The Escherichia coli acid stress response allows the survival of cells over a wide range of pH challenges: down to pH 2.0 with the extreme acid stress response and down to pH 4.0 – 5.0 with the mild acid stress response. The cell employs a number of different acid stress response systems, including a number of structurally related, pyridoxal-5′-phosphate (PLP)-dependent amino acid decarboxylases, including the glutamic acid, arginine, lysine, and ornithine decarboxylases. The decarboxylases are large multi-domain enzymes that exist as homodimers or higher-order oligomers and have various activity optima at different pH values. By the proton-consuming decarboxylation of a target amino acid, these enzymes provide a response to a wide range of pH challenges. The primary focus of this work is the elucidation of the X-ray crystal structure of the inducible lysine decarboxylase LdcI, a homodecameric enzyme that has distinct 5-fold symmetry. A combination of heavy-atom derivatization, anomalous scattering and molecular replacement techniques were used to determine the X-ray structure and the model was refined to a resolution of 2.0 Å. The structure of LdcI revealed that the protein co-crystallized with the stringent response alarmone ppGpp. The stringent response is activated under nutritional and stress conditions and reorganizes cellular transcription and metabolism from exponential-phase growth into stationary phase growth. The primary target of ppGpp is the RNA polymerase, but other classes of enzymes are known to be affected. ppGpp was found to be a potent inhibitor of LdcI both in vitro and in vivo and this role provides the first evidence of a linkage between the stringent response and acid stress response. Among the decarboxylases related to LdcI (the constitutive lysine, the ornithine and arginine decarboxylases), a number of these enzymes were similarly regulated by ppGpp.

Most free-living bacteria will encounter different environments and it is therefore critical to be able to rapidly adjust to new growth conditions in order to be competitively successful. Responding to changes requires efficient gene regulation in terms of transcription, RNA stability, translation and post-translational modifications. Studies of an extremely slow-growing mutant of Salmonella enterica, with a Glu125Arg mutant version of EF-Tu, revealed it to be trapped in a stringent response. The perceived starvation was demonstrated to be the result of increased mRNA cleavage of aminoacyl-tRNA synthetase genes leading to lower prolyl-tRNA levels. The mutant EF-Tu caused an uncoupling of transcription and translation, leading to increased turnover of mRNA, which trapped the mutant in a futile stringent response. To examine the essentiality of RNase E, we selected and mapped three classes of extragenic suppressors of a ts RNase E phenotype. The ts RNase E mutants were defective in the degradation of mRNA and in the processing of tRNA and rRNA. Only the degradation of mRNA was suppressed by the compensatory mutations. We therefore suggest that degradation of at least a subset of cellular mRNAs is an essential function of RNase E. Bioinformatically, we discovered that the mRNA of tufB, one of the two genes encoding EF-Tu, could form a stable structure masking the ribosomal binding site. This, together with previous studies that suggested that the level of EF-Tu protein could affect the expression of tufB, led us to propose three models for how this could occur. The stability of the tufB RNA structure could be affected by the elongation rate of tufB-translating ribosomes, possibly influenced by the presence of rare codons early in the in tufB mRNA. Using proteomic and genetic assays we concluded that two previously isolated RNAP mutants, each with a growth advantage when present as subpopulations on aging wild-type colonies, were dependent on the utilization of acetate for this phenotype. Increased growth of a subpopulation of wild-type cells on a colony unable to re-assimilate acetate demonstrated that in aging colonies, acetate is available in levels sufficient to sustain the growth of at least a small subpopulation of bacteria.

