Alarmone ppGpp Research Articles

Coupling of cell growth modulation to asymmetric division and cell cycle regulation in Caulobacter crescentus

In proliferating bacteria, growth rate is often assumed to be similar between daughter cells. However, most of our knowledge of cell growth derives from studies on symmetrically dividing bacteria. In many α-proteobacteria, asymmetric division is a normal part of the life cycle, with each division producing daughter cells with different sizes and fates. Here, we demonstrate that the functionally distinct swarmer and stalked daughter cells produced by the model α-proteobacterium Caulobacter crescentus can have different average growth rates under nutrient-replete conditions despite sharing an identical genome and environment. The discrepancy in growth rate is due to a growth slowdown associated with the cell cycle stage preceding DNA replication (the G1 phase), which initiates in the late predivisional mother cell before daughter cell separation. Both progenies experience a G1-associated growth slowdown, but the effect is more severe in swarmer cells because they have a longer G1 phase. Activity of SpoT, which produces the (p)ppGpp alarmone and extends the G1 phase, accentuates the cell cycle-dependent growth slowdown. Collectively, our data identify a coupling between cell growth, the G1 phase, and asymmetric division that C. crescentus may exploit for environmental adaptation through SpoT activity. This coupling differentially modulates the growth rate of functionally distinct daughter cells, thereby altering the relative abundance of ecologically important G1-specific traits within the population.

Direct detection of stringent alarmones (pp)pGpp using malachite green.

The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.

(p)ppGpp is required for virulence of Shigella flexneri.

Infection by the enteric pathogen Shigella flexneri requires transit through the gastrointestinal tract and invasion of and replication within the cells of the host colonic epithelium. This process exposes the pathogen to a range of diverse microenvironments. Furthermore, the unique composition and physical environment of the eukaryotic cell cytosol represents a stressful environment for S. flexneri, and extensive physiological adaptations are needed for the bacterium to thrive. In this work, we show that disrupting synthesis of the stringent response alarmone (p)ppGpp in S. flexneri diminished expression of key virulence genes, including ipaA, ipaB, ipaC, and icsA, and it reduced bacterial invasion and intercellular spread. Deletion of the (p)ppGpp synthase gene relA alone had no effect on S. flexneri virulence, but disruption of both relA and the (p)ppGpp synthase/hydrolase gene spoT resulted in loss of (p)ppGpp synthesis and virulence. While the relA spoT deletion mutant was able to invade a cultured human epithelial cell monolayer, albeit at reduced levels, it was unable to maintain the infection and spread to adjacent cells, as indicated by loss of plaque formation. Complementation with spoT on a plasmid vector restored plaque formation. Thus, SpoT alone is sufficient to provide the necessary level of (p)ppGpp for virulence. These results indicate that (p)ppGpp is required for S. flexneri virulence and adaptation to the intracellular environment, adding to the repertoire of signaling pathways that affect Shigella pathogenesis.

An atypical GdpP enzyme linking cyclic nucleotide metabolism to osmotic tolerance and gene regulation in Mycoplasma bovis

Nucleotide second messengers play an important role in bacterial adaptation to environmental changes. Recent evidence suggests that some of these regulatory molecular pathways were conserved upon the degenerative evolution of the wall-less mycoplasmas. We have recently reported the occurrence of a phosphodiesterase (PDE) in the ruminant pathogen Mycoplasma bovis, which was involved in c-di-AMP metabolism. In the present study, we demonstrate that the genome of this mycoplasma species encodes a PDE of the GdpP family with atypical DHH domains. Characterization of M. bovis GdpP (MbovGdpP) revealed a multifunctional PDE with unusual nanoRNase and single-stranded DNase activities. The alarmone ppGpp was found unable to inhibit c-di-NMP degradation by MbovGdpP but efficiently blocked its nanoRNase activity. Remarkably, MbovGdpP was found critical for the osmotic tolerance of M. bovis under K+ and Na+ conditions. Transcriptomic analyses further revealed the biological importance of MbovGdpP in tRNA biosynthesis, pyruvate metabolism, and several steps in genetic information processing. This study is an important step in understanding the role of PDE and nucleotide second messengers in the biology of a minimal bacterial pathogen.

