Post-translationale Modifikationen (PTMs) sind wesentlich für die Regulierung von zellulären Mechanismen. Um diese Prozesse besser zu verstehen, ist es essentiell Methoden für deren Erforschung zu entwickeln. In dieser Arbeit wurden zwei chemoproteomische Ansätze entwickelt, um die PTMs, Proteinphosphorylierung und Proteinpyrophosphorylierung zu untersuchen. Die Proteom-weite Erforschung von Proteinphosphorylierungen beruht gewöhnlich auf der LC-MS/MS-Analyse von enzymatisch verdauten Proteomen und da die Phosphorylierung von niedriger Abundanz ist, wird ein Phosphopeptid-Anreicherungsschritt benötigt. Die Identifizierung von bestimmten Phosphopeptiden ist allerdings abhängig von der gewählten Anreicherungsmethode. Die Entwicklung von neuen Prozeduren ist daher bedeutsam, um neue Phosphorylierungsstellen zu identifizieren. Im ersten Projekt wurde eine milde und selektive Phosphopeptid-Anreicherungsmethode entwickelt und optimiert. Die Methode zeigte die Fähigkeit Phosphopeptide anzureichern und somit das Potential, das Repertoire der vorherigen Methoden zu erweitern, um neue Phosphorylierungsstellen zu identifizieren. Proteinpyrophosphorylierung ist eine unlängst identifizierte PTM, die nicht-enzymatisch an Proteine angefügt wird und es ist nur wenig ist über ihre Funktion bekannt. Vorherige Studien wiesen darauf hin, dass diese Modifikation enzymatisch entfernt wird, allerdings sind die verantwortlichen Enzyme („Proteinpyrophosphatasen“) nicht bekannt. Hier wurde eine Peptidaffinitätsmethode entwickelt, um potentielle Pyrophosphatasen und weitere interagierende Proteine aus humanen Zellen zu identifizieren. Damit wurden 6 Phosphatasen als potentielle Pyrophosphatase-Kandidaten identifiziert und weitere interagierende Proteine gaben Aufschlüsse über die Funktion der Proteinpyrophosphorylierung. Dadurch wurde das Potential der Methode aufgezeigt, interagierende Proteine der Proteinpyrophosphorylierung zu identifizieren, um die zelluläre Rolle zu verstehen. / Post-translational modifications (PTMs) are crucial for the regulation of cellular mechanisms. To better understand these processes, the development of chemical tools to investigate them is of high importance. In this thesis, two chemoproteomics approaches were established to investigate the PTMs protein phosphorylation and protein pyrophosphorylation. The proteome-wide study of protein phosphorylation usually relies on LC-MS/MS analysis of enzymatically digested proteomes, requiring a phosphopeptide enrichment step, due to the low abundance of phosphorylation. However, the identification of certain sets of phosphopeptides is dependend on the choice of enrichment method. Therefore, the development of new workflows is important to identify new phosphorylation sites. In the first project, a mild and selective phosphopeptide enrichment method was developed and optimized. The method was able to enrich phosphopeptides and therefore, showed the potential to complement the repertoire of current methods to identify new phosphorylation sites. Protein pyrophosphorylation is a recently discovered PTM, which is non-enzymatically attached to proteins and there is only sparse knowledge about the function. Previous studies have indicated the enzymatic removal of this modification, but the responsible enzymes (‘protein pyrophosphatases’) are unknown. Here, a peptide affinity capture method was developed to identify potential pyrophosphatases and further interacting proteins from human cells. Therewith, 6 phosphatases were identified as potential pyrophosphatase candidates and further interacting proteins gave insights into the function and mechanisms of protein pyrophosphorylation. Thereby, the potential of this method was demonstrated to identify interacting proteins of protein pyrophosphorylation to understand the cellular role.