The Escherichia coli acid stress response allows the survival of cells over a wide range of pH challenges: down to pH 2.0 with the extreme acid stress response and down to pH 4.0 – 5.0 with the mild acid stress response. The cell employs a number of different acid stress response systems, including a number of structurally related, pyridoxal-5′-phosphate (PLP)-dependent amino acid decarboxylases, including the glutamic acid, arginine, lysine, and ornithine decarboxylases. The decarboxylases are large multi-domain enzymes that exist as homodimers or higher-order oligomers and have various activity optima at different pH values. By the proton-consuming decarboxylation of a target amino acid, these enzymes provide a response to a wide range of pH challenges. The primary focus of this work is the elucidation of the X-ray crystal structure of the inducible lysine decarboxylase LdcI, a homodecameric enzyme that has distinct 5-fold symmetry. A combination of heavy-atom derivatization, anomalous scattering and molecular replacement techniques were used to determine the X-ray structure and the model was refined to a resolution of 2.0 Å. The structure of LdcI revealed that the protein co-crystallized with the stringent response alarmone ppGpp. The stringent response is activated under nutritional and stress conditions and reorganizes cellular transcription and metabolism from exponential-phase growth into stationary phase growth. The primary target of ppGpp is the RNA polymerase, but other classes of enzymes are known to be affected. ppGpp was found to be a potent inhibitor of LdcI both in vitro and in vivo and this role provides the first evidence of a linkage between the stringent response and acid stress response. Among the decarboxylases related to LdcI (the constitutive lysine, the ornithine and arginine decarboxylases), a number of these enzymes were similarly regulated by ppGpp.

Seja no meio ambiente, dentro de um hospedeiro ou em outro habitat, bactérias estarão frequentemente enfrentando condições adversas, como exposição a compostos antibacterianos ou carência nutricional. Em situações como essas, as bactérias são capazes de ativar a chamada resposta estringente, modulada pelo alarmônio (p)ppGpp. O acúmulo de (p)ppGpp promove a inibição da transcrição de rRNAs e tRNAs e a supressão do processo de tradução, e a ativação de operons de biossíntese de aminoácidos. Sabe-se também hoje que a resposta estringente está relacionada a outras importantes carências nutricionais em Escherichia coli, como a falta de ácidos graxos, porém não se sabe se o mesmo ocorre em Bacillus subtilis ou em outras Grampositivas. (p)ppGpp atua também direta e indiretamente em vários outros processos celulares, como motilidade, resistência a antibióticos, virulência e persistência, indicando que (p)ppGpp é um regulador central que integra informação metabólica e respostas adaptativas. O presente trabalho buscou estudar a correlação da resposta estringente de B. subtilis com a carência de ácidos graxos e a busca por pequenas moléculas capazes de modular RelA (a principal proteína envolvida na síntese de (p)ppGpp) e impedir o acúmulo de (p)ppGpp. Para a indução da carência de ácidos graxos, foram utilizadas duas estratégias; uso da droga Cerulenina (inibidor de FabF) e mutantes condicionais no gene FabF. Observou-se que mutantes incapazes de ativar a resposta estringente (cepa ppGpp(0) ou RelAD264G) apresentaram grande perda de viabilidade celular durante a carência de ácidos graxos, ao passo que a cepa selvagem manteve sua viabilidade celular. A causa da morte se deu majoritariamente devido ao colapso do potencial de membrana. Apesar de não termos observado aumento de (p)ppGpp nas células selvagens durante a carência de ácidos graxos, observou-se uma redução da razão GTP/ATP, ao passo que na cepa ppGpp(0), a razão GTP/ATP aumentou, devido ao acúmulo de GTP. O uso da droga decoinina, capaz de reduzir os níveis intracelulares de GTP, resgatou parcialmente a viabilidade da cepa e impediu a perda do potencial de membrana, indicando que os níveis de GTP são importantes durante a carência de ácidos graxos em B. subtilis. Para a triagem de pequenas moléculas inibidoras do acúmulo de (p)ppGpp, foi utilizada uma biblioteca de 2320 diferentes compostos químicos, e buscou-se drogas capazes de reverter o fenótipo de crescimento lento de cepas de B. subtilis que acumulam (p)ppGpp (via mutação pontual; mutante RelAH77A e via tratamento com o indutor hidroxamato de arginina) em meio rico. A primeira etapa selecionou 40 moléculas capazes de resgatar o crescimento de células tratadas com arginina-hidroxamato, porém apenas uma, salicilanilida, foi capaz de também resgatar o crescimento da cepa RelAH77A. Todavia, apesar de ser capaz de acelerar o crescimento de B. subtilis esse efeito é limitado. Diversos análogos de salicilanilida foram testados, porém não apresentaram