Molecular Microbiology

The spheroid bacterium S. aureus is often used as a model of morphogenesis due to its apparent simple cell cycle. S. aureus has many cell division proteins that are conserved across bacteria alluding to common functions. However, despite intensive study we still do not know the roles of many of these components. Here we have examined the functions of the paralogues DivIVA and GpsB in the S. aureus cell cycle. Cells lacking gpsB display a more spherical phenotype than wild type, associated with a decrease in peripheral cell wall peptidoglycan synthesis. This correlates with an increased localisation of penicillin binding proteins at the developing septum, notably PBPs 2 and 3. Our results highlight the role of GpsB as an apparent regulator of cell morphogenesis in S. aureus.

Francisella tularensis is a highly infectious human pathogen that must replicate inside macrophage to cause disease. The ribosomes of F. tularensis can incorporate one of three different paralogs for the small ribosomal subunit protein bS21. One of these paralogs positively impacts translation of key virulence genes and promotes intramacrophage replication. Although ribosomal bS21 content influences F. tularensis virulence, the factors that control bS21 paralog production are not well understood. Here, we reveal that all three bS21 proteins influence the transcript abundance of the paralog important for virulence, bS21-2. In contrast, the other bS21 paralogs (bS21-1 and bS21-3) do not affect their own production. We further determined that the leader sequence of the bS21-2 mRNA is sufficient for bS21-mediated repression of mRNA abundance, suggesting that bS21-2 is autogenously regulated. Yet we determined that the increase in bS21-2-encoding mRNA is not reflected by increased protein production, suggesting that translation of this transcript is controlled by other factors. Finally, we found that bS21-2 exerts at least some of its effects on the bS21-2 transcript by decreasing its stability. Together, our findings suggest that F. tularensis integrates multiple signals into a regulatory network to control the appropriate production of each bS21 paralog, and particularly the paralog important for virulence, bS21-2. This regulatory network in turn may control ribosomal heterogeneity and virulence gene expression.

Biogenesis of the bacterial outer membrane is key to bacterial survival and antibiotic resistance. Central to this is the {beta}-barrel assembly machine (Bam) complex and its associated chaperones, which are responsible for transport, folding and insertion of outer membrane proteins (OMPs). The Escherichia coli Bam complex is composed of two essential subunits, BamA and BamD, and three non-essential accessory lipoproteins, BamB, BamC and BamE. Optimal Bam function is further dependent on the non-essential periplasmic chaperones DegP, Skp and SurA. Despite intensive study, the specific function of these non-essential Bam-associated proteins remains unknown. Here, we analysed {Delta}bamB, {Delta}bamC, {Delta}bamE, {Delta}surA, {Delta}skp and {Delta}degP knockout strains by phenotypic screening, conservation analysis and high-throughput genetics. We reveal that Bam complex activity is impacted by changes in outer membrane lipid composition and that enterobacterial common antigen is essential in the absence of the chaperone SurA. We also show components of peptidoglycan are conditionally essential with Bam accessory lipoproteins and that DNA replication control is perturbed in the absence of specific OMP assembly components. Together, our data indicates potential mechanisms for coordination of OMP biogenesis with other cellular growth processes such as LPS and peptidoglycan biogenesis, and DNA replication control.

Bacterias ability to maintain chromosomal integrity throughout their life cycle is crucial for their survival. In Caulobacter crescentus, the polar factor TipN has been proposed to be involved with the partitioning system ParABS. However, cells with tipN knocked out display subtle parS segregation defects. We hypothesized that TipNs role with parS segregation is obscured by other forces that are ParABS-independent. To test our hypothesis, we removed one of those forces - chromosome replication - and analyzed the role of TipN with ParA. We first demonstrate that ParA retains its ability to transport the centromeric region parS from the stalked pole to the opposite pole in the absence of chromosome replication. Our data revealed that in the absence of chromosome replication, TipN becomes essential for ParAs ability to transport parS. Furthermore, we identify a potential connection between the replication initiator DnaA and TipN. Although TipN is not essential for viability, tipN knockout cells lose viability when the regulation of DnaA levels is altered. Our data suggest that the DnaA-dependent susceptibility of tipN knockout cells is connected to parS segregation. Collectively, this work provides insights into the complex regulation involved in the coordination of chromosome replication and segregation in bacteria.

