Strategies for the engineering of allosteric proteins, which are proteins that bind ligands at a specific site other than the reaction site and affect the reaction activity, are still being perfected. There have been allosteric proteins successfully engineered based on the hypothesis that the two allosterically related sites are distinct, modular domains and use trial and error to construct and test novel protein domain fusions for allostery. This work uses laboratory evolution to engineer the peptide binding affinity of the protein binding domain of the allosteric E. coli protease DegS. The protein binding domain is a PDZ domain (named for Postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and Zonula occludens-1 (ZO-1)) that binds the C-terminus of unfolded outer membrane porins. Combinatorial libraries of PDZ domain variants were displayed anchored to the periplasmic membrane of E. coli. The cells were permeabilized and incubated with fluorescent peptide ligands. PDZ domains were screened by flow cytometry for binding to the target peptide ligands. The PDZ domain binding affinity was improved by 20-fold for the peptide ligand that represents the physiological ligand; and the PDZ domain binding affinity was expanded to accommodate a negatively charged residue in a novel peptide ligand. The E. coli anchored peripalsmic expression (APEx) methodology in conjunction with flow cytometry had not previously been used to modify the binding affinity of a PDZ domain. The selected PDZ domain variants were then fused to the wild-type DegS protease domain and analyzed to determine if allosteric activation was made more sensitive to the native ligand or altered to respond to the novel peptide ligand. Interestingly, the DegS fusion protein with the PDZ variant containing the most subtle mutations retained a degree of allostery for the physiological peptide ligand and obtained a degree of allostery for the novel activating peptide ligand. Other selected PDZ variants with additional and expected mutations in the ligand binding site did not respond allosterically to the peptide ligands and the respective DegS fusions were constitutively active, suggesting that the amino acid network linking the allosteric binding event to protease activity is intricately integrated. / text