efeito superior a salicilanilida para a reversão do fenótipo de crescimento lento de B. subtilis. Em adição, a droga não foi capaz de aumentar a sensibilidade dos organismos a diversos antibióticos testados, e aparentemente é incapaz de alterar os níveis internos de (p)ppGpp, porém é capaz de causar alterações nos níveis de ATP. Logo, acredita-se que o efeito observado para o crescimento das células seja devido a efeitos indiretos, possivelmente envolvendo alteração de outros nucleotídeos fosforilados. / In the environment, inside a host or other habitat, bacteria will always face adverse conditions, as for example exposure to antimicrobials or starvation. In situations like those, bacteria activate the stringent response, modulated by the alarmone (p)ppGpp. (p)ppGpp accumulation promotes inhibition of rRNA and tRNA transcription and suppression of translational process, at the same time that it activates several amino acid biosynthesis operons. It is known also that the stringent response it is related to other starvation stress in Escherichia coli, like lack of fatty acids, but there is no knowledge if the same occurs for Bacillus subtilis or other gram-positive bacteria. ppGpp acts directly and indirectly affecting several other cellular process, as motility, resistance to antibiotics, virulence and persistence, indicating that (p)ppGpp is a central regulator that integrates metabolic information and adaptive responses. This work aimed to study the correlation between the stringent response in B. subtilis with fatty acid starvation, and search for small moleculas capable of modulating RelA (the main enzyme responsible for ppGpp synthesis) and stop (p)ppGpp production. For fatty acid starvation induction, two strategies were used; use of the drug Cerulenin (inhibitor of the FabF protein) and conditional mutants of the FabF gene. We observed that mutants incapable of activating the stringent response (strains ppGpp(0) ou RelAD264G) presented great loss of viability during fatty acid starvation, whereas the wild-type strain keeps its viability. The main cause of death is due membrane rupture in some cells, but mainly due to membrane potential collapse. Although we did not observed increase of (p)ppGpp in wild-type strains during fatty acid starvation, we observed reduction in GTP/ATP ratios, a hallmark of (p)ppGpp production in gram-positive bacteria. In the strain ppGpp(0) GTP/ATP ratio increased, mainly due to GTP increase. Using the drug decoyinine, capable of reducing GTP levels, partially recued viability and protects cells of losing its membrane potential, indicating that GTP levels plays an important role during fatty acid starvation in B. subtilis. For the screening of small molecules capable of inhibit (p)ppGpp production, a library of 2320 different chemical compounds were used, and we looked for drugs capable of reverting the slow growth phenotype of B. subtilis strains with (p)ppGpp accumulation (using a mutant RelAH77A; and using a stringent response inductor, arginine hidroxamate). The first step selected for 40 molecules capable of rescuing the growth of cells treated with arginine hidroxamate, but only one drug, salicilanilyde could also rescue the growth of the strain RelAH77A. Although capable of rescuing growth of B. subtilis that accumulates (p)ppGpp, this rescue is limited. Several analogues of salicilanilyde were tested, but none were stronger than salicilanilyde itself in rescuing growth of slow growing strains of B. subtilis. In addition, the drug was not capable of increasing antibiotic sensibility and it is incapable of changing intracellular (p)ppGpp levels, but it does shifts ATP levels. Therefore, we believe that the observed effects of salicilanilyde is due indirect action, probably involving other phosphorylated nucleotides, rather than modifying (p)ppGpp levels

Pseudomonas aeruginosa is a ubiquitous, opportunistic pathogen that causes acute and chronic infections. Infection is typically initiated via motile and virulent strains. After exposure to stressors, acute infections make both genotypic and phenotypic switches to a chronic, sessile strain. This is due to intricate regulatory networks directing gene expression in response to stressors. One network, GacA/GacS, has been established to control virulence factors. The stringent response of bacteria is mediated by alarmones produced primarily by RelA which responds to starvation. To study the effect of the stringent response on the virulence switch. A series of experiments were run in both PAO1, a virulent strain, and PDO300, an acute strain, and RelA deletion mutants of each transcriptional fusions of GacA/GacA system were integrated in the wild-types and mutants. Alginate, swimming, twitching, and biofilm formation assays were performed on all. The preliminary data suggests that the stringent response influences the GacA/GacS system.

