guanosine-tetraphosphate and serine-hydroxamate

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.

Topics: Amino Acids, Aromatic; Escherichia coli; Escherichia coli Proteins; Glycine; Glyphosate; Guanosine Tetraphosphate; Ligases; Mutation; Serine

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) play a central role in the adaptation of bacterial and plant cells to nutritional and environmental stresses and in bacterial resistance to antibiotics. These compounds have historically been detected and quantified by two-dimensional thin-layer chromatography of

Topics: Chromatography, Liquid; Escherichia coli; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Hydrolysis; Isotopes; Kinetics; Mutation; Nucleotidyltransferases; Serine; Spectrometry, Mass, Electrospray Ionization

The stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro, implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo. Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.

Topics: Amino Acids; DNA Replication; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Guanosine Tetraphosphate; Monomeric GTP-Binding Proteins; Serine

The stringent response is the initial reaction of microorganisms to nutritional stress. During stringent response the small nucleotides (p)ppGpp act as global regulators and reprogram bacterial transcription. In this work, the genetic network controlled by the stringent response was characterized in the amino acid-producing Corynebacterium glutamicum.. The transcriptome of a C. glutamicum rel gene deletion mutant, unable to synthesize (p)ppGpp and to induce the stringent response, was compared with that of its rel-proficient parent strain by microarray analysis. A total of 357 genes were found to be transcribed differentially in the rel-deficient mutant strain. In a second experiment, the stringent response was induced by addition of DL-serine hydroxamate (SHX) in early exponential growth phase. The time point of the maximal effect on transcription was determined by real-time RT-PCR using the histidine and serine biosynthetic genes. Transcription of all of these genes reached a maximum at 10 minutes after SHX addition. Microarray experiments were performed comparing the transcriptomes of SHX-induced cultures of the rel-proficient strain and the rel mutant. The differentially expressed genes were grouped into three classes. Class A comprises genes which are differentially regulated only in the presence of an intact rel gene. This class includes the non-essential sigma factor gene sigB which was upregulated and a large number of genes involved in nitrogen metabolism which were downregulated. Class B comprises genes which were differentially regulated in response to SHX in both strains, independent of the rel gene. A large number of genes encoding ribosomal proteins fall into this class, all being downregulated. Class C comprises genes which were differentially regulated in response to SHX only in the rel mutant. This class includes genes encoding putative stress proteins and global transcriptional regulators that might be responsible for the complex transcriptional patterns detected in the rel mutant when compared directly with its rel-proficient parent strain.. In C. glutamicum the stringent response enfolds a fast answer to an induced amino acid starvation on the transcriptome level. It also showed some significant differences to the transcriptional reactions occurring in Escherichia coli and Bacillus subtilis. Notable are the rel-dependent regulation of the nitrogen metabolism genes and the rel-independent regulation of the genes encoding ribosomal proteins.

Topics: Bacterial Proteins; Corynebacterium glutamicum; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes, Bacterial; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Histidine; Ligases; Models, Genetic; Mutation; Oligonucleotide Array Sequence Analysis; Operon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Time Factors

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Topics: Adaptation, Physiological; Adenosine Triphosphate; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Guanosine Tetraphosphate; Guanosine Triphosphate; IMP Dehydrogenase; Ligases; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Ribosomal Proteins; Ribosomes; RNA, Bacterial; RNA, Ribosomal, 23S; RNA, Ribosomal, 5S; Sequence Alignment; Serine; Tetracycline; Thermus thermophilus; Thiostrepton; Transcription, Genetic; Uracil

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.