Vibrio cholerae, der Erreger der gastrointestinalen Erkrankung Cholera, ist ein Gram- negatives, fakultativ anaerobes gekrümmtes Stäbchenbakterium und zugleich der wohl bekannteste Vertreter der Familie Vibrionaceae. Es persisitiert die meiste Zeit in aquatischen Ökosystemen wie Flüssen, Seen oder Meeresküsten, wo das Bakterium meist mit Crustaceen oder anderen Organismen mit Chitin-haltigen Oberflächen assoziiert vorliegt. Über orale Aufnahme kontaminierter Lebensmittel oder von Wasser kann das Bakterium in den menschlichen Organismus gelangen und dort den oberen Dünndarmbereich kolonisieren, wo letztlich durch verschiedene Virulenzfaktoren, aber hauptsächlich durch das Cholera-Toxin, die Symptomatik der Cholera ausgelöst wird. V. cholerae ist somit sowohl in seiner natürlichen Umgebung, als auch im humanen Wirt höchst unterschiedlichen Umweltbedingungen ausgesetzt. Diese alternierenden Umweltreize stellen verschiedene Anforderungen an die Expressions- und Regulationsfähigkeiten von Proteinbiosynthesen des Bakteriums dar. Die Notwendigkeit einer raschen Adaption setzt daher vielfältige und komplexe Genregulationsmechanismen voraus. Im ersten Teil der hier vorliegenden Arbeit sollte die Genregulation des chs-Operons untersucht werden. Als Grundlage dienten hierbei Hinweise, nach welchen dieses Operon als putatives PTS eine Rolle für den Metabolismus von dem Chitin-Derivat Chitobiose spielen könnte. Zudem sollte der Einfluss des aus Escherichia coli bekannten Repressors Mlc auf die Expression des Operons tiefer gehend untersucht werden. Im Rahmen dieser Arbeit war es gelungen, das als ChsR benannte Protein eindeutig als spezifischen LacI-ähnlichen Repressor für das chs-Operon zu bestätigen. Weiter konnte auch eine cAMP-abhängige Expressionsinduktion bestätigt werden, welche sich allerdings nur bei inaktiven ChsR durchsetzen kann. Als spezifischer Induktor für den Repressor ChsR konnte Chitobiose (GlcN)2 identifiziert werden, welches zwar bei dem in dieser Arbeit verwendeten O1-Stamm SP27459-S nicht als alleinige Kohlenstoffquelle dienen kann, aber unter induktiven Konzentrationen die Repressoreigenschaft von ChsR inhibiert. Zugleich konnte ChsC als für den Import des Induktors Chitobiose verantwortliches Protein identifiziert werden. Weiter nicht eindeutig zu klären blieb der Einfluss von Mlc auf das chs-Operon. Zwar konnte der aktivierende Effekt von Mlc auf die chs-Expression durch Komplementation bestätigt werden, der genaue Mechanismus bleibt jedoch weiterhin unbekannt und bedarf weiterer Untersuchungen. Einzig der Einfluss von Mlc auf den Chitobiose-Import konnte ausgeschlossen werden. Im zweiten Teil dieser Arbeit sollte der weitaus komplexere Mechanismus der Virulenzgenregulation untersucht werden. Im Fokus stand hierbei der Hauptvirulenz-genregulator ToxR und dessen Abhängigkeit von der periplasmatischen Protease DegS. Anhand unterschiedlicher Experimente auf Promotoraktivitäts-, mRNA- und Proteinebene konnte eine Abnahme der ToxR-Aktivität in der degS-Knockout Mutante beobachtet werden, was auf eine Aktivierung von ToxR durch DegS schließen lässt. Weiter konnte eine Abhängigkeit der Aktivität von ToxR von der ebenfalls DegS-abhängigen RpoE-Signalkaskade ausgeschlossen werden. Auch konnte gezeigt werden, dass die Integrität von ToxR durch ToxS, nicht aber durch DegS bestimmt wird. Der exakte Mechanismus der DegS-induzierten ToxR-Aktivierung konnte im Rahmen dieser Arbeit nicht mehr ermittelt werden. Es wurden jedoch Hinweise darauf gewonnen, dass eine direkte ToxR-DegS-Interaktion im periplasmatischen Raum stattfinden könnte. Die in dieser Arbeit gewonnen Erkenntnisse hinsichtlich der ToxR-Regulation durch DegS bieten sowohl eine interessante neue Perspektive der Funktionsweise der periplasmatischen Protease DegS, als auch eine breite Grundlage für weitergehende Untersuchungen bezüglich der Aktivierung des wichtigsten Virulenzregulators ToxR in V. cholerae. / Vibrio cholerae, the causative agents of the gastrointestinal disease cholera, is a Gram-negative facultative anaerobic curved bacterium. It further is probably the best characterized member of the family Vibrionaceae. V. cholerae mainly persists in aquatic ecosystems such as rivers, lakes or sea-coasts where it is found associated with crustaceae and other organisms exposing chitin-containing surfaces. The bacterium infects the human organism via the oral uptake pathway by ingestion of contaminated food or water. Subsequently, it colonizes the upper part of the small intestine and there it eventually causes the typical symptoms of cholera. Thus, both in its natural surrounding and within the human host, V. cholerae faces dramatically alternating environmental conditions. These challenges exhibit different demands and flexibility to alteration of protein expression. This necessity for efficient adaption requires manifold and complex mechanisms of gene regulation. In the first part of the study presented here, the gene regulation of the chs-operon has been examined. In the forefront of this examination there were indications that this operon may play a role as a putative PTS for the metabolism of the chitin-derivate chitobiose. Furthermore, the influence of the in Escherichia coli well-known repressor Mlc on the expression of the operon has been determined. Within this study the protein termed ChsR could be confirmed as a specific LacI-similar repressor type protein for the chs-operon. Also, a cAMP-dependend induction of expression could be verified, which however, can only be achieved when ChsR is inactive. Chitobiose (GlcN)2 has been identified as the specific inductor for the repressor ChsR. This inductor substrate cannot be used as the only carbon-source for the O1-strain SP27459-S, but is able to act on the repressor ChsR under inductive concentrations to cause depression on the chs-operon. Furthermore, ChsC could be identified to be responsible for the import of the inductor chitobiose. The influence of Mlc on the chs-operon could not be elucidated. Even though the activating effect of Mlc on the chs-expression has been confirmed via complementation analysis, however the exact mechanism remains unknown and needs further investigations. Finally, an influence of Mlc on the import of chitobiose could be ruled out. In the second part of this study a far more complex mechanism of virulence gene expression has been investigated. The examinations concentrated on the main virulence regulator ToxR, which is involved in gene regulation of cholera-toxin genes and others, and its dependence on the periplasmatic protease DegS. On the basis of various experiments a decrease of ToxR-activity in a degS-knockout mutant could be observed on promoter-activity-, mRNA- and protein level, utilizing the ToxR dependent regulated porin OmpU. The obtained results clearly indicated that an activation of ToxR via interaction with DegS seems possible. Furthermore, a dependence of ToxR-activity on the DegS-dependent RpoE-signal cascade could be ruled out. Also it could be demonstrated that the integrity of ToxR is maintained by ToxS, but not by DegS. However, the exact mechanism of the DegS-induced activation of ToxR could not be determined within this study and should be investigated in future. So far only genetic derived indications have been gained that there is direct interaction between ToxR and DegS in the periplasmic space, a proof by protein/protein interaction is still lacking. The findings summarized in this study addressing the regulation of ToxR via DegS present an interesting new perspective of the function of the periplasmic protease DegS involved in affecting a general virulence regulatory pathway. Moreover, the data will serve as the basis for further investigations on the molecular mechanism of activation and signal transduction of the most important virulence factor ToxR in V. cholerae.

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.

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.