Salmonella spp. sind fakultative intrazelluläre Pathogene, die gastrointestinale und systemische Erkrankungen in einem umfassenden Wirtsbereich, einschließlich Tier und Mensch, hervorrufen. Salmonella benötigt verschiedene Virulenzgene für die Infektion welche auf sogenannten Salmonella Pathogenitäts-Inseln (SPI) kodiert sind. Hinzu kommt, dass auch zahlreiche im Salmonella Genom verstreuten Gene an verschiedenen Aspekten von Virulenz und Pathogenese beteiligt sind. In der vorliegenden Studie wurde die Funktion eines zuvor nicht beschriebenen putativen transkriptionellen Regulators (STM0029) charakterisiert und definiert. Dieser scheint für die Abwehr von zellulären bakterizid wirkenden Verbindungen und das Überleben des Bakteriums innerhalb einer intrazellulären Nische von entscheidender Bedeutung zu sein. Die STM0029-deletierte Mutante wies eine gesteigerte Sensitivität gegenüber antimikrobiellen Peptiden und bakteriziden Verbindungen auf. Dazu zählten α-Defensin-1, β- Defensin-1, β-Defensin-2, LL-37 und Polymyxin B sowie Komponenten des Komplementsystems. Unerwartet war die Beobachtung, dass die Expression von STM0029 durch das PmrA/B Zwei Komponenten System reprimiert vorlag, während das PhoP/Q Zwei Komponenten System keinen Einfluss auf die Expression von STM0029 zu scheinen hat. Beide Komponent Systeme spielen bekanntlich eine entscheidende Rolle bei der Expressionsregulation von Genen die für das intrazelluläre Überleben von Salmonella wichtig sind. Bemerkenswert ist, dass ein Set von Genen welche an der Biosynthese und/oder der Modifikation für das LPS O-Antigen sowie des Peptidoglykans in der bakteriellen Zellwand beteiligt ist, im STM0029 Deletionshintergrund herab reguliert vorlag. Dieses Ergebnis deutet darauf hin, dass das STM0029 Genprodukt die Persistenz des Pathogen in Wirtszellen beeinflusst. Möglicherweise geschieht dies durch das Umgehen von wirtseigenen Abwehrmechanismen. / Salmonella spp. are facultative intracellular pathogens, which cause gastrointestinal and systemic diseases in a broad range of hosts including animals and humans. In addition to virulence genes clustered within pathogenicity islands, numerous additional genes scattered throughout the genome are also involved in various aspects of Salmoenlla virulence and pathogenesis. In this study, I identified a Salmonella putative transcriptional regulator encoded by a previously uncharacterized open reading frame designated STM0029. Deletion of STM0029 altered the expression of genes involved in both the resistance to host bactericidal challenges, and bacterial cell wall biosynthesis in S. Tyhpimurium. The ΔSTM0029 strain showed a defect in the resistance to host antimicrobial peptides, including α-defensin-1, β-defensin-1, β-defensin-2, LL-37, and polymyxin B as well as serum challenges compared to the wildtype. Unexpectedly, expression of STM0029 was found to be repressed by the PmrA/B two component system, but appeared to be independent of the PhoP/Q two component system, both of which are well-known regulatory systems involved in the regulation of expression of genes involved in Salmonella intracellular survival. Notably, the expression of a set of genes involved in bacterial LPS O-antigen and peptidoglycan biosyntheses and modifications showed decreases in the absence of STM0029. These experimental results indicate that the STM0029 gene product in S. Typhimurium contributes to resistance against host cell defense mechanisms, likely through regulation of genes involved in LPS O-antigen and peptidoglycan biosynthesis and modifications.

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.

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In this thesis, I investigate dynamic and computational properties of prokaryotic signalling architectures commonly known as the Two Component Signalling networks and phosphorelays. The aim of this study is to understand the information processing capabilities of different prokaryotic signalling architectures by examining the dynamics they exhibit. I present original investigations into the dynamics of different phosphorelay architectures and identify network architectures that include a commonly found four step phosphorelay architecture with a capacity for tuning its steady state output to implement different signal-response behaviours viz. sigmoidal and hyperbolic response. Biologically, this tuning can be implemented through physiological processes like regulating total protein concentrations (e.g. via transcriptional regulation or feedback), altering reaction rate constants through binding of auxiliary proteins on relay components, or by regulating bi-functional activity in relays which are mediated by bifunctional histidine kinases. This study explores the importance of different biochemical arrangements of signalling networks and their corresponding response dynamics. Following investigations into the significance of various biochemical reactions and topological variants of a four step relay architecture, I explore the effects of having different types of proteins in signalling networks. I show how multi-domain proteins in a phosphorelay architecture with multiple phosphotransfer steps occurring on the same protein can exhibit multistability through a combination of double negative and positive feedback loops. I derive a minimal multistable (core) architecture and show how component sharing amongst networks containing this multistable core can implement computational logic (like AND, OR and ADDER functions) that allows cells to integrate multiple inputs and compute an appropriate response. I examine the genomic distribution of single and multi domain kinases and annotate their partner response regulator proteins across prokaryotic genomes to find the biological significance of dynamics that these networks embed and the processes they regulate in a cell. I extract data from a prokaryotic two component protein database and take a sequence based functional annotation approach to identify the process, function and localisation of different response regulators as signalling partners in these networks. In summary, work presented in this thesis explores the dynamic and computational properties of different prokaryotic signalling networks and uses them to draw an insight into the biological significance of multidomain sensor kinases in living cells. The thesis concludes with a discussion on how this understanding of the dynamic and computational properties of prokaryotic signalling networks can be used to design synthetic circuits involving different proteins comprising two component and phosphorelay architectures.

Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.

Les antibiotiques sont des molécules incontournables dans le traitement des infections bactériennes. L’émergence et la dissémination de la résistance aux antibiotiques chez la pathogène opportuniste Pseudomonas aeruginosa, ont amené l’Organisation Mondiale de la Santé à déclarer indispensable le développement de nouvelles approches thérapeutiques pour lutter contre cette bactérie. Bien que certaines alternatives aient été envisagées, la préservation de l’activité d’antibiotiques majeurs tels que les aminosides et la colistine est primordiale. La caractérisation des mécanismes de résistance à ces médicaments est nécessaire pour la mise au point de nouvelles molécules et mieux prendre en charge les patients. Dans ce contexte, nous montrons que des mutations dans le gène fusA1 (codant le facteur d’élongation EF-G1A) et dans l’opéron pmrAB (système à deux composants PmrAB) entrainent une augmentation de la résistance aux aminosides chez des mutants isolés au laboratoire et des souches issues de patients, atteints ou non, de mucoviscidose. Certaines substitutions d’acide aminé dans EF-G1A accroissent les niveaux de résistance de 2 à 16 fois aux quatre sous-classes d’aminosides. Par ailleurs, des changements d’acide aminé dans le système à deux composants PmrAB activent l’expression des gènes PA4773-PA4774-PA4775, et la production de norspermidine et de spermidine. La synthèse de ces polyamines va de pair avec une baisse de 4 à 16 fois de la sensibilité aux aminosides à noyan 2-désoxystreptamine bisubstitué en 4,6 (gentamicine, amikacine et tobramycine). De plus, il apparaît que la résistance des mutants pmrB à la colistine est en partie dépendante de la pompe d’efflux MexXY(OprM), un système impliqué dans la résistance naturelle, adaptative ou acquise aux aminosides. Enfin, nous montrons que les mutants pmrB surproduisent des alcaloïdes contenant un motif azétidine, par une voie de synthèse non-ribosomale et dépendante du quorum sensing. Ces alcaloïdes diminuent la virulence de P. aeruginosa dans le modèle Galleria mellonella. / Antibiotics are invaluable drugs to combat bacterial infections. Emergence and spread of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa have led the World Health Organization to consider as a crucial priority the development of new therapeutic approaches to fight this bacterium. In addition to other alternatives, preservation of activity of major antibiotics such as aminoglycosides and colistin is primordial. Consequently, characterization of the resistance mechanisms to these drugs is a prerequisite to design novel molecules, and improve patient care. In this context, we show that mutations in gene fusA1 (encoding elongation factor EF-G1A) and in operon pmrAB (two-component system PmrAB) lead to an increased resistance to aminoglycosides in in vitro-selected mutants and strains isolated from cystic fibrosis (CF) and non-CF patients. Certain amino acid substitutions in EF-G1A confer a 2- to 16-fold increased resistance to the four aminoglycoside subclasses. On the other hand, amino acid variations in two-component system PmrAB activate the expression of genes PA4773-PA4774-PA4775, and production of norspermidine and spermidine. This upregulated polyamine biosynthesis is associated with a 4- to 16-fold decreased susceptibility to 4,6-di-substituted deoxystreptamine aminoglycosides (gentamicin, amikacin and tobramycin). Moreover, our work reveals that the acquired resistance of pmrB mutants to colistin partially depends upon pump MexXY(OprM), a system that otherwise mediates intrinsic, adaptive and acquired resistance to aminoglycosides. Finally, we show that pmrB mutants overproduce azetidine-containing alkaloids by a quorum-sensing-regulated, nonribosomal peptide synthetase pathway. These alkaloids impair the virulence of P. aeruginosa in a Galleria mellonella infection model.