Topics: Amino Acids; Animals; Bacterial Proteins; Cystic Fibrosis; Drosophila melanogaster; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Mutation; Pancreatic Elastase; Pseudomonas aeruginosa; Pyocyanine; Serine; Sigma Factor; Virulence

Accumulation of 16S rRNA and production of guanosine polyphosphates (pppGpp and ppGpp) were studied during amino acid starvation in three wild-type strains of Helicobacter pylori. All strains exhibit a relaxed phenotype with respect to accumulation of 16S rRNA. This constitutes the first example of a wild-type eubacterium showing a relaxed phenotype. The guanosine polyphosphate levels do not rise as a result of amino acid starvation, as expected for relaxed organisms. However, in both growing and starved cells, basal levels of the two polyphosphates appeared to be present, demonstrating that the enzymatic machinery for guanosine polyphosphate production is present in this organism. These findings are discussed within the framework of the hypothesis that stringent control is a physiological control mechanism more important for the fitness of prokaryotes growing in the general environment than for those that inhabit protected niches.

Topics: Amino Acids; Bacterial Proteins; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Helicobacter pylori; Kinetics; Mupirocin; Protein Biosynthesis; RNA, Ribosomal, 16S; Serine; Species Specificity; Time Factors; Transcription, Genetic

The effects of mutations in the promoter of the histidine operon of Salmonella typhimurium were examined in vivo. The wild-type chromosomal copy of the his promoter was replaced with mutations in the -10 hexamer sequence and in the region between the -10 hexamer and the transcriptional start point-termed the discriminator sequence. The substitutions were performed with a phage M13 allele replacement system. Expression of the his operon is known to correlate with levels of guanosine 5',3'-bispyrophosphate (ppGpp) in vivo. Strains containing either the wild-type his promoter or his promoter mutations were grown in both nutrient-rich and minimal media under steady-state conditions known to alter intracellular levels of ppGpp in a predictable way. The effect of the presence or absence of the his attenuator was assessed under these conditions as well. Expression of the his operon was studied by measuring the differential rate of beta-galactosidase synthesis with a his-lac transcriptional fusion. Regulation of the his operon in the promoter mutants was also studied under conditions of a transient amino acid downshift induced by the addition of serine hydroxamate to cultures growing in nutrient-rich medium. These growth conditions cause elevated levels of ppGpp. The results provide physiological confirmation of previous evidence obtained with a coupled transcription-translation system in vitro which indicated that ppGpp regulates interaction of RNA polymerase at the his promoter. More specifically, the in vivo evidence shows that the region of the his promoter that includes the -10 hexamer and discriminator sequences is the target at which ppGpp stimulates transcription.

Topics: Amino Acids; Culture Media; Gene Expression Regulation, Bacterial; Guanosine Tetraphosphate; Histidine; Mutagenesis, Site-Directed; Mutation; Operon; Promoter Regions, Genetic; Salmonella typhimurium; Serine

The reversion rates of two isogenic Escherichia coli K-12 auxotrophs differing only in relA have been determined in the absence or presence of serine hydroxamate, which provokes the stringent response. Reversion rates of leuB- and argH- were significantly higher in the relA+ than in the relA- strain, and the reversion rates in both strains were enhanced by serine hydroxamate. A positive correlation was established between reversion rates and the synthesis of guanosine-5'-diphosphate-3'-diphosphate in the absence and presence of serine hydroxamate. It is proposed that mutation rates are dependent upon rates of transcription and upon the genes which regulate the level of the signal nucleotide, guanosine tetraphosphate.

Topics: 3-Isopropylmalate Dehydrogenase; Alcohol Oxidoreductases; Arginine; Escherichia coli; Guanosine Tetraphosphate; Ligases; Mutation; Serine

The stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of actIII occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis alone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).

