Alarmone ppGpp Research Articles

The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase Rel Sau , with Rel Sau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.

YtfK activates the stringent response by triggering the alarmone synthetase SpoT in Escherichia coli

The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions. This reprogramming of cell physiology is caused by the accumulation of the alarmone (p)ppGpp which, in Escherichia coli, depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. Although conditions that control SpoT-dependent (p)ppGpp accumulation have been described, the molecular mechanisms regulating the switching from (p)ppGpp degradation to synthesis remain poorly understood. Here, we show that the protein YtfK promotes SpoT-dependent accumulation of (p)ppGpp in E. coli and is required for activation of the stringent response during phosphate and fatty acid starvation. Our results indicate that YtfK can interact with SpoT. We propose that YtfK activates the stringent response by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis.

Structure, Function, and Evolution of the Pseudomonas aeruginosa Lysine Decarboxylase LdcA

The only enzyme responsible for cadaverine production in the major multidrug-resistant human pathogen Pseudomonas aeruginosa is the lysine decarboxylase LdcA. This enzyme modulates the general polyamine homeostasis, promotes growth, and reduces bacterial persistence during carbenicillin treatment. Here we present a 3.7-Å resolution cryoelectron microscopy structure of LdcA. We introduce an original approach correlating phylogenetic signal with structural information and reveal possible recombination among LdcA and arginine decarboxylase subfamilies within structural domain boundaries. We show that LdcA is involved in full virulence in an insect pathogenesis model. Furthermore, unlike its enterobacterial counterparts, LdcA is regulated neither by the stringent response alarmone ppGpp nor by the AAA+ ATPase RavA. Instead, the P.aeruginosa ravA gene seems to play a defensive role. Altogether, our study identifies LdcA as an important player in P.aeruginosa physiology and virulence and as a potential drug target.

Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA.

Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.

Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction.

The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein-ligand interactions.

Biochemical determinants of ObgE-mediated persistence.

Obg is a versatile GTPase that plays a pivotal role in bacterial persistence. We previously showed that the Escherichia coli homolog ObgE exerts this activity through transcriptional activation of a toxin-antitoxin module and subsequent membrane depolarization. Here, we assessed the role of G-domain functionality in ObgE-mediated persistence. Through screening of a mutant library, we identified five obgE alleles (with substitutions G166V, D246G, S270I, N283I and I313N) that have lost their persistence function and no longer activate hokB expression. These alleles support viability of a strain otherwise deprived of ObgE, indicating that ObgE's persistence function can be uncoupled from its essential role. Based on the ObgE crystal structure, we designed two additional mutant proteins (T193A and D286Y), one of which (D286Y) no longer affects persistence. Using isothermal titration calorimetry, stopped-flow experiments and kinetics, we subsequently assessed nucleotide binding and GTPase activity in all mutants. With the exception of the S270I mutant that is possibly affected in protein-protein interactions, all mutants that have lost their persistence function display severely reduced binding to GDP or the alarmone ppGpp. However, we find no clear relation between persistence and GTP or pppGpp binding nor with GTP hydrolysis. Combined, our results signify an important step toward understanding biochemical determinants underlying persistence.

Evolution of the bacterial nucleosidase PpnN and its relation to the stringent response.

In our recent publication (Zhang et al., 2019), we demonstrate an interesting mode of regulation of purine metabolism unique to Proteobacteria. In this microreview, we would like to reflect on the ideas put forward, with special focus on protein domain architecture of the enzyme involved, its orthologues in plants, and the implications of the differential effects observed between binding of the two alarmone molecules, ppGpp (guanosine 3′,5′-bisdiphosphate) and pppGpp (guanosine-5′-triphosphate-3′-diphosphate). In our previous work, we showed that the Escherichia coli nucleotide 5'-monophosphate nucleosidase, PpnN, which is conserved in Proteobacteria, cleaves its preferred substrate, guanosine monophosphate (GMP), at a much higher rate in the presence of both pppGpp and ppGpp (Figure 1A). Structural analysis reveals that binding of pppGpp leads to a conformational change in the protein that exposes its active site, suggesting this is the reason for the observed increase in activity. Finally, point mutation of the alarmone-interacting residues show a defect in binding, resulting in (i) increased basal catalytic activity of PpnN and higher competitive fitness of E. coli in an environment with fluctuating nutrient levels, and (ii) increased bacterial sensitivity towards antibiotics. In contrast, complete loss of the ppnN gene has the inverse effect, i.e. reduced competitive growth and improved antibiotic tolerance. We used these observations to propose a model in which E. coli uses PpnN to balance the need of fitness (fast growth) against tolerance towards antibiotics to improve survival.

