptation to amino acid starvation (Liu et al., 2015Liu K. Myers A.R. Pisithkul T. Claas K.R. Satyshur K.A. Amador-Noguez D. Keck J.L. Wang J.D. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.Mol. Cell. 2015; 57: 735-749Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). ppGpp inhibits enzymes that utilize pRpp as a substrate for purine synthesis in both E. coli, where PurF is a target (Wang et al., 2019Wang B. Dai P. Ding D. Del Rosario A. Grant R.A. Pentelute B.L. Laub M.T. Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.Nat. Chem. Biol. 2019; 15: 141-150Crossref PubMed Scopus (65) Google Scholar), and in B. subtilis, where the purine salvage enzymes HPRT and XPRT are targets (Anderson et al., 2020Anderson B.W. Hao A. Satyshur K.A. Keck J.L. Wang J.D. Molecular mechanism of regulation of the purine salvage enzyme XPRT by the alamones pppGpp, ppGpp, and pGpp.J. Mol. Biol. 2020; 432: 4108-4126Crossref Scopus (7) Google Scholar). Thus, preserving appropriate levels and use of pRpp during the stringent response could be crucial for Gram-positive bacteria as well. As much as we have learned, there are further degrees of complexity. In many cases, (p)ppGpp affects not only the activity of an enzyme but its transcription as well. How effectively ppGpp inhibits the enzymes of purine metabolism depends not only on the level of ppGpp but the levels and binding affinities of other nucleotides. Thus, the relative levels of these nucleotides at a given point of time can influence the cellular decision making and hence growth and survival. Finally, most studies thus far have focused on the transition into starvation or stress. What are the critical regulatory points for emerging from starvation? The approaches highlighted in recent papers, including the Wang paper, give us the tools to start to understand these intricate but fundamental metabolic switches. The pace of recent work on ppGpp gives us hope that it will not take another 50 years to fully understand what this molecule is telling us about bacterial physiology. ppGpp Coordinates Nucleotide and Amino-Acid Synthesis in E. coli During StarvationWang et al.Molecular CellAugust 27, 2020In BriefWang et al. demonstrate that ppGpp, the universally conserved growth regulator in bacteria, directly inhibits the purine nucleotide salvage enzyme Gsk. By characterizing a Gsk variant that is insensitive to ppGpp, they demonstrate that inhibiting purine nucleotide synthesis is required during starvation to maintain levels of the metabolite pRpp, which is required for synthesizing histidine and tryptophan. Full-Text PDF