The relA/spoT-homologous gene in Streptomyces coelicolor encodes both ribosome-dependent (p)ppGpp-synthesizing and -degrading activities.
ABSTRACT: Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.
Project description:We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.
Project description:The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.
Project description:In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.
Project description:Arabidopsis RPP5 is a member of a large class of pathogen resistance genes encoding nucleotide-binding sites and leucine-rich repeat domains. Yeast two-hybrid analysis showed that RPP5 specifically interacts with At-RSH1, an Arabidopsis RelA/SpoT homolog. In Escherichia coli, RelA and SpoT determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotides of the bacterial stringent response. Functional analysis in E. coli and in Streptomyces coelicolor A3 (2) showed that At-RSH1 confers phenotypes associated with (p)ppGpp synthesis. We characterized two additional Arabidopsis RelA/SpoT homologs, At-RSH2 and At-RSH3. At-RSH genes may regulate a rapid plant (p)ppGpp-mediated response to pathogens and other stresses.
Project description:Stringent response mediated by modified guanosine nucleotides is conserved across bacteria and is regulated through the Rel/Spo functions. In Escherichia coli, RelA and SpoT proteins synthesize the modified nucleotides ppGpp and pppGpp, together referred to as (p)ppGpp. SpoT is also the primary (p)ppGpp hydrolase. In this study, using hypomorphic relA alleles, we provide experimental evidence for SpoT-mediated negative regulation of the amplification of RelA-dependent stringent response. We investigated the kinetics of ppGpp degradation in cells recovering from stringent response in the complete absence of SpoT function. We found that, although greatly diminished, there was slow ppGpp degradation and growth resumption after a lag period, concomitant with decrease in ppGpp pool. We present evidence for reduction in the ppGpp degradation rate following an increase in pppGpp pool, during recovery from stringent response. From a genetic screen, the nudix hydrolases MutT and NudG were identified as over-expression suppressors of the growth defect of ?spoT and ?spoT ?gppA strains. The effect of over-expression of these hydrolases on the stringent response to amino acid starvation and basal (p)ppGpp pool was studied. Over-expression of each hydrolase reduced the strength of the stringent response to amino acid starvation, and additionally, perturbed the ratio of ppGpp to pppGpp in strains with reduced SpoT hydrolase activity. In these strains that do not accumulate pppGpp during amino acid starvation, the expression of NudG or MutT supported pppGpp accumulation. This lends support to the idea that a reduction in the SpoT hydrolase activity is sufficient to cause the loss of pppGpp accumulation and therefore the phenomenon is independent of hydrolases that target pppGpp, such as GppA.
Project description:Deletion of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives of S. coelicolor relA that could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducible tipA promoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in a relA null mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion of relA had little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A second S. coelicolor RelA homologue (RshA, with 42% identity to S. coelicolor RelA) was identified in the genome sequence. However, deletion of rshA had no effect on the ability of the relA mutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction of tipAp::rshA in the relA mutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in the relA mutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression of rshA.
Project description:During amino acid starvation the Escherichia coli stringent response factor RelA recognizes deacylated tRNA in the ribosomal A-site. This interaction activates RelA-mediated synthesis of alarmone nucleotides pppGpp and ppGpp, collectively referred to as (p)ppGpp. These two alarmones are synthesized by addition of a pyrophosphate moiety to the 3' position of the abundant cellular nucleotide GTP and less abundant nucleotide GDP, respectively. Using untagged native RelA we show that allosteric activation of RelA by pppGpp increases the efficiency of GDP conversion to achieve the maximum rate of (p)ppGpp production. Using a panel of ribosomal RNA mutants, we show that the A-site finger structural element of 23S rRNA helix 38 is crucial for RelA binding to the ribosome and consequent activation, and deletion of the element severely compromises (p)ppGpp accumulation in E. coli upon amino acid starvation. Through binding assays and enzymology, we show that E. coli RelA does not form a stable complex with, and is not activated by, deacylated tRNA off the ribosome. This indicates that in the cell, RelA first binds the empty A-site and then recruits tRNA rather than first binding tRNA and then binding the ribosome.
Project description:During the diauxic shift, Escherichia coli exhausts glucose and adjusts its expression pattern to grow on a secondary carbon source. Transcriptional profiling studies of glucose-lactose diauxic transitions reveal a key role for ppGpp. The amount of ppGpp depends on RelA synthetase and the balance between a strong SpoT hydrolase and its weak synthetase. In this study, mutants are used to search for synthetase or hydrolase specific regulation. Diauxic shifts experiments were performed with strains containing SpoT hydrolase and either RelA or SpoT synthetase as the sole source of ppGpp. Here, the length of the diauxic lag times is determined by the presence of ppGpp, showing contributions of both ppGpp synthetases (RelA and SpoT) as well as its hydrolase (SpoT). A balanced ppGpp response is key for a proper adaptation during diauxic shift. The effects of one or the other ppGpp synthetase on diauxic shifts are abolished by addition of amino acids or succinate, although by different mechanisms. While amino acids control the RelA response, succinate blocks the uptake of the excreted acetate via SatP. Acetate is converted to Acetyl-CoA through the ackA-pta pathway, producing Ac-P as intermediate. Evidence of control of the ackA-pta operon as well as a correlation between ppGpp and Ac-P is shown. Finally, acetylation of proteins is shown to occur during a diauxic glucose-lactose shift.
Project description:Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and, based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. This work is an important step forward towards understanding the complex regulatory network underlying the oxidative stress response of F. tularensis.
Project description:Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.