Basal level of ppGpp coordinates Escherichia coli cell heterogeneity and ampicillin resistance and persistence

The universal stringent response alarmone ppGpp (guanosine penta and tetra phosphates) plays a crucial role in various aspects of fundamental cell physiology (e.g., cell growth rate, cell size) and thus bacterial tolerance to and survival of external stresses, including antibiotics. Besides transient antibiotic tolerance (persistence), ppGpp was recently found to contribute to E. coli resistance to ampicillin. How ppGpp regulates both the persistence and resistance to antibiotics remains incompletely understood. In this study, we first clarified that the absence of ppGpp in E. coli (ppGpp0 strain) resulted in a decreased minimal inhibition concentration (MIC) value of ampicillin but, surprisingly, a higher persistence level to ampicillin during exponential growth in MOPS rich medium. High basal ppGpp levels, thus lower growth rate, did not produce high ampicillin persistence. Importantly, we found that the high ampicillin persistence of the ppGpp0 strain is not due to dormant overnight carry-over cells. Instead, the absence of ppGpp produced higher cell heterogeneity, propagating during the regrowth and the killing phases, leading to higher ampicillin persistence. Consistently, we isolated a suppressor mutation of the ppGpp0 strain that restored the standard MIC value of ampicillin and reduced its cell heterogeneity and the ampicillin persistence level concomitantly. Altogether, we discussed the fundamental role of basal level of ppGpp in regulating cell homogeneity and ampicillin persistence.

Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Guanosine tetraphosphate (ppGpp) is a new player in Brassica napus L. seed development

Rapeseed oil, constituting 12% of global vegetable oil production, is susceptible to quality degradation due to stress-induced incomplete seed degreening, fatty acid oxidation, or poor nutrient accumulation. We hypothesise that the hyperphosphorylated nucleotide alarmone ppGpp (guanosine tetraphosphate), acts as a pivotal regulator of these processes, given its established roles in nutrient management, degreening, and ROS regulation in leaves. Using qPCR, UHPLC-MS/MS, and biochemical methods, our study delves into the impact of ppGpp on seed nutritional value. We observed a positive correlation between ppGpp levels and desiccation, and a negative correlation with photosynthetic pigment levels. Trends in antioxidant activity suggest that ppGpp may negatively influence peroxidases, which are safeguarding against chlorophyll decomposition. Notably, despite increasing ppGpp levels, sugars, proteins and oils appear unaffected. This newfound role of ppGpp in seed development suggests it regulates the endogenous antioxidant system during degreening and desiccation, preserving nutritional quality. Further validation through mutant-based research is needed.

Control of transcription elongation and DNA repair by alarmone ppGpp

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

Bacteriophage antidefense genes that neutralize TIR and STING immune responses

Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.

Sound the (Smaller) Alarm: The Triphosphate Magic Spot Nucleotide pGpp.

It has recently become evident that the bacterial stringent response is regulated by a triphosphate alarmone (pGpp) as well as the canonical tetra- and pentaphosphate alarmones ppGpp and pppGpp [together, (p)ppGpp]. Often dismissed in the past as an artifact or degradation product, pGpp has been confirmed as a deliberate endpoint of multiple synthetic pathways utilizing GMP, (p)ppGpp, or GDP/GTP as precursors. Some early studies concluded that pGpp functionally mimics (p)ppGpp and that its biological role is to make alarmone metabolism less dependent on the guanine energy charge of the cell by allowing GMP-dependent synthesis to continue when GDP/GTP has been depleted. However, recent reports that pGpp binds unique potential protein receptors and is the only alarmone synthesized by the intestinal pathogen Clostridioides difficile indicate that pGpp is more than a stand-in for the longer alarmones and plays a distinct biological role beyond its functional overlap (p)ppGpp.

Glyphosate affects persistence and tolerance but not antibiotic resistance

Glyphosate is a herbicide widely used in food production that blocks the synthesis of aromatic amino acids in plants and in microorganisms and also induces the accumulation of the alarmone (p)ppGpp. The purpose of this study was to investigate whether glyphosate affects the resistance, tolerance or persistence of bacteria towards three different classes of antibiotics and the possible role of (p)ppGpp in this activity. Glyphosate did not affect the minimum inhibitory concentration of the tested antibiotics, but enhanced bacterial tolerance and/or persistence towards them. The upshift in ciprofloxacin and kanamycin tolerance was partially dependent on the presence of relA that promotes (p)ppGpp accumulation in response to glyphosate. Conversely, the strong increase in ampicillin tolerance caused by glyphosate was independent of relA. We conclude that by inducing aromatic amino acid starvation glyphosate contributes to the temporary increase in E. coli tolerance or persistence, but does not affect antibiotic resistance.

The structure of DarB in complex with RelNTD reveals nonribosomal activation of Rel stringent factors.