The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus.

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.

The expanded specificity and physiological role of a widespread N-degron recognin

All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacterium Salmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

Bacterial nucleosidase is regulated by the alarmone (p)ppGpp

Bacterial nucleosidase is regulated by the alarmone (p)ppGpp

Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus

Natural competence for genetic transformation refers to the natural ability of various bacteria to take up exogenous DNA from their surroundings and to incorporate internalized genetic information into their genomes. By promoting bacterial diversification and adaptability, this process represents a major driving force in bacterial evolution. Micrococcus luteus was one of the first organisms used to study natural transformation in bacteria. Since then, however, only very little information about this phenomenon has been reported in M. luteus or in any member of the Actinobacteria phylum (low-GC Gram-positive bacteria). Previous work in our group indicated major differences between the transformation apparatus of M. luteus and the transformation machinery described for various Gram-negative and Gram-positive model bacteria belonging to the phyla Proteobacteria and Firmicutes (high-GC Gram-positive bacteria). This prompted us to initiate a study concerning the regulation mechanism of competence development in M. luteus. In this report, we identify amino acids as a nutritional factor that influences competence in a concentration-dependent manner. By using a transcriptional reporter strain for one of the late competence genes, we demonstrate how increasing concentrations of both amino acids mixtures and single amino acids supplemented to the growth medium affect transformability on transcriptional and post-transcriptional level. Furthermore, we revisit previously generated auxotrophic mutants to show that the transformation machinery is turned down during a state of extreme hunger for amino acids presumably as a part of a general response to auxotrophy. Finally, by generating and analysing knockout mutants for two predicted stringent response enzymes, we provide evidence for the involvement of the alarmone (p)ppGpp as a putative mediator of the effects on transformation development caused by amino acids. As a member of the Actinobacteria phylum, M. luteus could serve as a model for other representatives of the phylum, including a number of important human pathogens.

Abstract 2667: Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ

Abstract The capacity of tumor cells to survive various stresses is an essential property that endows them invulnerability. One of the bacterial key adaptive strategies against nutrient deprivation is stringent response. In bacteria, nutrient deprivation leads to an elevation of a signaling alarmone, guanosine tetraphosphate ((p)ppGpp) to repress the protein synthesis and proliferation activity in order to maintain cell viability. The accumulation of (p)ppGpp is partially caused by the repression of its hydrolase, SpoT. However, this strategy still remains elusive in humans, but the human ortholog of SpoT-MESH1 has been discovered. MESH1 also has the hydrolase activity on (p)ppGpp in vitro, but human cells lack the required alarmone (p)ppGpp and its synthetase, which restricts the discovery of stringent response in humans. Unexpectedly, our recent data suggests that MESH1 depletion allows cell survival under cystine deprivation, an extremely oxidative stress, yet with a slow proliferation rate. These correspond to bacterial stringent response phenotypes. Moreover, our gene expression profiling shows that MESH1 silencing triggers a global transcriptional reprogramming with a similarity to bacterial transcriptional responses of stringent response. Altogether, these evidences inspire us to speculate on the existence of human stringent response with similar cell protective functions, albeit in the absence of (p)ppGpp. In this study, I aim to identify the molecular pathways and regulatory mechanisms that MESH1 adopts to explain the MESH1-related stringent response phenotypes in human cancer cell lines. One interesting phenomenon we observed from our genome-wide RNA screen is that MESH1 silencing significantly represses TAZ RNA level. TAZ serves as a key co-activator involved in the HIPPO pathway that has been well studied to regulate cell proliferation, cell stemness, and etc, which are perfectly relevant to the MESH1-related stringent response phenotypes. Therefore, I will specifically determine the functional roles of TAZ repression upon MESH1 removal in the expression of stringent response features by genetic tools. Furthermore, I will characterize the transcriptional control of TAZ and other gene signatures of human stringent response. We observed that MESH1 depletion significantly represses the acetyl-CoA:CoA ratio and further inhibits the histone acetylation activity that presumably turns off/on certain gene transcription. Based on these facts, I will determine the importance of histone modification on the transcriptional reprogramming upon MESH1 silencing. Successful completion of this study will equip us with more thorough understandings of stress response mechanisms in humans so that we can improve the treatment for stress-related diseases, such as cancer, by providing a novel therapeutic target, MESH1. Note: This abstract was not presented at the meeting. Citation Format: Tianai Sun, Chien-Kuang Ding, Jen-Tsan A. Chi. Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2667.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis of Salmonella enterica serovar Typhi.