Protein-Arrays sind das Mittel der Wahl, um eine Vielzahl von Proteinen parallel zu untersuchen. Ziele dieser Untersuchungen sind meistens Proteininteraktionsnetzwerke zu entdecken oder besser verstehen zu können. Bisher wurden die benötigten Proteine fast ausschließlich mit biologischen Methoden gewonnen. Diese bieten allerdings keinen generellen Zugang zu posttranslational-modi-fizierten (PTM)-Proteinen. Somit war es bisher nicht möglich den Einfluss von PTMs auf Protein-Protein-Interaktionen (PPIs) im Arrayformat zu untersuchen. Die chemische Synthese kann dagegen Proteine mit ortsspezifischen PTMs liefern. Daher ist es verwunderlich, dass bislang noch keine Berichte über chemisch hergestellte PTM-Protein-Arrays existieren, besonders da PTMs meist entscheidend für proteomische Interaktionsnetzwerke sind. In der vorliegenden Arbeit wird eine Methodik beschrieben, die es ermöglicht PTM-modifizierte Protein-Domänen-Arrays auf der Oberfläche zu synthetisieren und zu analysieren. Mit der Methodik wurden 20 SH3-Domänen synthetisiert und 64 PPIs gemessen. Neben vier Hefe-SH3-Domänen wurden je acht humane (Phospho)SH3-Domänen der Abl- und Arg(Abl2)-Tyrosinkinase synthetisiert und funktionell untersucht. Es wurde gefunden, dass die Ligandenspezifität von Abl-SH3-Domänen durch Phosphorylierung feinreguliert wird. Je nach Phosphorylierungsmustern wurde die Affinität für spezifische Liganden erhöht oder erniedrigt. Der Ursprung dieser Phosphoregulierung wurde für die Abl-SH3-Domäne mit Hilfe der NMR-Spektroskopie und durch Zellexperimente versucht zu entschlüsseln und weiter validiert. / Protein-arrays are the method of choice to investigate a variety of proteins in a parallel fashion. Objectives of these studies are mostly to discover or to investigate protein interaction networks. So far, the necessary proteins were almost exclusively gained by biological methods. Unfortunately, generic access to proteins bearing post-translational modifications (PTM) is not provided by these techniques. Therefore, it was not possible to investigate the impact of PTMs on protein-protein-interactions (PPIs) on arrays so far. Chemical synthesis in contrast offers proteins with site-specific PTM incorporation. In this context, it is surprising, that chemical methods of PTM-protein array synthesis remained virtually unexplored, especially since these modifications are usually crucial for proteomic interaction networks. In this thesis, a methodology is described, that allows to synthesize and functional analyse post-translationally modified protein domain arrays on the surface. By using this methodology, 20 SH3 domains were synthesized and 64 protein-pep-tide interactions were measured. In addition to 4 yeast SH3 domains, 8 human (phospho) SH3 domains of the Abl and Arg(Abl2) tyrosine kinase were synthesized and functionally investigated. The experiments revealed that phosphorylation might serve as a means to fine tune the ligand recognition. Depending on the phosphorylation pattern the affinity to specific interaction partners were enhanced or reduced. The origin of this phosphoregulation was further investigated for the Abl SH3 domain by means of NMR spectroscopy and cellular experiments.

Legionella pneumophila ist der Erreger der Legionärskrankheit. Die Pathogenität des Bakteriums basiert auf seiner Fähigkeit innerhalb menschlicher Lungenzellen zu überleben und sich zu vermehren. Demzufolge ist L. pneumophila nicht nur interessant als wichtiges Pathogen, sondern kann auch als Sonde verwendet werden, um allgemeine intrazelluläre Ereignisse zu untersuchen. Ein Beispiel hierfür ist die, durch das Pathogen gestörte, intrazelluläre Kommunikation zwischen den Organellen des endoplasmatischen Retikulums (ER) und dem Golgi Apparat (GA). In der vorliegenden Studie schlagen wir ein neues Modell vor, wie das Bakterium erfolgreich seine replikative Nische, die Legionella Vakuole (LV), innerhalb des Zytosols aufbauen könnte, um seine Ausbreitung zu garantieren. Um die Mechanismen für die erfolgreiche Ausbeutung der Wirtszelle gezielt untersuchen zu können, haben wir mit Hilfe von siRNA spezifisch verschiedene Wirtszellproteinen herunterreguliert und den Einfuß der Abwesenheit dieser Proteine auf die Vermehrung von L. pneumophila gemessen. Die Ergebnisse wiesen darauf hin, dass die LV möglicherweise den Golgi Apparat imitiert und auf diese Weise den zellulären Vesikeltransport umleitet. Diese Theorie wurde durch in silico Ergebnisse unterstützt, die in der Proteinsequenz des Legionella Effektor-Proteins LidA, das auf der Vakuole lokalisiert ist, ein SNARE-ähnliches Motiv zeigte. Dies weist auf ein auf der Vakuole lokalisiertes SNARE-Erkennungsmotiv hin, das notwendig sein könnte, um zelluläre Transportvesikel zu koppeln. Aus dem Wissen heraus, dass L. pneumophila in der Lage ist, die Aktivierung der zellulären Proteine Arf1 und Rab1 durch Phosphorylierung und Dephosphorylierung zu regulieren, machten