The YmgB-SpoT interaction triggers the stringent response in Escherichia coli.

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion.

Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.

Proteome Dynamics during Antibiotic Persistence and Resuscitation.

During antibiotic persistence, bacterial cells become transiently tolerant to antibiotics by restraining their growth and metabolic activity. Detailed molecular characterization of antibiotic persistence is hindered by low count of persisting cells and the need for their isolation. Here, we used sustained addition of stable isotope-labeled lysine to selectively label the proteome during hipA-induced persistence and hipB-induced resuscitation of Escherichia coli cells in minimal medium after antibiotic treatment. Time-resolved, 24-h measurement of label incorporation allowed detection of over 500 newly synthesized proteins in viable cells, demonstrating low but widespread protein synthesis during persistence. Many essential proteins were newly synthesized, and several ribosome-associated proteins such as RaiA and Sra showed high synthesis levels, pointing to their roles in maintenance of persistence. At the onset of resuscitation, cells synthesized the ribosome-splitting GTPase HflX and various ABC transporters, restored translation machinery, and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. IMPORTANCE While bactericidal antibiotics typically require actively growing cells to exploit their function, persister cells are slowly replicating which makes them tolerant to the lethal action of antimicrobials. Here, we used an established in vitro model of bacterial persistence based on overexpression of the paradigm toxin-antitoxin (TA) system hipA/hipB to devise a generic method for temporal analysis of protein synthesis during toxin-induced persistence and antitoxin-mediated resuscitation. Our time-resolved, 24-h measurement of label incorporation demonstrated low but widespread protein synthesis during persistence. At the onset of resuscitation, cells restored translation machinery and resumed metabolism by inducing glycolysis and biosynthesis of amino acids. Our study provides the first global analysis of protein synthesis in persisting and resuscitating bacterial cells, and as such, presents an unprecedented resource to study the processes governing antibiotic persistence.

NirD curtails the stringent response by inhibiting RelA activity in Escherichia coli.

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 Escherichia coli, 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 E. coli. 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.

YtfK activates the stringent response by triggering the alarmone synthetase SpoT in Escherichia coli.

The stringent response is a general bacterial stress response that allows bacteria to adapt and survive adverse conditions. This reprogramming of cell physiology is caused by the accumulation of the alarmone (p)ppGpp which, in Escherichia coli, depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. Although conditions that control SpoT-dependent (p)ppGpp accumulation have been described, the molecular mechanisms regulating the switching from (p)ppGpp degradation to synthesis remain poorly understood. Here, we show that the protein YtfK promotes SpoT-dependent accumulation of (p)ppGpp in E. coli and is required for activation of the stringent response during phosphate and fatty acid starvation. Our results indicate that YtfK can interact with SpoT. We propose that YtfK activates the stringent response by tilting the catalytic balance of SpoT toward (p)ppGpp synthesis.

HipA-Mediated Phosphorylation of SeqA Does not Affect Replication Initiation in Escherichia coli.

The SeqA protein of Escherichia coli is required to prevent immediate re-initiation of chromosome replication from oriC. The SeqA protein is phosphorylated at the serine-36 (Ser36) residue by the HipA kinase. The role of phosphorylation was addressed by mutating the Ser36 residue to alanine, which cannot be phosphorylated and to aspartic acid, which mimics a phosphorylated serine residue. Both mutant strains were similar to wild-type with respect to origin concentration and initiation synchrony. The minimal time between successive initiations was also unchanged. We therefore suggest that SeqA phosphorylation at the Ser36 residue is silent, at least with respect to SeqA's role in replication initiation.

The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance.

The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high-copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.

Peptide-nucleotide antibiotic Microcin C is a potent inducer of stringent response and persistence in both sensitive and producing cells.

Microcin C (McC) is a peptide-nucleotide antibiotic that inhibits aspartyl-tRNA synthetase. Here, we show that McC is a strong inducer of persistence in Escherichia coli. Persistence induced by McC is mediated by (p)ppGpp and requires chromosomally encoded toxin-antitoxin modules. McC-producing cells have increased persistence levels due to a combined effect of McC imported from the cultured medium and intracellularly synthesized antibiotic. McC-producing cells also induce persistence in sensitive cells during co-cultivation, underscoring complex interactions in bacterial communities where an antagonistic compound produced by one community member can benefit other members by increasing their ability to withstand antibiotics.

Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases.

The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.

Molecular mechanism of bacterial persistence by HipA.

HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.