Topics: Amino Acids; Anthraquinones; Anti-Bacterial Agents; Genes, Bacterial; Guanosine Tetraphosphate; Kinetics; Promoter Regions, Genetic; RNA, Bacterial; RNA, Ribosomal; Serine; Single-Strand Specific DNA and RNA Endonucleases; Streptomyces; Transcription, Genetic

Igarashi et al. (K. Igarashi, N. Fujita, and A. Ishihama, Nucleic Acids Res. 17:8755-8765, 1989) reported that the omega (omega) subunit of Escherichia coli RNA polymerase was required for stringent control as judged by in vitro transcription assays in the presence and absence of guanosine 3',5'-bispyrophosphate (ppGpp). This conclusion predicts that a deletion of the omega gene (designated rpoZ or spoS) should show a relaxed RNA control phenotype in vivo. However, we find that wild-type stringent control of stable RNA accumulation is unaffected by a spoS null allele that abolishes cellular production of the omega protein. We conclude that omega protein is not necessary for the operation of the stringent RNA control response.

Topics: DNA-Directed RNA Polymerases; Escherichia coli; Guanosine Tetraphosphate; RNA, Bacterial; Serine; Transcription, Genetic

Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.

Topics: Base Sequence; Blotting, Western; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanosine Tetraphosphate; Molecular Sequence Data; Mutation; Operon; Plasmids; Promoter Regions, Genetic; Serine; Transcription, Genetic

We have analyzed the correlation of attenuator-independent expression of the Salmonella typhimurium histidine operon in vivo with levels of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate. Amino acid downshift caused by serine hydroxamate addition increased his expression in a relA+ strain and decreased his expression in a relA mutant, whereas levels of guanosine 5'-diphosphate-3'-diphosphate varied in parallel with the changes in his expression in the two strains. In several experiments, overall variations in his expression ranged from 20- to 60-fold after downshift. The mild downshift allowed growth of the cultures to continue at near-preshift rates. Serine hydroxamate addition was also used to analyze the effect of amino acid downshift on induced expression of wild-type and mutant lac promoters. There was a 12-fold difference in lac expression when a relA+-relA1 pair was subjected to mild starvation but only a 3-fold difference when the strains carried the lacZpL8UV5 promoter mutation. These results suggest that guanosine 5'-diphosphate-3'-diphosphate stimulates gene expression in vivo at the level of transcription initiation.

Topics: Amino Acids; Genotype; Guanine Nucleotides; Guanosine Tetraphosphate; Histidine; Kinetics; Mutation; Operon; Promoter Regions, Genetic; Salmonella typhimurium; Serine; Species Specificity

The accumulation of RNA and protein and the kinetics of nucleoside triphosphate and guanosine polyphosphate pools during amino acid starvation and carbon source downshift were investigated in Streptomyces hygroscopicus. RNA accumulation was controlled stringently during both amino acid starvation and carbon source downshift. The pool size of ppGpp increased dramatically under these conditions. However, the intracellular concentrations of nucleoside triphosphates were low and the concentration of guanosine polyphosphates was much lower than in Escherichia coli. The possible significance of this phenomenon in the regulation is discussed.

Topics: Adenosine Triphosphate; Amino Acids; Autoradiography; Bacterial Proteins; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Guanosine Triphosphate; Methylglucosides; Methylglycosides; Nucleotides; RNA, Bacterial; Serine; Streptomyces

Lack of three different amino acids or treatment with the analogue DL-serine hydroxamate does not induce the accumulation of ppGpp and pppGpp, the 3'-pyrophosphates of GDP and GTP, respectively, in Rhizobium meliloti strain 41. Surprisingly, RNA accumulation is controlled under the above mentioned conditions stringently. Moreover, no significant RNA accumulation was found during chloramphenicol, tetracycline, and streptomycin treatment, suggesting that R. meliloti, unlike any other bacteria in investigated so far, is not able to accumulate RNA without ongoing protein synthesis. On the other hand, lack of carbon source and ammonium starvation result in a significant ppGpp accumulation.

Topics: Adenosine Triphosphate; Amino Acids; Bacterial Proteins; Chloramphenicol; Guanine Nucleotides; Guanosine Tetraphosphate; Guanosine Triphosphate; Kinetics; Phenylalanine; Rhizobium; RNA, Bacterial; Serine