Glyphosate induces the synthesis of ppGpp.

Glyphosate, the most widely used herbicide in both agricultural and urban areas is toxic for plants and for many bacterial species. The mechanism of action of glyphosate is through the inhibition of the EPSP synthase, a key enzyme in the biosynthetic pathway of aromatic amino acids. Here we show that glyphosate induces the stringent response in Escherichia coli. Bacteria treated with glyphosate stop growing and accumulate ppGpp. Both growth arrest and ppGpp accumulation are restored to normal levels upon addition of aromatic amino acids. Glyphosate-induced ppGpp accumulation is dependent on the presence of the (p)ppGpp synthetase RelA. However, unlike other cases of amino acid starvation, pppGpp could not be discerned. In a gppA background both ppGpp and pppGpp accumulated when exposed to glyphosate. Conversely, the wild-type strain and gppA mutant treated with serine hydroxamate accumulated high levels of both ppGpp and pppGpp. Altogether, the data indicate that glyphosate induces amino acid starvation resulting in a moderate accumulation of ppGpp and a reversible stringent response.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

Overexpression of RelA/SpoT homologs, PpRSH2a and PpRSH2b, induces the growth suppression of the moss Physcomitrella patens.

Two genes encoding RelA/SpoT homologs, PpRSH2a and PpRSH2b, which are involved in the synthesis of bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp) for the stringent response, were isolated from the moss, Physcomitrella patens. A complementary analysis of PpRSH2a and PpRSH2b in Escherichia coli showed that these genes had ppGpp biosynthetic activity. The recombinant PpRSH2a and PpRSH2b were also shown to synthesize ppGpp in vitro. Both proteins were localized to the chloroplasts of P. patens. Expression of the PpRSH genes was induced upon treatment with abscisic acid or abiotic stresses, such as dehydration and UV irradiation. Overexpression of PpRSH2a and PpRSH2b caused suppression of the growth in response to 1% (w/v) of glucose. The present study suggests the existence of a mechanism to regulate the growth of P. patens, which is governed by plant RSH in chloroplasts.

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis.

Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

Differential regulation by ppGpp versus pppGpp in Escherichia coli.

Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5'-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-σ(70) holoenzyme with ppGpp and pppGpp. We find that both nucleotides bind to a site at the interface between ß' and ω subunits.

A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress?

Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp(0) phenotypes. The null mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

Positive allosteric feedback regulation of the stringent response enzyme RelA by its product.

During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.

[Characterization of the Rela/SpoT homologue slr1325 (syn-rsh) of the cyanobacterium Synechocystis sp. PCC6803].

OBJECTIVE: Nucleotide guanosine-3', 5'-(bis) pyrophosphate (ppGpp) synthesized by (ppGpp) synthesase RelA or bifunctional ppGpp synthase/degradase RelA/SpoT, mediates bacterial stringent response to various stressful conditions. Here we characterized the slr1325 (syn-rsh) gene encoding a RelA/SpoT homolog (Syn-RSH) of the cyanobacterium Synechocystis sp. PCC6803. METHODS: We performed phenotypic complement test using Escherichia coli strain with(p) ppGpp-synthesis defect to determine Syn-RSH function(s), and employed chromatographic analysis of 32P-labeled cellular mononucleotides to detect the accumulation of ppGpp in Escherichia coli strains expressing Syn-RSH and in Synechocystis sp. PCC6803. RESULTS: Syn-RSH expression in E. coli relA/spoT double mutant was able to restore the cell growth arrest; Chromatographic analysis of 32P-labeled cellular mononucleotides revealed that Syn-RSH expression resulted in the synthesis of ppGpp in E. coli strain with relA and spoT mutant mutation. Additionally, Synechocystis cells accumulated a low level of ppGpp under laboratory growth conditions. CONCLUSION: Syn-RSH possesses ppGpp synthase/degradase activities, and ppGpp is required for Synechocystis cell viability under normal growth conditions.

Signalling by the global regulatory molecule ppGpp in bacteria and chloroplasts of land plants.

The hyperphosphorylated guanine ribonucleotide ppGpp mediates the stringent response in bacteria. Biochemical and genetic studies of this response in Escherichia coli have shown that the biosynthesis of ppGpp is catalysed by two homologous enzymes, RelA and SpoT. RelA is activated in response to amino acid starvation, and SpoT responds to abiotic physical stress beside nutritional stress. All free-living bacteria, including Gram-positive firmicutes, contain RelA-SpoT homologues (RSH). Further, novel ppGpp biosynthetic enzymes, designated small alarmone synthetases (SASs), were recently identified in a subset of bacteria, including the Gram-positive organism Bacillus subtilis, and were shown to consist only of a ppGpp synthetase domain. Studies suggest that these SAS proteins contribute to ppGpp signalling in response to stressful conditions in a manner distinct from that of RelA-SpoT enzymes. SAS proteins currently appear to always occur in addition to RSH enzymes in various combinations but never alone. RSHs have also been identified in chloroplasts, organelles of photosynthetic eukaryotes that originated from endosymbiotic photosynthetic bacteria. These chloroplast RSHs are exclusively encoded in nuclear DNA and targeted into chloroplasts. The findings suggest that ppGpp may regulate chloroplast functions similar to those regulated in bacteria, including transcription and translation. In addition, a novel ppGpp synthetase that is regulated by Ca²âº as a result of the presence of two EF-hand motifs at its COOH terminus was recently identified in chloroplasts of land plants. This finding indicates the existence of a direct connection between eukaryotic Ca²âº signalling and prokaryotic ppGpp signalling in chloroplasts. The new observations with regard to ppGpp signalling in land plants suggest that such signalling contributes to the regulation of a wider range of cellular functions than previously anticipated.