<p>A survival distribution is developed for exponential two-component systems that can survive as long as at least one of the two components in the system function. It is assumed that the two components are initially independent and non-identical. If one of the two components fail (repair is impossible), the surviving component is subject to a different failure rate due to the stress caused by the failure of the other.</p> <p>In this paper, we consider such an exponential two-component system failure model when the observed failure time data are (1) complete, (2) Type-I censored, (3) Type-I censored with partial information on component failures, (4) Type-II censored and (5) Type-II censored with partial information on component failures. In these situations, we discuss the maximum likelihood estimates (MLEs) of the parameters by assuming the lifetimes to be exponentially distributed. The exact distributions (whenever possible) of the MLEs of the parameters are then derived by using the conditional moment generating function approach. Construction of confidence intervals for the model parameters are discussed by using the exact conditional distributions (when available), asymptotic distributions, and two parametric bootstrap methods. The performance of these four confidence intervals, in terms of coverage probabilities are then assessed through Monte Carlo simulation studies. Finally, some examples are presented to illustrate all the methods of inference developed here.</p> <p>In the case of Type-I and Type-II censored data, since there are no closed-form expressions for the MLEs, we present an iterative maximum likelihood estimation procedure for the determination of the MLEs of all the model parameters. We also carry out a Monte Carlo simulation study to examine the bias and variance of the MLEs.</p> <p>In the case of Type-II censored data, since the exact distributions of the MLEs depend on the data, we discuss the exact conditional confidence intervals and asymptotic confidence intervals for the unknown parameters by conditioning on the data observed.</p> / Thesis / Doctor of Philosophy (PhD)

Background: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.

Das Biopolymer-produzierende Knallgasbakterium Ralstonia eutropha H16 gilt mit seinem außergewöhnlichen Stoffwechsel als vielversprechender Produktionsstamm für die weiße Biotechnologie. Es wächst auf einer Vielzahl organischer Substrate sowie chemolithoautotroph mit H2 und CO2 als einzige Energie- bzw. Kohlenstoffquelle. Unter anaeroben Bedingungen ist es zudem zur Denitrifikation befähigt. In dieser Arbeit wurde das Proteinprofil von R. eutropha unter chemolithoautotrophen sowie anaeroben Bedingungen mittels GeLC-MS/MS untersucht. Beide Proteomstudien offenbarten, dass die Nutzung unterschiedlicher Elektronendonoren bzw. -akzeptoren mit zahlreichen Veränderungen im Proteinbestand der Zellen einherging. Hierbei waren neben Proteinen metabolischer und Transportprozesse auch jene der Zellbewegung betroffen. Die Ergebnisse stellen im Vergleich zu vorangegangenen Studien den bisher umfassendsten Überblick zum Proteinbestand beim H2-basierten sowie anaeroben Wachstum in R. eutropha dar. Von besonderer Bedeutung war dabei das Einbinden der Analyse der Membran als Ort wichtiger Energie- und Transportprozesse. Besonderes Interesse galt einem unter H2/CO2-Bedingungen abundanten Zweikomponentensystem. Sequenzvergleiche zeigten Ähnlichkeit zum Regulationssystem der Katabolitrepression des Biphenylabbaus in Acidovorax sp. KKS102. Die Deletion des Response-Regulator-Gens führte zu vielfältigen Wachstumseffekten auf Substraten wie Fructose, Glycerin sowie auf H2/CO2. Der pleiotrope Phänotyp sowie die Ergebnisse von Genexpressionsstudien und der Suche nach Regulator-Bindestellen lassen eine globale Rolle des Systems im Energie- und/oder Kohlenstoffmetabolismus von R. eutropha H16 annehmen. Histidin-Kinase und Response Regulator wurden in GloS bzw. GloR umbenannt. Die vorliegende Arbeit zeigt eindrucksvoll das Potential der Proteomik als Teil der funktionellen Genomik für den Anstoß neuer Forschungsansätze zur Evaluierung des biotechnologischen Potentials von Mikroorganismen. / Due to its remarkable metabolism the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16 is ranked as a promising production strain for white biotechnology. It grows on a wide range of organic substrates as well as lithoautotrophically on H2 and CO2 as sole energy and carbon source, respectively. Under anaerobic conditions it thrives by denitrification. This thesis focused on characterizing the protein profiles of lithoautotrophically and anaerobically grown R. eutropha cells. Proteome analyses revealed an extensive protein repertoire adapting the organism to alternative electron donors and acceptors, respectively. Changes concerned proteins involved in metabolic and transport processes as well as in cell movement. Compared to previous studies the results reported here offer the most comprehensive proteomic survey regarding the H2-based as well as anaerobic lifestyle of R. eutropha so far. In this context analyzing the cell membrane as a place for a number of energy, transport and signal transduction processes was of particular importance. Special interest aroused the identification of a two-component system upregulated on H2/CO2. Sequence analysis offered high similarity to the regulatory system for catabolite control of biphenyl degradation in Acidovorax sp. KKS102. Deletion of the response regulator gene led to versatile growth effects on substrates such as fructose and glycerol as well as H2/CO2. This pleiotrophic phenotype as well as the results of gene expression studies and the search for regulator binding sites suggests that the two-component system is a global player in energy and/or carbon metabolism in R. eutropha and possibly other bacteria. Thus, histidine kinase and response regulator have been renamed GloS/R. Since their characterization was initiated by proteomic data this study impressively elucidates the power of functional genomics in terms of revealing new research approaches to evaluate the biotechnological use of microbes.