Adaptation to any undesirable change in the environment dictates the survivability of many microorganisms. Such changes generate a quick and suitable response, which guides the physiology of bacteria. Stringent response is one of the mechanisms that can be called a survival strategy under nutritional starvation in bacteria and was first observed in E. coli upon amino acid starvation, when bacteria demonstrated an immediate downshift in the rRNA and tRNA levels (Stent and Brenner 1961). Mutations that rendered bacteria insensitive to amino acid levels were mapped to an ‘RC gene locus’, later termed relA because of the relAxed behavior of the bacteria (Alfoldi et al. 1962). Later on, Cashel and Gallant, showed that two “magic spots” (MSI and MSII) were specifically observed in starved cells when a labeled nucleotide extract of these cells was separated by thin layer chromatography (Cashel and Gallant 1969). These molecules were found to be polyphosphate derivatives of guanosine, ppGpp and pppGpp (Cashel and Kalbacher 1970; Sy and Lipmann 1973), and were shown to be involved in regulating the gene expression in the bacterial cell, demonstrating a global response, thus fine-tuning the physiology of the bacterium. Two proteins in E. coli, RelA and SpoT, carry out the synthesis and hydrolysis of these molecules, respectively, and maintain their levels in the cell (Cashel et al. 1996; Chatterji and Ojha 2001). On the other hand, Gram-positive organisms have only one protein Rel carrying out the functions of both RelA and SpoT (Mechold et al. 1996; Martinez-Costa et al. 1998; Avarbock et al. 1999). Although Rel or RelA/SpoT has been studied from several systems in detail pertaining to the physiological adaptation, less information is available on the egulation of the protein activity under different conditions. Our studies show that the RelMsm is composed of several domains (HD, RSD, TGS and ACT) with distinct function. HD and RSD domains, present in the N-terminal half of the protein, harbor catalytic sites for the hydrolysis and the synthesis of (p)ppGpp, respectively. TGS and ACT domains, on the other hand, are present at the C-erminal half of the protein and have regulatory function. It, therefore, appears that a communication exists between these domains, to regulate protein activity. It was shown earlier, while studying Rel from S.equisimilis, that there exists an interaction between the C-terminal and the N- terminal of the protein which determines the kind of activity (synthesis/hydrolysis), the protein should demonstrate (Mechold et al. 2002). Later, the N-terminal half crystal structure of the same protein suggested an inter-domain “cross-talk” between the HD and the RSD domain that controls the synthesis/hydrolysis switch depending on cellular conditions (Hogg et al. 2004). In the present work, studies have been carried out to understand a Gram- positive Rel in greater detail and to find out how the opposing activities of Rel are regulated so that a futile cycle of synthesis and hydrolysis of (p)ppGpp, at the expense of ATP, can be avoided. The work has been divided into several chapters describing studies on various aspects of the protein. Chapter 1 outlines the history of the stringent response and summarizes the information available about the stringent response in various systems including plants. Several roles that (p)ppGpp plays in different bacteria have been examined. A special mention on the crystal structure of RelSeq has been made with respect to the regulation of activity. Also, the information available regarding the effects of (p)ppGpp on RNA polymerase has been documented. Role of ppGpp in plants has been discussed in great detail with special emphasis on abiotic stresses. Since different functional domains have been identified in RelMsm, the protein has been divided into two halves and they have been discussed separately in the form of two chapters. Chapter 2 describes the N-terminal half of the Rel protein of M. smegmatis in greater detail. Out of the several domains identified, the role of the two domains present in the N-terminal half of the protein has been studied. The N-terminal half shows both synthesis and hydrolysis activities. Importantly, we find that the protein is active even in the absence of accessory factors such as ribosome and uncharged tRNA, unlike RelA of E. coli. Moreover, deletion of the C-terminal