The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven Serine/Threonine protein kinases (STPKs) phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated but the role of its phosphorylation in regulating its activity has been unclear. In this study we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two regulators of peptidoglycan in Mtb, FhaA and Wag31. We also show that a phospho-mimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulators FhaA, Wag31, CwlM, PknB and PknA, and that the corresponding mutation in Mycobacterium smegmatis (Msmeg) causes mis-regulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.

Trypanosoma brucei and related kinetoplastid parasites possess unique RNA processing pathways, including in their mitochondria, that regulate metabolism and development. Altering RNA composition or conformation through nucleotide modifications is one such pathway, and modifications including pseudouridine regulate RNA fate and function in many organisms. We surveyed pseudouridine synthase (PUS) orthologs in Trypanosomatids, with a particular interest in mitochondrial enzymes due to their potential importance for mitochondrial function and metabolism. T. brucei mt-LAF3 is an ortholog of human and yeast mitochondrial PUS enzymes, and a mitoribosome assembly factor, but structural studies differ in their conclusion as to whether it has PUS catalytic activity. Here, we generated T. brucei cells that are conditionally null for mt-LAF3 and showed that mt-LAF3 loss is lethal and disrupts mitochondrial membrane potential ({Delta}{Psi}m). Addition of a mutant gamma-ATP synthase allele to the conditionally null cells permitted {Delta}{Psi}m maintenance and cell survival, allowing us to assess primary effects on mitochondrial RNAs. As expected, these studies showed that loss of mt-LAF3 dramatically decreases levels of mitochondrial 12S and 9S rRNAs. Notably, we also observed decreases in mitochondrial mRNA levels, including differential effects on edited vs. pre-edited mRNAs, indicating that mt-LAF3 is required for mitochondrial rRNA and mRNA processing, including of edited transcripts. To assess the importance of PUS catalytic activity in mt-LAF3 we mutated a conserved aspartate that is necessary for catalysis in other PUS enzymes and showed it is not essential for cell growth, or maintenance of {Delta}{Psi}m and mitochondrial RNA levels. Together, these results indicate that mt-LAF3 is required for normal expression of mitochondrial mRNAs in addition to rRNAs, but that PUS catalytic activity is not required for these functions. Instead, our work, combined with previous structural studies, suggests that T. brucei mt-LAF3 acts as a mitochondrial RNA-stabilizing scaffold.

Streptococci may enter a physiological state called competence, during which they express a specific set of genes required for exogenous DNA uptake and its subsequent integration into the genome through homologous recombination. This process, termed natural transformation, facilitates the horizontal acquisition of genetic material, potentially conferring adaptive advantages that enhance bacterial survival under selective pressures. To make homologous DNA available in the surrounding environment, Streptococcus pneumoniae expresses a cell wall hydrolase (CbpD) that lyses and kills closely related species. This process has been coined fratricide, and the acting hydrolase a fratricin. A significant fraction of streptococcal species does not encode a CbpD-like protein, but another competence-induced peptidoglycan hydrolase LytF. It has been speculated that LytF serves the same purpose as CbpD, however, our investigations into the role of LytF in Streptococcus sanguinis revealed no evidence supporting LytF as a fratricin. Instead, we show that LytF is involved in natural transformation by promoting DNA uptake. An essential part of DNA uptake is the competence-induced type IV pilus, which facilitates DNA uptake by pulling nearby DNA toward the cell. By immunoblotting and microscopy imaging, we found that LytF increases the extracellular levels of the major pilus component ComGC, suggesting that LytF may modify peptidoglycan to promote pilus extrusion across the cell wall, thereby enhancing the efficiency of DNA uptake. ImportanceStreptococci are a significant cause of severe infections in both humans and animals. They are particularly adept at acquiring new genes through horizontal gene transfer as they can become competent for natural transformation. This allows them to quickly adapt to selective pressure and spread genes involved in virulence and antibiotic resistance. In Streptococcus sanguinis, the competence-induced peptidoglycan hydrolase LytF has been reported to stimulate natural transformation. Our study adds to the understanding of this process by demonstrating that LytF promotes extrusion of the transformation pilus required for DNA uptake.