How Salmonella enterica serovar Typhi (S.Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S.Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S.Typhi Ty2 strain. In an iron overload mouse model of S.Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S.Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

(p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch.

The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity of PpnN both invitro and invivo causing accumulation of several types of nucleobases during stress. The structure of PpnN reveals a tetramer with allosteric (p)ppGpp binding sites located between subunits. pppGpp binding triggers a large conformational change that shifts the two terminal domains to expose the active site, providing a structural rationale for the stimulatory effect. We find that PpnN increases fitness and adjusts cellular tolerance to antibiotics and propose a model in which nucleotide levels can rapidly be adjusted during stress by simultaneous inhibition of biosynthesis and stimulation of degradation, thus achieving a balanced physiological response to constantly changing environments.

(p)ppGpp: the magic governor of bacterial growth economy.

A fundamental question in microbiology is how bacterial cells manage to coordinate gene expression with cell growth during adapting to various environmental conditions. Although the cellular responses to changing environments have been extensively studied using transcriptomic and proteomic approaches, it remains poorly understood regarding the molecular strategy enabling bacteria to manipulate the global gene expression patterns. The alarmone (p)ppGpp is a key secondary messenger involved in regulating various biochemical and physiological processes of bacterial cells. However, despite of the extensive studies of (p)ppGpp signaling in stringent response during the past 50years, the connection between (p)ppGpp and exponential growth remains poorly understood. Our recent work demonstrates that (p)ppGpp is strongly involved in regulating cell growth of Escherichia coli through balancing the cellular investment on metabolic proteins and ribosomes, highlighting itself as a magic governor of bacterial global resource allocation. In this mini-review, we briefly summarize some historical perspectives and current progress of the relation between (p)ppGpp and bacterial exponential growth. Two important future directions are also highlighted: the first direction is to elucidate the cellular signal that triggers (p)ppGpp accumulation during poor growth conditions; the second direction is to investigate the relation between (p)ppGpp and exponential growth for bacterial species other than E. coli.

TRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Genesis of Antibiotic Resistance (AR) XLIX: Mechanism(s) of Sepsis‐induced Increase in Mortality Cobblestone Antibiotic Resistance Pandemic (ARP)

NCT01663701: is a clinical trial with the principle hypothesis that early fluid resuscitation will significantly decrease in‐hospital mortality in patients with severe sepsis and hypotension. Based on the observations from NCT01663701, 209 adults with sepsis and hypotension presenting to an emergency department in Zambia, a 6‐hour sepsis protocol emphasizing administration of intravenous fluids, vasopressors, and blood transfusion significantly increased in‐hospital mortality compared with usual care (48.1% vs 33.0%, respectively)(Verbatim). Here, we present a rationale for the plausible reason for such an outcome of 48.1% mortality in sepsis protocol group as opposed to expected decrease in the mortality rate with a “time zero” of admittance to emergency room (ER) and 28 days. Based on the intake criteria of admission to ER, observations and limitations of this investigation, it is hypothesized that “Colonization Pressure” (CP) of “Persisters (Prs)” is the most ostensible factor for the sepsis and septic shock induced increase in mortality rate from “time zero” of admittance to ER. “Persisters”(Prs) are antibiotic‐sensitive bacterial populations have a small fraction (~10−6) of slow or non‐growing, antibiotic‐tolerant cells”. Stochastic switch of specific toxin‐antitoxin (TA) expression and alarmone (p) ppGpp has been implicated in evolution of AR. Additional contributing factors could be a. Lack of “Antibiogram” for P. aeruginosa, S. aureus, C. difficile (C. diff), b. failure to implementation of “Antibiotic Stewardship Program”; c. lack of information on the “CP” data on a daily basis. It is suggested that sustained “low tissue perfusion index (PI)”; “Oxygen Saturation of arterial hemoglobin (SpO2)” would hypothetically generate a chronic hypoxic state enabling the emergence of anaerobic gram negative bacilli (GNB)” acquiring antibiotic resistance via multidrug‐resistant (MDR); extensively drug‐resistant (XDR), Gain of function (GOF) mutations and Pandrug‐resistant (PDR) escalating the horizontal gene transfer. Chronic tissue hypoxia would likely to increase the “CP” of GNB due to the sub‐therapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and suboptimal infection control in the setting of outbreaks such as NCT01663701. Lack of information on utilization antifungals via antifungal stewardship is critical to reduce the morbidity & mortality by limiting emergence of MDR in the Intensive Care Units (ICU). Candida bloodstream infections (BSI) / Candidemia and candidiasis in (C.albicans, C.glabrata, C.auris with MDR and XDR attributes with higher “CP” indices transmitted via horizontal transmission (~30%) posing serious challenges in caring for chronically ill (HIV patients) in the long term care facilities, ICU, and also across the globe. Taken together, the observed 48% mortality in NCT01663701 is a consequence of solidification of factors beyond the realm of current clinical practices caring for chronically ill infectious diseases patients (ex. HIV) across the globe leading to ARP.Support or Funding InformationSupported by professional development funds from SWTJC and CME activities of Subburaj Kannan MDThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Chemical Inhibitor of Cell Growth Reduces Cell Size in Bacillus subtilis.