half of the protein leads to a much higher synthetic activity, clearly indicating that the C-terminus is involved in regulating the activity of the protein. Both TGS and ACT domains (the two domains found in the C-terminal half of the protein) have been found to play a regulatory role. The results also indicate that all the deleted constructs are active both in vitro and in vivo. Chapter 3 discusses the C-terminal half of the protein and its role in the multimerization observed in RelMsm. We show that multimerization of Rel protein is due to the inter-molecular disulfide cross-linking. Furthermore, we find that the monomer is the active species in vivo. One of the fascinating points about the C- terminal half is that it is largely unstructured. Additionally, the C-terminal half cannot complement the N-terminal part of the protein when provided in trans, demonstrating further, the requirement of an intact protein for bringing about regulation of Rel activity. This requirement in cis suggests the presence of an intra-molecular communication between the N- and the C-termini, as a mediator of protein regulation. Further, presence of uncharged tRNA increases pppGpp synthesis and down-regulates its hydrolysis in the wildtype protein. However, the uncharged tRNA-mediated regulation is absent in the deleted construct with only the N-terminus half, indicating that uncharged tRNA binds to the C-terminal half of the protein. Several cysteine mutants have been constructed to understand their role in the regulation of Rel activity. The results suggest that one cysteine, present at the C-terminus, is required for intra-molecular cross-talk and the uncharged tRNA-mediated regulation. A detailed characterization of the communication between the two halves of the protein has been attempted in Chapter 4. Surface plasmon resonance experiments carried out on the different cysteine mutants discussed in Chapter 3, for uncharged tRNA binding indicate that all the mutants bind to uncharged tRNA with near-equal affinities as the wildtype protein. This study suggests that the non-responsiveness for tRNA seen in one of the cysteine mutants is due to the loss of inter-domain interaction, while the binding of protein to accessory factors is unaffected. Fluorescence resonance energy transfer has been carried out to observe domain movement in the presence of accessory factors. Distances between the different domains scattered in this ~90 kDa protein, measured by FRET technique, are suggestive of an inter-domain cross-talk, specifically between C338 and C692, thereby regulating the activity of this enzyme. We show, for the first time, that the product of this protein, (p)ppGpp can bind to the C-terminal half making it unstructured, and can, therefore, regulate the protein activity. Chapter 5 is an effort to characterize the promoter of rel from M. tuberculosis. This study was undertaken in order to develop an expression system in mycobacteria. The +1 transcription and the translation start sites have been identified. The –10 hexamer for the RNA polymerase binding has also been mapped using site-directed mutagenesis and is found to be TATCCT. This promoter is also unusually close to the +1 transcription start site. The promoter is specific for mycobacteria and does not function in E. coli. Additionally, the promoter is found to be constitutive in M. smegmatis; however, the possibility of it being regulated in M. tuberculosis cannot be ruled out. Appendix section discusses, in short, the phylogenetic analysis of the mycobacterial Rel sequences. Diagrams of the plasmids used in this study have been provided. Mass spectra recorded for the in vitro synthesized and purified pppGpp and the trypsin digest of the full-length Rel protein have also been given. O O O O