Regulated gene expression in response to metabolite sensing is a fundamental process for cellular adaptation and survival. Cells have developed diverse strategies to detect and respond to various metabolites in their environment. Here, we have identified a post-transcriptional mechanism in Rhodobacter capsulatus that integrates the periplasmic Cu concentration into the translational control of the copper detoxifying enzyme CutO. This is achieved through Cu-induced stalling of the nascent CutF protein inside the ribosomal peptide tunnel during co-translational secretion. Stalling at a C-terminal proline-rich motif overrides the function of elongation factor P (EF-P) and allows melting of an mRNA stem-loop that shields the cutO ribosome-binding site. Thus, CutF acts as a transmembrane Cu sensor that controls CutO production via ribosomal stalling. Considering that CutF is a member of the widely distributed bacterial DUF2946 protein family, the mechanism identified here likely represents a conserved bacterial strategy for adapting to toxic heavy metals.

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS involved in the control of heme metabolism in Leptospira spirochetes. However, the signals and molecular means by which HemKR is switched on/off, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Here we show that 5-aminolevulinic acid (ALA), a committed porphyrin biosynthesis precursor, triggers the shutdown of the HemKR pathway by stimulating the phosphatase activity of HemK towards phosphorylated HemR. HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Furthermore, HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signalling systems. Such tolerance could be vital during infection in pathogenic Leptospira species, which comprise a conserved HemKR TCS. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model.

Two-component systems (TCSs), consisting of a histidine kinase (HK) and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a HK of the TCS KdpDE, which controls production of the K+ pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high K+ levels, using not its C-terminal HK domain but an N-terminal atypical serine kinase (ASK) domain. Sequence analysis of KdpDs from different species highlights that some KdpDs comprise solely ASK and Usp domains. We show that, while Escherichia coli KdpDs ASK responds directly to K+ levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial K+ homeostasis.

Copper is essential to most living beings but also toxic. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. The 3-gene operon bp2923-bfrG-bp2921 is down-regulated by copper and notably encodes a TonB-dependent transporter in Bordetella pertussis. We show that the protein encoded by bp2923, which is a member of the DUF2946 family, represents a new type of upstream Open Reading Frame (uORF) involved in post-transcriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologues of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has unveiled an original mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator.

Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridium difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We have identified and characterised a penicillin binding protein (PBP) that is required for cortex synthesis in C. difficile. Surprisingly this PBP was also found to contribute to cephalosporin resistance, indicating an additional role in the synthesis of vegetative cell wall. This is the first description of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

YabG is a sporulation-specific protease that is conserved among sporulating bacteria. C. difficile YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in yabG were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the yabG locus contribute to C. difficile spore germination, we introduced these mutations into an isogenic background. Spores derived from C. difficile yabGC207A (catalytically inactive), C. difficile yabGA46D, C. difficile yabGG37E, and C. difficile yabGP153L strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with E. coli lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabGA46D showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in yabG expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.

The ability to clear transcription complexes from DNA is especially important after DNA damage that produces replication stress. Using the bacterium E. coli, we show here that mutations in RNA polymerase that reduce termination, inhibitors of Rho-dependent termination, and inversion of a highly transcribed ribosomal RNA operon both enhance sensitivity to the quinolone ciprofloxacin (CPX); and identify two transcription factors, SspA and RapA, that impact these effects in opposite ways.We demonstrate that the rapA promoter is induced by CPX, independent of the LexA/RecA SOS response but is dependent on DnaA. Previous work has shown that RapA is expressed highest in rapidly growing cells whereas SspA levels respond to starvation.The factors have opposing effects on tolerance to chronic exposure to CPX, with RapA promoting cell growth and SspA inhibiting it. Functional SspA is also required for the CPX toxicity of the rRNA operon inversion; in sspA{Delta} mutants it has no negative consequence. In otherwise wild-type cells, loss of RapA has little effect except in strains lacking RNase HI, the enzyme that removes RNA/DNA hybrids from DNA. However, in cells lacking SspA, RapA strongly promotes survival, suggesting that SspA must block positive effects of RapA on tolerance. The RapA requirement for CPX tolerance is not relieved by RNase HI overexpression and therefore RapA must be not be merely preventing R-loop formation. RapA also in some way promotes the use of RNA loops to initiate DNA replication in the absence of DnaA. We propose that SspA stabilizes stalled or post-termination RNAP/DNA complexes and that the presence of SspA prevents RapA release of these complexes.

