La ppGpp et la pppGpp, qui sont synthétisées/hydrolysées par les RelA/Spot homologs (RSH), jouent un rôle centrale dans l’adaptation des bactéries contre la privation des nutriments et les stress environnementaux. Les enzymes RSH et ppGpp ont été découverts dans le chloroplaste. Il a été récemment démontré que ppGpp joue un rôle comme répresseur globale de l’expression de gènes chloroplastiques. Certains stresses environnementaux et hormones induisent l’accumulation de ppGpp chez les plantes, cependant le mécanisme moléculaire n’est pas encore connu. Ici nous nous sommes intéressés à découvrir les facteurs qui interagissent avec les RSH, et qui donc sont susceptible de réguler le métabolisme du ppGpp. En utilisant un crible double-hybridation de levure nous avons identifiées des proteines qui interagissent avec les RSH y compris l’acyl carrier protein (ACP) et des GTPases associées au ribosome. ACP et RSH1 semblent être indispensables pour l’accumulation de ppGpp induite par la carence de la biosynthèse des acides gras, tandis que le ppGpp et un GTPase associé au ribosome contribuent à la résistance contre le heat-shock. Nous avons aussi effectué du co-immunoprécipitation spectrométrie de masse avec RSH1. Plusieurs protéines ont été identifiées y compris des protéines associées au nucléoid et des protéines liées à la signalisation chloroplastique, indiquant que RSH1 pourrait etre impliqué dans la machinerie de transcription chloroplastique. Nos résultats montrent que chez les plantes le ppGpp joue un rôle non seulement comme chez les bacteriés mais aussi participe à de nombreux processus biologiques qui sont spécifiques aux plantes. / Guanosine tetra-phosphate and penta-phosphate (ppGpp and pppGpp), which are synthesized/hydrolyzed by RelA/Spot homolog (RSH) enzymes, play a central role in the adaptation of bacteria to nutrient limitation or other stresses. Both RSH enzymes and ppGpp are present in the chloroplasts of plants. Recent studies have shown that ppGpp acts as a global repressor of chloroplast gene expression. Certain environmental stresses and hormones induce ppGpp accumulation in chloroplasts, however the molecular mechanisms underlying the activation of ppGpp signalling in response to such stimuli is essentially unknown. We searched for factors that interact with RSH enzymes and so could play a role in activating ppGpp signalling. Using a targeted yeast two hybrid screen several proteins were identified that interact with RSH enzymes including acyl carrier protein (ACP) and ribosome associated GTPases. ACP and RSH1 appear to be required for ppGpp induction in response to fatty acid biosynthesis depletion, while ppGpp and an RSH-interacting GTPase contribute to the resistance of plants to heat shock. We also performed non-targeted co-immunoprecipitation mass spectrometry (CoIP-MS) of RSH1. New RSH interaction candidates were identified, including plastid nucleoid associated proteins and chloroplast signalling proteins, suggesting that RSH1 may be associated with the plastid transcription machinery. Our results give new insights into ppGpp signalling, and show that some elements are conserved between plants and bacteria, while others are implicated in plant-specific biological processes.

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

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.