Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus.
ABSTRACT: 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).
Project description:Bacteria respond to stressful growth conditions through a conserved phenomenon of stringent response mediated by synthesis of stress alarmones ppGpp and pppGpp [referred to as (p)ppGpp]. (p)ppGpp synthesis is known to occur by ribosome-associated RelA. In addition, a dual-function protein, SpoT (with both synthetase and hydrolase activities), maintains (p)ppGpp homeostasis. The presence of (p)ppGpp is also known to contribute to antibiotic resistance in bacteria. <i>Mycobacterium smegmatis</i> possesses Arr, which inactivates rifampin by its ADP ribosylation. Arr has been shown to be upregulated in response to stress. However, the roles Arr might play during growth have remained unclear. We show that Arr confers growth fitness advantage to <i>M. smegmatis</i> even in the absence of rifampin. Arr deficiency in <i>M. smegmatis</i> resulted in deficiency of biofilm formation. Further, we show that while Arr does not interact with the wild-type <i>Escherichia coli</i> ribosomes, it interacts with them when the <i>E. coli</i> ribosomal protein L11 (a stringent response regulator) is replaced with its homolog from <i>M. smegmatis</i> The Arr interaction with <i>E. coli</i> ribosomes occurs even when the N-terminal 33 amino acids of its L11 protein were replaced with the corresponding sequence of <i>M. smegmatis</i> L11 (<i>Msm-Eco</i>L11 chimeric protein). Interestingly, Arr interaction with the <i>E. coli</i> ribosomes harboring <i>M. smegmatis</i> L11 or <i>Msm-Eco</i>L11 results in the synthesis of ppGpp <i>in vivo</i> Our study shows a novel role of antibiotic resistance gene <i>arr</i> in stress response.<b>IMPORTANCE</b><i>Mycobacterium smegmatis</i>, like many other bacteria, possesses an ADP-ribosyltransferase, Arr, which confers resistance to the first-line antituberculosis drug, rifampin, by its ADP ribosylation. In this report, we show that in addition to its known property of conferring resistance to rifampin, Arr confers growth fitness advantage to <i>M. smegmatis</i> even when there is no rifampin in the growth medium. We then show that Arr establishes species-specific interactions with ribosomes through the N-terminal sequence of ribosomal protein L11 (a stringent response regulator) and results in ppGpp (stress alarmone) synthesis. Deficiency of Arr in <i>M. smegmatis</i> results in deficiency of biofilm formation. Arr protein is physiologically important both in conferring antibiotic resistance as well as in mediating stringent response.
Project description:The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress response or "stringent" response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the stringent response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA+ CgtA+ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the stringent response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.
Project description:Microorganisms continuously monitor their surroundings and adaptively respond to environmental cues. One way to cope with various stress-related situations is through the activation of the stringent stress response pathway. In <i>Pseudomonas aeruginosa</i> this pathway is controlled and coordinated by the activity of the RelA and SpoT enzymes that metabolize the small nucleotide secondary messenger molecule (p)ppGpp. Intracellular ppGpp concentrations are crucial in mediating adaptive responses and virulence. Targeting this cellular stress response has recently been the focus of an alternative approach to fight antibiotic resistant bacteria. Here, we examined the role of the stringent response in the virulence of <i>P. aeruginosa</i> PAO1 and the Liverpool epidemic strain LESB58. A ?<i>relA</i>/?<i>spoT</i> double mutant showed decreased cytotoxicity toward human epithelial cells, exhibited reduced hemolytic activity, and caused down-regulation of the expression of the alkaline protease <i>aprA</i> gene in stringent response mutants grown on blood agar plates. Promoter fusions of <i>relA</i> or <i>spoT</i> to a bioluminescence reporter gene revealed that both genes were expressed during the formation of cutaneous abscesses in mice. Intriguingly, virulence was attenuated <i>in vivo</i> by the ?<i>relA</i>/?<i>spoT</i> double mutant, but not the <i>relA</i> mutant nor the ?<i>relA</i>/?<i>spoT</i> complemented with either gene. Treatment of a cutaneous <i>P. aeruginosa</i> PAO1 infection with anti-biofilm peptides increased animal welfare, decreased dermonecrotic lesion sizes, and reduced bacterial numbers recovered from abscesses, resembling the phenotype of the ?<i>relA</i>/?<i>spoT</i> infection. It was previously demonstrated by our lab that ppGpp could be targeted by synthetic peptides; here we demonstrated that <i>spoT</i> promoter activity was suppressed during cutaneous abscess formation by treatment with peptides DJK-5 and 1018, and that a peptide-treated <i>relA</i> complemented stringent response double mutant strain exhibited reduced peptide susceptibility. Overall these data strongly indicated that synthetic peptides target the <i>P. aeruginosa</i> stringent response <i>in vivo</i> and thus offer a promising novel therapeutic approach.
Project description:The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. In Bacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that in B. subtilis, a relA mutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that the relA mutant is trapped in a SigD "on" state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility in B. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.
Project description:CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.
Project description:The bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram-negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase-binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ?dksA and (p)ppGpp-deficient ?spoT?relA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA-seq was conducted using ?dksA and ?spoT?relA mutant strains grown in hrp-inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB-dependent transporters. Phenotypically, the ?dksA and ?spoT?relA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non-host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc.
Project description:When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ?relA?relV and ?relA?relV?spoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ?relA?spoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp(0) mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ?relA?spoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp(0) mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine.
Project description: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:Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In <i>Escherichia coli</i>, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in <i>E. coli</i>. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.
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.