Most bacteria are encased in a rigid cell wall peptidoglycan (PG) meshwork. Cell growth requires the activities of both PG synthases and PG hydrolases that cleave bonds within the meshwork enabling its expansion. PG hydrolase activity must be carefully regulated to prevent excessive damage to this protective layer leading to catastrophic lysis. Here, I provide evidence for a novel type of regulation mediated by lipid-linked glycopolymer precursors. The Gram-positive bacterium Bacillus subtilis encodes two functionally redundant PG hydrolases, LytE and CwlO, that are required for growth. Here, I demonstrate that loss of LytR-CpsA-Psr (LCP) enzymes, which enzymatically transfer lipid-linked glycopolymers onto PG, leads to a requirement for lytE for growth. Genetic analysis suggests that this requirement is mediated by the accumulation of these membrane-anchored precursors, where they may interfere with PG hydrolase activity. These results are consistent with models in which polymer transfer influences the position or timing of PG hydrolysis.

The bacterium Listeria monocytogenes can grow in the cytoplasm of infected human cells, but there it relies on specific biosynthetic pathways for intracellular nutrient supply. We previously found that the glycine cleavage system (GCS) is needed for intracellular growth. The GCS decarboxylates glycine for generation of 1C-tetrahydrofolates (1C-THF), folate dependent one carbon donors needed for biosynthesis of other metabolites. We continued our studies on the GCS and show that a L. monocytogenes {Delta}gcvPAB mutant, lacking the GCS glycine dehydrogenase, is attenuated without resembling the phenotype of classical virulence factor mutants. The {Delta}gcvPAB mutant also grew poorly in synthetic medium, explained by the presence of glycine that was toxic for this strain. Selection of glycine resistant suppressors yielded a survivor, in which the N-and C-terminal parts of the formate-tetrahydrofolate ligase (fhs) gene, which is naturally separated into two parts by a premature stop codon in the L. monocytogenes reference strain EGD-e, were reassembled into a full-length open reading frame. Like the GCS, Fhs also feeds the 1C-THF pool and its restoration cured the virulence defects of the {Delta}gcvPAB mutant. Another suppressor had a mutated glyA gene, encoding serine hydroxymethyltransferase, and combinatorial deletions of gcvPAB and glyA in fhs0 and fhs+ backgrounds demonstrated a role of GlyA in 1C-THF metabolism. Our results show that three pathways feed the 1C-THF pool to support growth and virulence of L. monocytogenes and represent the first example of the spontaneous reactivation of a L. monocytogenes gene that is inactivated by a premature stop codon.

Essential peptidoglycan synthases, like penicillin binding proteins 2 and 3 (PBP2/PBP3) of Escherichia coli, define shape by orchestrating cell elongation and division, respectively. Despite being intensively studied as drug targets, the regulatory rules governing their production remain poorly understood. During infection, the closely related pathogen Salmonella enterica serovar Typhimurium downregulates PBP2/PBP3 production and replace them with alternative peptidoglycan synthases, PBP2SAL/PBP3SAL, absent in E. coli. The bases for such switch in morphogenetic proteins are unknown. Here, we show that the S. Typhimurium regulator OmpR triggers PBP2SAL and PBP3SAL expression responding solely to acid pH and define a shared motif present in upstream regions of the PBP2SAL- and PBP3SAL-coding genes sufficient for such control. The elimination of PBP2/PBP3 in infection conditions is however multifactorial, requiring acidity, high osmolarity and being favoured by OmpR and the Prc protease. Remarkably, we found that E. coli loses the essential PBP3 required for cell division when exposed to both acidity and high osmolarity, the environmental cues encountered by intracellular S. Typhimurium. Therefore, OmpR played a central role in the evolution of this pathogen when co-opting the regulation of PBP2SAL/PBP3SAL and, consequently, promoting a new morphogenetic cycle that made possible increasing progeny inside acidic eukaryotic phagosomes. SignificanceSome enzymes that participate in peptidoglycan metabolism are present exclusively in bacterial pathogens and modify its structure to limit immune recognition. The intracellular pathogen Salmonella enterica serovar Typhimurium is the only example known to date in which a "substitution" of essential peptidoglycan enzymes involved in cell division and elongation takes place during infection. The data presented here support instability of PBP3 in environments with acidity and high osmolarity as a probable selective pressure that promoted the fixation of alternative morphogenetic enzymes. This was possible due to the control that OmpR exerted over these new foreign functions. The acquisition of enzymes like PBP2SAL and PBP3SAL therefore represent a "quantum leap" evolutionary event in S. Typhimurium that made possible the colonization of acidic intracellular niches.