Bien que divers mécanismes coopèrent pour empêcher les erreurs lors de la synthèse des protéines chez les bactéries, des erreurs traductionnelles de type « frameshift » (ETFs) ou « faux-sens » peuvent avoir lieu. En particulier, les ETFs ont été détectées à de faibles niveaux lors de la phase de croissance exponentielle et à des niveaux plus élevés durant la phase de croissance stationnaire chez Escherichia coli et Bacillus subtilis. Ces observations ont conduit les chercheurs à revoir le rôle de la "réponse stringente" dans la survenue des ETFs, qui constitue l’un des mécanismes clé de l'adaptation bactérienne aux changements nutritionnels. Elle découle de l'interaction entre un ribosome en cours de traduction et la protéines RelA/SpoT ce qui permet de détecter les ARNs de transfert (ARNts) non chargés et résulte en la production d'une molécule appelée (p)ppGpp . Dans une souche mutante relA incapable de synthétiser le (p)ppGpp, les ETFs sont fortement augmentées.Dans ce contexte, notre objectif principal a été de revisiter le rôle de la réponse stringente dans le contrôle des erreurs traductionnelles et de clarifier le rôle des deux métabolites antagonistes GTP et (p)ppGpp. Par exemple, le GTP stimule l'initiation de la traduction (en ciblant le facteur d'initiation IF2) alors que le (p)ppGpp inhibe l'initiation de la traduction (en rentrant en concurrence avec le GTP pour se fixer sur IF2).A cette fin, nous avons utilisé le modèle des bactéries à Gram positif B. subtilis, conçu trois systèmes rapporteurs distincts pour détecter les ETFs et construit une souche incapable de synthétiser du (p)ppGpp (appelée "(p)ppGpp0"). Nous avons observé qu'au cours de la croissance dans des milieux pauvres, les ETFs augmentent en l'absence de (p)ppGpp durant la phase exponentielle et que, contrairement à la souche sauvage, la souche (p)ppGpp0 présente un pic d’ETFs en milieu riche pendant la transition à la phase stationnaire. En contrôlant les niveaux intracellulaires de GTP dans la souche (p)ppGpp0, nous avons montré que l'abondance de GTP est le facteur qui déclenche l'apparition des ETFs. Néanmoins, après une "faible" induction de la biosynthèse du GTP conduisant à des taux de croissance sous-optimaux, le niveau d’ETFs forme toujours un pic lors de la transition vers la phase stationnaire, ce qui montre que le mode d'action du (p)ppGpp pour prévenir l'apparition des ETFs ne repose pas uniquement sur son action inhibitrice de la biosynthèse du GTP. Nous nous sommes alors concentrés sur l'effet inhibiteur du (p)ppGpp sur IF2 et avons mimé son action en injectant des drogues connues pour inhiber l'initiation de la traduction. Nous avons ainsi démontré qu'en réduisant l'initiation de la traduction lors de l'épuisement des aminoacyl-ARNts, la souche "(p)ppGpp0" est capable de contrôler de façon optimale le taux d’ETFs lors de la transition vers la phase stationnaire.Dans une deuxième partie, nous avons étudié comment la transcription et la traduction sont affectées par les variations du niveau de GTP et de (p)ppGpp. Nous avons observé que les gènes possédant un "+1" de transcription (TSS, « transcription start site ») composé de deux guanines (gènes artificiels et ARNs ribosomaux) ont vu leur taux de transcription positivement corrélés au taux de croissance à l'inverse des gènes possédant un TSS composé de deux adénines. Cette différence est encore plus prononcée pour la souche (p)ppGpp0 cultivée en milieu riche lors de l'ajout de guanosine (ce qui conduit à un niveau élevé de GTP).En conclusion, nous avons démontré que le (p)ppGpp contrôle le niveau d'erreurs traductionnelles lors de la croissance en régime permanent en abaissant les niveaux de GTP et lors d’un changement nutritionnel en inhibant spécifiquement l'initiation de la traduction, assurant une allocation parcimonieuse des ressources au sein de la bactérie. / Even though diverse mechanisms cooperate to prevent protein synthesis errors in bacteria, missense and translational frameshift errors (TFEs) can occur . In particular, TFEs were detected at low levels in the exponential growth phase and at higher levels in the stationary phase in both Escherichia coli and Bacillus subtilis. This observation led researchers to revisit the role of the “stringent response” in the occurrence of TFEs since it is the key mechanism involved in the bacterial adaptation to nutritional downshifts. It relies on the interaction between the RelA/SpoT proteins and the translating ribosomes, which leads to the detection of uncharged tRNAs and to the production of an alarmone called (p)ppGpp. In a relA mutant strains unable to synthesize (p)ppGpp, translational errors are highly increased.In this context, the main goal of our work was to revisit the role of the stringent response in the translational error control and to clarify the role of the two key, antagonistic metabolites GTP and (p)ppGpp. Indeed, while GTP enhances translation initiation (targeting the initiation factor IF2) and elongation (targeting the elongation