Rel stringent factors are bifunctional ribosome-associated enzymes that catalyze both synthesis and hydrolysis of the alarmones (p)ppGpp. Besides the allosteric control by starved ribosomes and (p)ppGpp, Rel is regulated by various protein factors depending on specific stress conditions, including the c-di-AMP-binding protein DarB. However, how these effector proteins control Rel remains unknown. We have determined the crystal structure of the DarB2:RelNTD2 complex, uncovering that DarB directly engages the SYNTH domain of Rel to stimulate (p)ppGpp synthesis. This association with DarB promotes a SYNTH-primed conformation of the N-terminal domain region, markedly increasing the affinity of Rel for ATP while switching off the hydrolase activity of the enzyme. Binding to c-di-AMP rigidifies DarB, imposing an entropic penalty that precludes DarB-mediated control of Rel during normal growth. Our experiments provide the basis for understanding a previously unknown mechanism of allosteric regulation of Rel stringent factors independent of amino acid starvation.

The many roles of cyclic di-AMP to control the physiology of Bacillus subtilis.

The dinucleotide cyclic di-AMP (c-di-AMP) is synthesized as a second messenger in the Gram-positive model bacterium Bacillus subtilis as well as in many bacteria and archaea. Bacillus subtilis possesses three diadenylate cyclases and two phosphodiesterases that synthesize and degrade the molecule, respectively. Among the second messengers, c-di-AMP is unique since it is essential for B. subtilis on the one hand but toxic upon accumulation on the other. This role as an "essential poison" is related to the function of c-di-AMP in the control of potassium homeostasis. C-di-AMP inhibits the expression and activity of potassium uptake systems by binding to riboswitches and transporters and activates the activity of potassium exporters. In this way, c-di-AMP allows the adjustment of uptake and export systems to achieve a balanced intracellular potassium concentration. C-di-AMP also binds to two dedicated signal transduction proteins, DarA and DarB. Both proteins seem to interact with other proteins in their apo state, i.e. in the absence of c-di-AMP. For DarB, the (p)ppGpp synthetase/hydrolase Rel and the pyruvate carboxylase PycA have been identified as targets. The interactions trigger the synthesis of the alarmone (p)ppGpp and of the acceptor molecule for the citric acid cycle, oxaloacetate, respectively. In the absence of c-di-AMP, many amino acids inhibit the growth of B. subtilis. This feature can be used to identify novel players in amino acid homeostasis. In this review, we discuss the different functions of c-di-AMP and their physiological relevance.

Алармоны как фактор персистенции бактерий

The treatment of bacterial infections with antibiotics is significantly complicated because of the adaptive mechanisms employed by bacteria. Chronic and recurrent infections are often linked to bacterial persis-tence, biofilm formation, and antibiotic tolerance. These processes result in reduced metabolic activity, rendering bacteria insensitive to conventional antibiotics that primarily target actively growing cells. The stringent re-sponse, regulated by (p)ppGpp alarmone molecules, serves as a stress adaptation mechanism. It is conserved across numerous bacterial species and plays an important role in long-term survival under nutrient-depleted con-ditions. (p)ppGpp alarmones also play a significant role in bacterial persistence and the formation of biofilms. The pursuit of novel antibacterial agents that specifically target (p)ppGpp synthesis, thereby inhibiting the strin-gent response, presents a promising strategy in the battle against bacterial infections. In this context, alarmone synthetase inhibitors emerge as promising candidates for clinical application, as they have demonstrated their effectiveness in suppressing bacterial survival mechanisms, inhibiting biofilm formation, and reducing antibiotic tolerance and bacterial persistence.

Structure of SpoT reveals evolutionary tuning of catalysis via conformational constraint

Stringent factors orchestrate bacterial cell reprogramming through increasing the level of the alarmones (p)ppGpp. In Beta- and Gammaproteobacteria, SpoT hydrolyzes (p)ppGpp to counteract the synthetase activity of RelA. However, structural information about how SpoT controls the levels of (p)ppGpp is missing. Here we present the crystal structure of the hydrolase-only SpoT from Acinetobacter baumannii and uncover the mechanism of intramolecular regulation of ‘long’-stringent factors. In contrast to ribosome-associated Rel/RelA that adopt an elongated structure, SpoT assumes a compact τ-shaped structure in which the regulatory domains wrap around a Core subdomain that controls the conformational state of the enzyme. The Core is key to the specialization of long RelA-SpoT homologs toward either synthesis or hydrolysis: the short and structured Core of SpoT stabilizes the τ-state priming the hydrolase domain for (p)ppGpp hydrolysis, whereas the longer, more dynamic Core domain of RelA destabilizes the τ-state priming the monofunctional RelA for efficient (p)ppGpp synthesis.

The alarmone ppGpp selectively inhibits the isoform A of the human small GTPase Sar1.