SUMMARYCells have evolved a metabolic control of DNA replication to respond to a wide range of nutritional conditions. Accumulating data suggest that this poorly understood control depends, at least in part, on Central Carbon Metabolism (CCM). In Bacillus subtilis, the glycolytic pyruvate kinase (PykA) is intricately linked to replication. This 585 amino-acid-long enzyme comprises a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to produce pyruvate and ATP, and a C-terminal domain of unknown function. Interestingly, the C-terminal domain termed PEPut interacts with Cat and is homologous a domain that, in other metabolic enzymes, are phosphorylated at a conserved TSH motif at the expense of PEP and ATP to drive sugar import and catalytic or regulatory activities. To gain insights into the role of PykA in replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown in a medium where the metabolic activity of PykA is dispensable for growth. Measurements of replication parameters (ori/ter ratio, C period and fork speed) and of the pyruvate kinase activity showed that PykA mutants exhibit replication defects resulting from side chain modifications in the PykA protein rather than from a reduction of its metabolic activity. Interestingly, Cat and PEPut have distinct commitments in replication: while Cat impacts positively and negatively replication fork speed, PEPut stimulates initiation through a process depending on Cat-PEPut interaction and growth conditions. Residues binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and belonging to the TSH motif of PEPut were found important for the commitment of PykA in replication. In vitro, PykA affects the activities of replication enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for initiation and elongation and genetically linked to pykA. Our results thus connect replication initiation and elongation to CCM metabolites (PEP, ATP and ADP), critical Cat and PEPut residues and to multiple links between PykA and the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed with a moonlighting activity that senses the concentration of signaling metabolites and interacts with replication enzymes to convey information on the cellular metabolic state to the replication machinery and adjust replication initiation and elongation to metabolism. This defines a new type of replication regulator proposed to be part of the metabolic control that gates replication in the cell cycle.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Cyclic di-AMP (c-di-AMP) is an essential second messenger in Listeria monocytogenes, but its accumulation is detrimental as it disrupts cell wall homeostasis and attenuates virulence. The mechanisms underlying this toxicity remain poorly understood. To understand the molecular basis of this toxicity, we performed a forward genetic screen to identify suppressor mutations that restore {beta}-lactam resistance in a {Delta}pdeA {Delta}pgpH ({Delta}PDE) mutant, which accumulates high c-di-AMP and is susceptible to cell wall-targeting {beta}-lactam antibiotics. We found that the majority of suppressor mutants carried mutations in the mreB gene, which encodes the bacterial actin-like cytoskeletal protein, MreB, that directs lateral peptidoglycan synthesis during cell elongation. These mutations restored {beta}-lactam resistance and ex vivo virulence while still retaining high intracellular c-di-AMP levels. Microscopy analyses indicate that these suppressor mutations reduce MreB activity, as evidenced by cell widening, and that they phenocopy sublethal treatment with the MreB inhibitor A22. Consistently, A22 treatment also rescued {beta}-lactam sensitivity in the {Delta}PDE mutant, supporting a functional link between MreB activity and c-di-AMP toxicity. Mechanistically, c-di-AMP accumulation impaired cell division/septation and reduced peptidoglycan synthesis under cell wall stress, whereas MreB mutations restored both transglycosylation and transpeptidation activities and promoted cell division. These effects were independent of potassium homeostasis, suggesting a distinct pathway linking c-di-AMP to cell wall regulation in L. monocytogenes. Together, our findings demonstrate that dysregulated MreB activity contributes to cell wall defects at elevated c-di-AMP levels and highlight the importance of coordinating cytoskeletal dynamics with cell division to maintain cell envelope integrity.

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. Further, 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.