In Mycobacterium abscessus, the Stringent Factor Rel Regulates Metabolism but Is Not the Only (p)ppGpp Synthase.

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the nontuberculous mycobacterial pathogen, Mycobacterium abscessus (Mab). We found that deletion of rel does not ablate (p)ppGpp synthesis and that rel does not provide a survival advantage in several stress conditions or in antibiotic treatment. Transcriptional data show that RelMab is involved in regulating expression of anabolism and growth genes in the stationary phase. However, it does not activate transcription of stress response or antibiotic resistance genes and actually represses transcription of many antibiotic resistance genes. This work shows that there is an unannotated (p)ppGpp synthetase in Mab. IMPORTANCE In this study, we examined the functional roles of the stringent factor Rel in Mycobacterium abscessus (Mab). In most species, stringent factors synthesize the alarmone (p)ppGpp, which globally alters transcription to promote growth arrest and survival under stress and in antibiotic treatment. Our work shows that in Mab, an emerging pathogen that is resistant to many antibiotics, the stringent factor Rel is not solely responsible for synthesizing (p)ppGpp. We find that RelMab downregulates many metabolic genes under stress but does not upregulate stress response genes and does not promote antibiotic tolerance. This study implies that there is another critical but unannotated (p)ppGpp synthetase in Mab and suggests that RelMab inhibitors are unlikely to sensitize Mab infections to antibiotic treatment.

The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis.

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis.

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2'-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

Activation of RelA by pppGpp as the basis for its differential toxicity over ppGpp in Escherichia coli.

The nucleotide derivatives (p)ppGpp, comprising ppGpp and pppGpp, are important signalling molecules that control various facets of gene regulation and protein synthesis in Escherichia coli. Their synthesis is catalysed by RelA (in response to amino acid limitation) and by SpoT (in response to the limitation of carbon source or fatty acids). SpoT is also a hydrolase for degradation of both ppGpp and pppGpp, while GppA catalyses the conversion of pppGpp to ppGpp. Here we provide evidence to show that pppGpp exerts heightened toxicity compared to that by ppGpp. Thus, gppA spoT double mutants exhibited lethality under conditions in which the single mutants were viable. The extent of RelA-catalysed (p)ppGpp accumulation in the gppA spoT strain was substantially greater than that in its isogenic gppA+ derivative. The data is interpreted in terms of a model in which toxicity of pppGpp in the gppA spoT mutants is mediated by its activation of RelA so as to result in a vicious cycle of (p)ppGpp synthesis.

Synthesis of ppGpp impacts type IX secretion and biofilm matrix formation in Porphyromonas gingivalis.

In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors. The stringent response, mediated by the alarmone (p)ppGpp, is central to the stress response in many bacteria; yet, there is limited knowledge regarding the role of (p)ppGpp signaling in bacteria belonging to the phylum Bacteroidetes. Like its counterparts in the gut (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis), Porphyromonas gingivalis persists in close association with its human host. Given the potential for numerous perturbations in the oral cavity, and the fact that P. gingivalis can enter and replicate within host cells, we hypothesized that (p)ppGpp is a key signaling molecule for stress adaptation and persistence. Here, we show that accumulation of ppGpp in P. gingivalis is governed by two homologous enzymes, designated Rel, and RshB, and that ppGpp signaling affects growth rate, survival, biofilm formation, production of outer membrane vesicles, and expression of genes encoding type IX secretion structural and cargo proteins. Overall, our findings provide a potential mechanism by which biofilm formation and virulence of P. gingivalis are integrated via ppGpp signaling, a regulatory mechanism central to bacterial survival in dynamic environments.

The stringent response of marine bacteria - assessment of (p)ppGpp accumulation upon stress conditions.