Transport of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi is mediated by coat protein complex II (COPII). The assembly and disassembly of COPII vesicles is regulated by the molecular switch Sar1, which is a small GTPase and a component of COPII. Usually a small GTPase binds GDP (inactive form) or GTP (active form). Mammals have two Sar1 isoforms, Sar1a and Sar1b, that have approximately 90% sequence identity. Some experiments demonstrated that these two isoforms had distinct but overlapping functions. Here we found another instance of differing behavior: the alarmone ppGpp could bind to and inhibit the GTPase activity of human Sar1a but could not inhibit the GTPase activity of human Sar1b. The crystal structures of Sar1a⋅ppGpp and Sar1b⋅GDP have been determined. Superposition of the structures shows that ppGpp binds to the nucleotide-binding pocket, its guanosine base, ribose ring and 5'-diphosphate occupying nearly the same positions as for GDP. However, its 3'-diphosphate points away from the active site and, hence, away from the surface of the protein. The overall structure of Sar1a⋅ppGpp is more similar to Sar1b⋅GDP than to Sar1b⋅GTP. We also find that the Asp140-Arg138-water-ligand interaction net is important for the binding of ppGpp to Sar1a. This study provides further evidence showing that there are biochemical differences between the Sar1a and Sar1b isoforms of Sar1.

Molecular Characterization of Clinical Rel Mutations and Consequences for Resistance Expression and Fitness in Staphylococcus aureus.

The stringent response (SR) is a universal stress response that acts as a global regulator of bacterial physiology and virulence, and is a contributor to antibiotic tolerance and resistance. In most bacteria, the SR is controlled by a bifunctional enzyme, Rel, which both synthesizes and hydrolyzes the alarmone (p)ppGpp via two distinct catalytic domains. The balance between these antagonistic activities is fine-tuned to the needs of the cell and, in a "relaxed" state, the hydrolase activity of Rel dominates. We have previously shown that two single amino acid substitutions in Rel (that were identified in clinical isolates from persistent infections) confer elevated basal concentrations of (p)ppGpp and consequent multidrug tolerance in Staphylococcus aureus. Here, we explore the molecular details of how these mutations bring about this increase in cellular (p)ppGpp and investigate the wider cellular consequences in terms of resistance expression, resistance development, and bacterial fitness. Using enzyme assays, we show that both these mutations drastically reduce the hydrolase activity of Rel, thereby shifting the balance of Rel activity in favor of (p)ppGpp synthesis. We also demonstrate that these mutations induce high-level, homogeneous expression of β-lactam resistance and confer a significant fitness advantage in the presence of bactericidal antibiotics (but a fitness cost in the absence of antibiotic). In contrast, these mutations do not appear to accelerate the emergence of endogenous resistance mutations in vitro. Overall, our findings reveal the complex nature of Rel regulation and the multifaceted implications of clinical Rel mutations in terms of antibiotic efficacy and bacteria survival.

Effect of a single amino acid substitution G98D in a ribosome-associated essential GTPase, CgtA, on the growth and morphology of Vibrio cholerae

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae.

RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis

The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO4 in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO4 induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO4. These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.

Role of alarmone (p)ppGpp in the regulation of indole formation depending on glucose content in <i> Escherichia coli </i>

Signaling molecules such as indole (product of tryptophan catabolism) and (p)ppGpp (stringent response regulator) are involved in regulation of physiological processes in bacterial cells aimed to adapt to antibiotics and stresses. However, question of existence of relationship between the stringent response and indole signaling requires more detailed investigation.The aim. To study effect of stringent response regulator (p)ppGpp on indole production in Escherichia coli depending on glucose content.Materials and methods. In this work, we studied the dynamics of indole accumulation in batch cultures of parent E. coli BW25141 ((p)ppGpp+ strain) and deletion mutant BW25141∆relA∆spoT ((p)ppGpp0 strain) in glucose-mineral tryptophan-free M9 medium, as well as with 2 mM tryptophan addition. In order to study effect of starvation stress on bacterial cell ability to synthesize indole, we used a model of growth limitation by carbon substrate at two glucose concentrations, 0.1 % and 0.4 %.Results. We have shown here that (p)ppGpp absence in E. coli cells reduces their ability to produce indole in the tryptophan-free medium and significantly slows down the rate of its accumulation in the tryptophan-containing one. Low glucose concentration (0.1 %) leads to decrease in indole production by (p)ppGpp+ cells in the tryptophan-free medium. The presence of indole synthesis precursor, tryptophan, in growth medium, on the contrary, increases the production of indole at lower glucose concentration in both (p)ppGpp+ and (p)ppGpp0 strains demonstrating direct dependence of delay time for onset of indole formation on glucose content, which is more pronounced in the culture of deletion mutant unable of synthesizing (p) ppGpp. The data obtained can be interpreted as result of complex regulatory effect of catabolic repression and the stringent response caused by alarmone (p)ppGpp action on expression level of tnaCAB operon responsible for indole biosynthesis.