factor EF-Tu) , (p)ppGpp inhibits GTP biosynthesis (reducing the enzyme activity of Gmk, HprT and GuaB) and translation initiation (competing with GTP on IF2).For this purpose, we used the Gram positive model bacterium B. subtilis, designed three distinct reporter systems to detect TFEs and built a strain unable to synthesize (p)ppGpp (called “(p)ppGpp0”). We observed that during growth in poor media TFEs were increased in the absence of (p)ppGpp in the exponential phase (i.e. steady-state growth) and that by contrast to the wild type, the (p)ppGpp0 strain exhibited a TFE burst during the transition in rich medium to the stationary phase. By controlling intracellular levels of GTP in the (p)ppGpp0 strain, we showed that GTP abundance is the trigger factor of TFEs occurrence. Nevertheless, upon a "weak" induction of GTP biosynthesis leading to sub-optimal growth rates, the TFEs rate still peaked during the transition to the stationary phase, which demonstrated that the mode of action of (p)ppGpp to prevent TFEs occurrence did not only rely on its inhibition of GTP biosynthesis. We then focused on the (p)ppGpp inhibitory effect on IF2 and mimicked its action by injecting drugs known to inhibit translation initiation. Hence, we demonstrated that by reducing translation initiation (injecting drugs) upon aminoacyl-tRNAs depletion (p)ppgGp0 wild-strain type cells is are able to optimally control the rate of TFEs in the transition to the stationary phase. The same conclusion is obtained even in presence of a high GTP level.In a second part, we studied how transcription and translation are affected by variations in GTP and (p)ppGpp abundances. We observed that genes possessing a transcription start site (TSS) made of two guanines were more importantly transcribed at higher growth rates than genes possessing a TSS made of two adenines. This difference was even more pronounced for (p)ppGpp0 strains grown in rich medium upon guanosine addition (leading to a high level of GTP). Moreover, the ribosomal RNAs (rrns; for which the TSS is a guanine) synthesis level seemed to be positively correlated to GTP levels during exponential growth in poor and rich media as observed by the modulation of GTP biosynthesis.In conclusion, we demonstrated that (p)ppGpp controls the occurrence of translational errors during steady-state growth by decreasing GTP levels and during a nutritional downshift by specifically inhibiting translation initiation ensuring a parsimonious , which also globally affects resource allocation.

Les infections en biofilm, notamment de dispositifs médicaux, mettent fréquemment en échec les traitements antibiotiques, imposant le retrait du matériel. Pseudomonas aeruginosa s’est imposé comme le pathogène-type des infections en biofilm. Pour explorer les déterminants de l’échec du traitement antibiotique en biofilm, un modèle de biofilm in vitro à P. aeruginosa exposé à des doses supra-inhibitrices d’antibiotiques a été développé. En culture planctonique, une bithérapie deciprofloxacine et d’amikacine permettait de prévenir la sélection de mutants résistants pour des souches de P. aeruginosa de sensibilité diminuée à la ciprofloxacine ou à l’amikacine par surexpression d’efflux. En biofilm, l’association de la ciprofloxacine et de l’amikacine, administrées simultanément ou séquentiellement, n’était pas supérieure aux monothérapies, permettant une réduction bactérienne, mais pas d’éradication complète du biofilm. Quelles que soient les souches (sauvages ou exprimant un efflux) et l’antibiotique, l’échec microbiologique en biofilm était lié à la sélection de cellules persistantes, tolérantes aux antibiotiques. La ciprofloxacine induisait des modifications importantes de la structure du biofilm avec une réduction considérable des exopolysaccharides, composants majeurs de la matrice. L’étude transcriptomique de gènes potentiellement impliqués dans la persistance suggérait que l’activation précoce de la réponse stringente pourrait être une des voies principales de la tolérance en biofilm sous ciprofloxacine. Enfin, la présence de « small colony variants » au sein du biofilm, dotés d’une capacité accrue de formation de biofilm, témoignait de la diversité des populations en biofilm. Ces travaux participent ainsi à une meilleure compréhension des mécanismes d’échappement aux antibiotiques de P. aeruginosa en biofilm. / Biofilm device-related