wir uns auf die Suche nach Proteinen, die auf Infektion hin modifiziert werden. Die Kommunikation von Wirt und Pathogen über Phosphorylierung ist bekannt im Bezug auf pathogenspezifische Modifikation des Zytoskeletts und Signalkaskaden in der Anti-Apoptose. Für diese Studie wurde ein Antikörper verwendet, der spezifisch phosphorylierte Tyrosinreste erkennt. Dies resultierte in der Detektion einer Serin-Threonin-Kinase in der Amöbe Acanthamöba castellanii, die an einem Tyrosinrest phosphoryliert ist. Diese Amöben-Kinase wies in silico Homologie zu der humanen GS-Kinase 3 des Wnt-Signalwegs, bekannt aus der Forschung der embronalen Entwicklung bei Drosophila, auf. Der letzte Teil dieser Arbeit konzentrierte sich auf die, durch eine L. pneumophila-Infektion ausgelöste, anti-apoptotische Signalkaskade. Es ist bekannt, dass auf eine Infektion hin NF-kappaB aktiviert wird. Dies führt dazu, dass p65 in den Zellkern wandert und dort als Transkriptionsfaktor aktiv wird. Diese Translokation geschieht in 2 zeitversetzten Phasen. Eine Aktivierungsspitze wird nach dem Kontakt mir bakteriellem Flagellin gemessen, gefolgt, von einer dauerhaften Aktivierung, abhängig von einem funktionierenden Dot/ Icm Typ-IV-Translokationssystem. In dieser Arbeit stießen wir auf eine L. pneumophila Mutante, die den Dot/ Icm-Effektor SdbA nicht bildet, und die daraufhin NF-appaB nicht aktivieren kann. Diese Mutante war ebenfalls nicht in der Lage, sich in Epithelzellen zu vermehren. Dies ist außergewöhnlich, da das L. pneumophila Effektor Repertoire so redundant ist, dass die Abwesenheit eines einzigen Effektors selten einen so starken Einfluss auf die Replikation hat. All diese Ergebnisse zeigen zusammengenommen, auf wie vielen verschiedenen Ebenen L. pneumophila in der Lage ist, seine Wirtszelle zu manipulieren, um einerseits die nötige Nische für seine Vermehrung zu etablieren und andererseits die Zelle am Selbstmord zu hindern. Dies geschieht durch Imitation zellulärer Prozesse. / Legionella pneumophila is the causative agent of Legionnaires´ disease. The bacterium’s pathogenicity is based on its ability to survive and multiply efficiently inside human alveolar cells. Therefore, L. pneumophila is not only an important pathogen, but can also be used as a probe to investigate host cell function as for example, in the cellular trafficking pathway. In this study, we establish a new model of how this pathogen efficiently constructs its replicative niche, the Legionella containing vacuole (LCV), inside the host cytosol, enabling its dissemination. To investigate the mechanisms that lead to effective exploitation of the host cell, we down-regulated specific host cellular proteins via siRNA technology and measured the subsequent impact on L. pneumophila replication. The results suggest that the LCV mimicks the Golgi apparatus and via this mechanism hijacks host cellular vesicular trafficking. The L. pneumophila secreted effector protein LidA, located within the LCV, is shown to have a SNARE-like motif, suggesting a SNARE like sole connected to the LCV. Since it is known that cellular signalling proteins are controlled via phosphorylation and dephosphorylation, we went on to search for specifically modulated host cell proteins after L. pneumophila infection. The cross-talk of the pathogen with its host via phosphorylation has been connected to several sub-cellular activities leading to, for instance, cytoskeleton rearrangement and signalling events including anti-apoptosis pathways. Here we used a phosphorylated tyrosine antibody resulting in the detection of an amoeba serine-threonine-kinase, phosphorylated at its tyrosine residue. This kinase shows homologies to the human GSK3 of the wnt-signalling pathway. (“Wnt“ is merged from the names of the homologues genes Wg (Drosophila melanogaster) and Int (mouse) both employed in evolutionary developement.) The final part of this work concentrated on anti-apoptotic signalling events induced upon L. pneumophila infection. It is known that during L. pneumophila infection the activation of NF-kappaB and subsequent translocation of p65 from the cytosol into the nucleus follows a biphasic pattern. One short peak of activation is induced upon contact with bacterial flagellin, succeeded by a permanent Dot/ Icm type IV secretion system-dependent activation. In this study, we found the L. pneumophila mutant lacking the Dot/ Icm effector SdbA to be unable to activate NF-kappaB. This mutant also showed impaired growth in epithelial cells. This is remarkable due to the high redundancy of the L. pneumophila effector system, meaning deletion of a single effector rarely has such a big impact on replication. Taken together this work demonstrates, the manifold ways in which L. pneumophila on the one hand side establishes its niche to ensure replication and on the other hand side to bars its host cell from suicide. All of this is managed by mimicking cellular processes.