Microorganisms are particularly adapted to alterations in their environment. One of the global regulatory mechanisms involved in these adaptations is the stringent response. The unusual nucleotides, guanosine penta and tetraphosphates, (p)ppGpp act as alarmones of this response, heralding nutrient limitation and stressors. Marine bacteria encounter numerous stresses of sparse nutrient supplies and changes in physicochemical conditions. The aim of this work was to assess whether the stress conditions common in marine environment can induce the stringent response and what is a kinetic of this process. The representative bacterial species, Shewanella baltica, Acinetobacter johnsonii, Vibrio harveyi, and Escherichia coli were subjected to a variety of stressors. We analyzed the kinetics of (p)ppGpp synthesis by labeling in vivo nucleotides and analysis by thin layer chromatography. The (p)ppGpp accumulation followed the elevated temperature and amino acid starvation for all bacteria tested. The carbon and nitrogen limitation resulted in the response limited to V. harveyi and S. baltica. The DNA damaging agents induced the (p)ppGpp production in all strains, while osmotic stress did not result in significant alarmone synthesis. The representative marine bacteria species were shown to induce with varying extent the stringent response upon the onset of stress and limitation conditions. Importantly, the in vivo labeling and subsequent separation of the nucleotides by thin layer chromatography serves as a valid method for the analysis of the stringent response and (p)ppGpp accumulation in environmental bacteria.

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.

The Ps and Qs of alarmone synthesis in Staphylococcus aureus.

During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.

The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.

Triggering the stringent response: signals responsible for activating (p)ppGpp synthesis in bacteria.

The stringent response is a conserved bacterial stress response mechanism that allows bacteria to respond to nutritional challenges. It is mediated by the alarmones pppGpp and ppGpp, nucleotides that are synthesized and hydrolyzed by members of the RSH superfamily. Whilst there are key differences in the binding targets for (p)ppGpp between Gram-negative and Gram-positive bacterial species, the transient accumulation of (p)ppGpp caused by nutritional stresses results in a global change in gene expression in all species. The RSH superfamily of enzymes is ubiquitous throughout the bacterial kingdom, and can be split into three main groups: the long-RSH enzymes; the small alarmone synthetases (SAS); and the small alarmone hydrolases (SAH). Despite the prevalence of these enzymes, there are important differences in the way in which they are regulated on a transcriptional and post-translational level. Here we provide an overview of the diverse regulatory mechanisms that are involved in governing this crucial signalling network. Understanding how the RSH superfamily members are regulated gives insights into the varied important biological roles for this signalling pathway across the bacteria.

Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli.

Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA-spoT- mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli.

Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation in Mycobacterium smegmatis.

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using RelMSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to RelMSM by isothermal titration calorimetry. Enzyme kinetics was followed where K0.5 was found to be increased with the concomitant reduction of Vmax value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.

(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair.

UNLABELLED: The l-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcm mutant produced persisters for an extended period in stationary phase, and a ΔglpD mutation drastically increased persisters in a Δpcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the Δpcm ΔglpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcm mutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage. IMPORTANCE: Protein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.

Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone.

Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named "(p)ppGpp"] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) from Bacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp-but not ppGpp-positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

Differential regulation by ppGpp versus pppGpp in Escherichia coli.

Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5'-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-σ(70) holoenzyme with ppGpp and pppGpp. We find that both nucleotides bind to a site at the interface between ß' and ω subunits.

Dependence of RelA-mediated (p)ppGpp formation on tRNA identity.

The bacterial stringent response is a cellular response to amino acid limitations and is characterized by the accumulation of the alarmone polyphosphate guanosine ((p)ppGpp). A key molecular event leading to (p)ppGpp synthesis is the binding of a deacylated tRNA to the vacant A-Site of a ribosome. The resulting ribosomal complex is recognized by and activates RelA, the (p)ppGpp synthetase. Activated RelA catalyzes (p)ppGpp formation until the deacylated tRNA passively dissociates from the ribosomal A-Site. In this report, we have investigated a novel role for the identity of A-Site bound tRNA in RelA-mediated (p)ppGpp synthesis. A comparison in the stimulation of RelA activity was made using ribosome complexes with either a tightly or weakly binding deacylated tRNA occupying the A-Site. In vitro analysis reveals that ribosome complexes formed with tight binding tRNA(Val) stimulate RelA activity at lower concentrations than that required for ribosome complexes formed with the weaker binding tRNA(Phe). The data suggest that the recovery from the stringent response may be dependent on the identity of the amino acid that was initially limiting for the bacteria.

RelA regulates virulence and intracellular survival of Francisella novicida.

Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.