infections can lead to antibiotic failure requiring frequent removal of medical device. Pseudomonas aeruginosa has emerged as the typical pathogen for biofilm infections. To explore the determinants of antibiotic failure in biofilm, an in vitro P. aeruginosa biofilm model exposed to suprainhibitory antibiotic concentrations was developed. In planktonic culture, the ciprofloxacin and amikacin combination prevented the selection of resistant mutants in ciprofloxacin and amikacinlow-level resistant P. aeruginosa strains overexpressing efflux. In biofilm, the ciprofloxacin and amikacin combination, used simultaneously or sequentially, didn’t show superior effects compared to monotherapies. Despite an initial bacterial reduction, biofilm eradication was not obtained. Regardless of wild-type or efflux strains and antibiotic regimen used, antibiotic failure was related to the selection of antibiotic-tolerant cells named “persisters”. Ciprofloxacin induced significant alterations in the biofilm structure, notably a considerable reduction in the exopolysaccharides of the matrix. The transcriptomic analysis of genes, potentially involved in persistence, suggested that early activation of the stringent response might be one of the main pathways for ciprofloxacin tolerance in biofilm. Finally, the emergence of "small colony variants" within the biofilm, characterized by enhanced ability to form biofilm, attested to biofilm heterogeneity. This work therefore contributes to a better understanding of how P. aeruginosa biofilms escape antibiotic.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.

La production de protéines hétérologues permet de développer une nouvelle génération de vaccins. La bactérie Escherichia coli est l’un des organismes hôtes les plus utilisés pour la production de protéines hétérologues, appelées également protéines recombinantes. Le déclenchement de la production de protéine altère la croissance bactérienne en réponse à la réallocation des ressources métaboliques vers la synthèse de la protéine ; ce qui peut conduire à l’arrêt complet de la croissance. Le maintien de la croissance bactérienne durant la production de la protéine recombinante est pourtant essentiel pour améliorer significativement la quantité et la fonctionnalité des protéines produites. Dans une démarche rationnelle visant à développer un système biologique robuste et performant pour la production d’une grande diversité de protéines recombinantes chez E. coli, les contraintes métaboliques liées à leur production ont été quantifiées. A partir de ces résultats, le système d’expression T7 a été intégré à la régulation métabolique et traductionnelle de la bactérie E. coli BL21 (DE3) afin d’adapter la vitesse de production avec les capacités métaboliques de la souche. Ce nouveau système biologique de production a ainsi permis d’augmenter considérablement les quantités de protéines produites et offre la possibilité de développer de nouveaux procédés performants de production semi-continus et continus en milieu chimiquement défini. / The production of heterologous proteins offers the ability to develop a new generation of vaccines. The most used organism for the production of heterologous proteins, also called recombinant proteins, is the bacterium Escherichia coli. However, the induction of the production often alleviates the bacterial growth by the new allocation of metabolic resources toward the production of the recombinant protein. Even, this may also lead to growth arrest. The production of high quantities of functional recombinant proteins requires a good balance between of bacterial growth and production of the recombinant protein.In order to rationally develop a robust and an efficient biological system for the production of a large variety of recombinant proteins in E. coli, the metabolic constraints associated to their production have been quantified. From this observation, the T7 expression system has been integrated into the metabolic and translational regulation of the E. coli BL21 (DE3) in order to adjust as perfect as possible the protein production rate to the metabolic capacities of the strain. This new biological production system has made it possible to significantly increase the quantities of proteins produced and opens up the possibility of developing performant semi-continuous and continuous production processes in a chemically defined medium.