RelA regulates virulence and intracellular survival of Francisella novicida.

Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis.

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.

Role of RelA of Streptococcus mutans in global control of gene expression.

The production of (p)ppGpp by Streptococcus mutans UA159 is catalyzed by three gene products: RelA, RelP, and RelQ. Here, we investigate the role of the RelA (Rel) homologue of S. mutans in the stringent response and in the global control of gene expression. RelA of S. mutans was shown to synthesize pppGpp in vitro from GTP and ATP in the absence of added ribosomes, as well as in vivo in an Escherichia coli relA-spoT mutant. Mupirocin (MUP) was shown to induce high levels of (p)ppGpp production in S. mutans in a relA-dependent manner, with a concomitant reduction in GTP pools. Transcription profiling after MUP treatment of S. mutans revealed that 104 genes were upregulated and 130 were downregulated (P < or = 0.001); mainly, genes for macromolecular biosynthesis, translation, and energy metabolism were downregulated. When a derivative of UA159 carrying a complete deletion of the relA gene was treated with MUP, 72 genes were upregulated and 52 were downregulated (P < or = 0.001). The expression of 50 genes (P < or = 0.001) was commonly affected by MUP treatment in the two strains, suggesting that S. mutans can mount a relA-independent response to MUP. Consistent with the gene expression profiling, RelA was shown to play major roles in the regulation of phenotypic traits that are required for establishment, persistence, and virulence expression by this oral pathogen. Thus, RelA is the major (p)ppGpp synthase controlling the stringent response in S. mutans, and it coordinates the expression of genes and phenotypes that contribute to the pathogenic potential of the organism.

RelA-dependent (p)ppGpp production controls exoenzyme synthesis in Erwinia carotovora subsp. atroseptica.

In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen Erwinia carotovora subsp. atroseptica. A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type E. carotovora subsp. atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule, N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in E. carotovora subsp. atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.

The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2).

BACKGROUND: Regulation of production of the translational apparatus via the stringent factor ppGpp in response to amino acid starvation is conserved in many bacteria. However, in addition to this core function, it is clear that ppGpp also exhibits genus-specific regulatory effects. In this study we used Affymetrix GeneChips to more fully characterize the regulatory influence of ppGpp synthesis on the biology of Streptomyces coelicolor A3(2), with emphasis on the control of antibiotic biosynthesis and morphological differentiation. RESULTS: Induction of ppGpp synthesis repressed transcription of the major sigma factor hrdB, genes with functions associated with active growth, and six of the thirteen conservons present in the S. coelicolor genome. Genes induced following ppGpp synthesis included the alternative sigma factor SCO4005, many for production of the antibiotics CDA and actinorhodin, the regulatory genes SCO4198 and SCO4336, and two alternative ribosomal proteins. Induction of the CDA and actinorhodin clusters was accompanied by an increase in transcription of the pathway regulators cdaR and actII-ORF4, respectively. Comparison of transcriptome profiles of a relA null strain, M570, incapable of ppGpp synthesis with its parent M600 suggested the occurrence of metabolic stress in the mutant. The failure of M570 to sporulate was associated with a stalling between production of the surfactant peptide SapB, and of the hydrophobins: it overproduced SapB but failed to express the chaplin and rodlin genes. CONCLUSION: In S. coelicolor, ppGpp synthesis influences the expression of several genomic elements that are particularly characteristic of streptomycete biology, notably antibiotic gene clusters, conservons, and morphogenetic proteins.

Effective symbiosis between Rhizobium etli and Phaseolus vulgaris requires the alarmone ppGpp.

The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the sigma(N)-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific sigma(N) copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.

The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

Inducible expression, enzymatic activity, and origin of higher plant homologues of bacterial RelA/SpoT stress proteins in Nicotiana tabacum.

All living cells possess adaptive responses to environmental stress that are essential to ensuring cell survival. For motile organisms, this can culminate in avoidance or attractile behavior, but for sessile organisms such as plants, stress adaptation is a process of success or failure within the confines of a given environment. Nearly all bacterial species possess a highly evolved system for stress adaptation, known as the "stringent response." This ancient and ubiquitous regulatory response is mediated by production of a second messenger of general stress, the nucleotide guanosine-3',5'-(bis)pyrophosphate (ppGpp), which mediates reprogramming of the global transcriptional output of the cell. Accumulation of ppGpp is stress-induced through the enzymatic activation of the well known bacterial ppGpp synthetases, RelA and SpoT. We have recently discovered homologues of bacterial relA/spoT genes in the model plant Nicotiana tabacum. We hypothesize that these homologues (designated RSH genes for RelA/SpoT homologues) serve a stress-adaptive function in plants analogous with their function in bacteria. In support of this hypothesis, we find 1) inducibility of tobacco RSH gene expression following treatment with jasmonic acid; 2) bona fide ppGpp synthesis activity of purified recombinant Nt-RSH2 protein, and 3) a wide spread distribution of RSH gene expression in the plant kingdom. Phylogenetic analyses identifies a distinct phylogenetic branch for the plant RSH proteins with two subgroups and supports ancient symbiosis and nuclear gene transfer as a possible origin for these stress response genes in plants. In addition, we find that Nt-RSH2 protein co-purifies with chloroplasts in subcellular fractionation experiments. Taken together, our findings implicate a direct mode of action of these ppGpp synthetases with regard to plant physiology, namely regulation of chloroplast gene expression in response to plant defense signals.

Dissection of the mechanism for the stringent factor RelA.

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.