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

In plants, calcium is a ubiquitous signaling molecule and changes in cytosolic calcium levels reported in response to various abiotic and biotic stresses like salt stress, drought, pathogen attack and phytohormone signaling. Any calcium- mediated signal transduction process involves the establishment of a signal-specific change in the cytosolic calcium concentration termed as ‗calcium signature‘ which is decoded by the specific group of proteins called ‗calcium sensors‘ (eg: Calmodulin (CaM) and Ca2+ - regulated kinases). Plants have a novel group of kinases designated as Ca2+- dependent protein kinases (CDPK; EC 2.7.1.37). CDPKs are biochemically distinct from other Ca2+- dependent kinases, such as Ca2+- and phospholipid- dependent protein kinases, as they are activated directly by Ca2+-and are independent of CaM. They exist as monomeric serine/threonine protein kinases and consist of four domains namely an amino-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). CDPKs represent a unique class of Ca2+ sensors, having protein kinase as well as CaM-LD in a single polypeptide chain, enabling them to couple the calcium sensor directly to its responder (kinase). In the absence of calcium signature, CDPKs activity is inhibited by the autoinhibitory domain, which acts as a pseudo-substrate of kinase domain and thus blocks the active site of the enzyme. In the presence of calcium signature, CDPKs undergo conformational changes leading to removal of the inhibition. Besides plants, CDPKs are also reported in few protozoans viz Plasmodium falciparam, Paramecium and Taxoplasma. However, CDPKs are not found in the eukaryotic genome of yeast, nematodes, fruitflies and humans. In the current study, we have cloned CDPK1 gene from Cicer arietinum (CaCDPK1) in pRSET-A expression vector and expressed it in Escherichia coli BL21pLysS strain. However, while expressing the recombinant CaCDPK1 in E.coli, most of the recombinant CaCDPK1 protein was expressed as insoluble form. Therefore, we focused our efforts on optimizing the culture conditions for achieving the maximum yield of soluble recombinant CaCDPK1. Expression of the soluble CaCDPK1 was achieved by optimizing the different conditions like IPTG concentrations, temperature and growth time after induction. Maximum amount of soluble expression of recombinant CaCDPK1 was achieved by inducing the bacterial culture with 0.1 mM IPTG at 0.6 OD and growing it further for 4 h at 25°C. As with several other CDPKs, CaCDPK1 was found to get autophosphorylated in a calcium-dependent manner. To find the significance of autophosphorylation, we measured the substrate phosphorylation activity of the native and autophosphorylated CaCDPK1, which revealed that the autophosphorylation enhances the kinase activity of CaCDPK1 by 2-fold. Autophosphorylation was linearly dependant on concentrations of the enzyme suggesting that the autophosphorylation in CaCDPK1 occurs via an intra-molecular mechanism. Further analysis of autophosphorylation shows that autophosphorylation happens before substrate phosphorylation and provides calcium -independent substrate phosphorylation property. It also reduces the lag phase for activation of the enzyme and utilizes both ATP and GTP as phosphor-donor, but ATP is preferred over GTP. Autophosphorylation was found to occur at serine and threonine residues. The MALDI MS/MS analysis of the cold ATP autophosphorylated CaCDPK1 showed Thr- 339, Ser- 357, and Ser- 367 residues could be the potential autophosphorylation sites in CaCDPK1. Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress conditions. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported in this study. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 1.3 nM) when compared to PA (K½ = 56 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response. In the current study we also investigated CaCDPK1 interactions with calcium ions to address the Ca2+ -induced conformational changes in CaCDPK1 by using circular dichroism (CD), fluorescence spectroscopy and isothermal titration (ITC). Isothermal calorimetric analysis of calcium binding to CaCDPK1 shows a biphasic curve with two Kd of 27 nM and 1.72 µM respectively. The fluorescence measurements showed quenching in fluorescence intensity with a 5 nm red shift. The plot of changes in intensity against calcium concentrations again showed a biphasic curve, indicating that there may be more than one kind of Ca2+ binding sites. 8-anilinonaphthalene-1-sulfonic acid (ANS) binding showed that calcium bound form of CaCDPK1 exposes hydrophobic surfaces which may act as binding sites for other proteins. CD analysis of CaCDPK1 showed that it‘s an alpha helical rich protein and its helical content increases after binding to calcium. Taken all together this study describes the successful heterologous expression of Cicer arietinum CDPK isoform 1 in E.coli. and demonstrates that the autophoshorylation happens via an intra-molecular mechanism and it increases the kinase activity of CaCDPK1 at least by 2-fold. We also report here that CaCDPK1 prefers ATP as phosphodonor over GTP. The present study also shows the activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its Vmax and affinity towards the exogenous substrate, histone III-S. The current study also shows that calicum binding induces conformational changes in CaCDPK1 and the all four EF hand motifs of CaCDPK1 do not function in an equivalent manner.