Bacteria exhibit complex responses to biologically active small molecules. These responses include reductions in transcriptional and translational efficiency, alterations in metabolic flux, and in some cases, dramatic changes in growth and morphology. Here, we describe Min-1, a novel small molecule that inhibits growth of Gram-positive bacteria by targeting the cell envelope. Subinhibitory levels of Min-1 inhibits sporulation in Streptomyces venezuelae and reduces growth rate and cell length in Bacillus subtilis. The effect of Min-1 on B. subtilis cell length is significant at high growth rates sustained by nutrient-rich media but drops off when growth rate is reduced during growth on lessenergy-rich carbon sources. In each medium, Min-1 has no impact on the proportion of cells containing FtsZ-rings, suggesting that Min-1 reduces the mass at which FtsZ assembly is initiated. The effect of Min-1 on size is independent of UDP-glucose, which couples cell division to carbon availability, and the alarmone ppGpp, which reduces cell size via its impact on fatty acid synthesis. Min-1 activates the LiaRS stress response, which is sensitive to disruptions in the lipid II cycle and the cell membrane, and also compromises cell membrane integrity. Therefore, this novel synthetic molecule inhibits growth at high concentrations and induces a short-cell phenotype at subinhibitory concentrations that is independent of known systems that influence cell length, highlighting the complex interactions between small molecules and cell morphology.

(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli

Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced. We show that (p)ppGpp is produced and required for a pertinent stress response during interference with outer membrane biogenesis and ADP synthesis specifically. When these processes were perturbed via the transcriptional repression of essential genes, wild type E. coli MG1655 ceased growing and entered a semi-dormant state, whereas isogenic (p)ppGpp0 cells continued to grow uncontrollably to the point of lysis. Furthermore, in vivo measurements revealed that the ATP levels were intrinsically offset in (p)ppGpp0 cells, further indicating a role for the alarmone in cellular energy homeostasis. In summary, our investigation suggests that (p)ppGpp acts as a coordinator of cell growth in response to imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis, elucidating novel roles for (p)ppGpp in bacterial physiology.

Structural basis for (p)ppGpp-mediated inhibition of the GTPase RbgA

Efficient adaptation to environmental changes is pivotal for all bacterial cells. Almost all bacterial species depend on the conserved stringent response system to prompt timely transcriptional and metabolic responses according to stress conditions and nutrient depletion. The stringent response relies on the stress-dependent synthesis of the second messenger nucleotides and alarmones (p)ppGpp, which pleiotropically target and reprogram processes that consume cellular resources, such as ribosome biogenesis. Here we show that (p)ppGpp acts on the ribosome biogenesis GTPase A (RbgA) of Gram-positive bacteria. Using X-ray crystallography, hydrogen-deuterium exchange MS (HDX-MS) and kinetic analysis, we demonstrate that the alarmones (p)ppGpp bind to RbgA in a manner similar to that of binding by GDP and GTP and thereby act as competitive inhibitors. Our structural analysis of Staphylococcus aureus RbgA bound to ppGpp and pppGpp at 1.8 and 1.65 Å resolution, respectively, suggested that the alarmones (p)ppGpp prevent the active GTPase conformation of RbgA by sterically blocking the association of its G2 motif via their 3'-pyrophosphate moieties. Taken together, our structural and biochemical characterization of RbgA in the context of the alarmone-mediated stringent response reveals how (p)ppGpp affects the function of RbgA and reprograms this GTPase to arrest the ribosomal large subunit.