Bacillus subtilis est la bactérie modèle des bactéries Gram-positif à bas pourcentage en GC et possède un intérêt marqué en biotechnologie. Par ailleurs, la phosphorylation des protéines est un mécanisme de régulation essentiel chez les bactéries qui reste encore largement à explorer. B. subtilis possède plusieurs ser/thr kinases potentielles (PrkA, YbdM, YabT et PrkC, qui a été déjà largement caractérisée), mais très peu de substrats de ces kinases ont été mis en évidence. Récemment, des études phosphoprotéomiques ont permis d’identifier de nombreux peptides phosphorylés sur des sérines ou des thréonines chez B. subtilis, incluant: a) deux régulateurs globaux de la phase de transition, DegS et AbrB et b) RecA, qui joue un rôle essentiel dans la réparation des cassures double-brin de l’ADN et la recombinaison. Des tests de phosphorylation in vitro nous ont permis d’identifier les ser/thr kinases capables de phosphoryler DegS, RecA et AbrB. La phosphorylation de DegS sur son résidu sérine 76 par la kinase YbdM influence, in vitro et in vivo, son activité kinase vis à vis de son substrat DegU. L’expression chez B. subtilis d’un allèle codant la protéine DegS-S76D (la sérine étant remplacée par un aspartate phosphomimétique) perturbe l’ensemble des processi cellulaires régulés par le système à deux composants DegS/DegU. Ces résultats suggèrent un lien entre la phosphorylation de DegS sur sa sérine 78 et le niveau de phosphorylation de son substrat DegU, cette modification post-traductionnelle représentant un degré supplémentaire de régulation pour ce système à deux composants. Au cours du démarrage de la sporulation, B. subtilis exprime une ser/thr kinase atypique, YabT, qui localise au septum et est activée grâce à la liaison de séquences ADN non spécifiques. YabT activée phosphoryle RecA sur sa sérine 2, ce qui induit la formation de foci RecA. Dans une souche exprimant une protéine RecA non phosphorylable (RecA-S2A) ou inactivée pour yabT, la formation de spores en présence de lésions de l’ADN est diminuée. Ces résultats suggèrent une homologie fonctionnelle au cours du développement entre la phosphorylation de RecA chez B. subtilis et la phosphorylation de son homologue eukaryote Rad51, qui permet leur recrutement sur des lésions de l’ADN. Nous proposons donc que la phosphorylation de RecA serve de signal pour promouvoir la formation de foci au cours de la sporulation. In vitro, le régulateur transcriptionnel AbrB est phosphorylé par les kinases YabT, YbdM et PrkC, L’utilisation de protéines mutées AbrB-S86A (non phosphorylable) et AbrB-S86D (forme phosphomimétique) nous a permis de montrer que la phosphorylation d’AbrB diminue son affinité pour l’ADN cible. L’expression chez B. subtilis des protéines AbrB-S86A et –S86D perturbe des phénomènes mis en place au cours de la phase stationnaire comme la production d’exoprotéases, la compétence et la sporulation via la dérégulation des gènes et opérons AbrB-dépendants correspondants. Nous proposons donc que la phosphorylation d’AbrB par les Hanks-kinases constitue un mécanisme de contrôle supplémentaire nécessaire à l’inactivation de ce régulateur transcriptionnel, qui peut être activateur ou répresseur, pendant la phase de transition. / Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

No description available.

No description available.