Search

The flagellum is of great importance for Salmonella enterica serovar Typhimurium to establish a successful infection by enabling directed movement. Flagellar biosynthesis is tightly regulated in a transcriptional hierarchy with the flagellar master regulatory operon flhDC on top. Recently, two novel regulators of flhDC transcription were discovered, RflM and HilD. In the first part of this thesis, RflM-dependent repression of flhDC transcription and the role of RcsB in this process was investigated. RcsB is the response regulator of the RcsCDB phosphorelay system and known to interact with auxiliary proteins to regulate target gene expression upon binding. Accordingly, results of this thesis demonstrated that RcsB and RflM form a heterodimeric protein complex. The RcsB-RflM protein complex represses flhDC transcription by binding to a RcsB/RflM box downstream of the P1-flhDC transcriptional start site. Thereby, RflM mediates target specificity of unphosphorylated RcsB for flhDC repression and increases its binding affinity, whereas RcsB serves to stabilize RflM. Since FlhDC itself controls transcription of rflM, RcsB/RflM-dependent repression of flhDC transcription is proposed to constitute a mechanism that fine-tunes the initiation of flagellar biosynthesis independent of external stimuli. In the second part of this thesis, the mode of action of HilD-dependent activation of flhDC transcription was investigated. HilD belongs to a family of DNA-binding proteins and is a major activator of the Spi-1 injectisome. Here, it was demonstrated that HilD operates by binding upstream of the P5-flhDC transcriptional start site, which adds another level of cross-talk between the flagellar and Spi-1 regulons. In the third part of this thesis, the gene regulation dynamics of RcsB/RflM- and HilD-mediated transcriptional regulation of flhDC was addressed. Single cell analyses showed that flhDC is growth phase-dependent heterogeneously expressed resulting in two subpopulations: flhDC-On and flhDC-Off. The negative FlhDC-RflM feedback loop enables downregulation of the P1-flhDC-dependent flhDC-On subpopulation during early exponential growth. The double positive FlhDC-FliZ-HilD feedback loop facilitates expression of flhDC during late exponential growth from P5-flhDC. Taken together, this allows S. Typhimurium to fine-tune flagellar motility and Spi-1-mediated host cell invasion and to rapidly adapt to changing environmental conditions during the infection process., Das Flagellum spielt für Salmonella enterica serovar Typhimurium eine große Rolle bei der Infektion, da es eine zielgerichtete Bewegung ermöglicht. Die Flagellen-Biosynthese ist durch eine transkriptionelle Hierarchie streng reguliert, wobei sich der Hauptregulator der Flagellensynthese flhDC am Anfang dieser Kaskade befindet. Vor kurzem wurden zwei neue Regulatoren der flhDC Transkription entdeckt: RflM und HilD. Im ersten Teil dieser Arbeit wurden die RflM-abhängige Repression der flhDC Transkription und die Rolle von RcsB dabei untersucht. RcsB ist der Responseregulator des RcsCDB Phosphorelays und bekannt dafür mit Hilfsproteinen die Expression von Zielgenen zu regulieren. Dementsprechend wurde in dieser Arbeit gezeigt, dass RcsB und RflM einen heterodimeren Proteinkomplex bilden. Der RcsB-RflM Komplex bindet downstream des P1-flhDC Transkriptionsstarts, um die flhDC Transkription zu hemmen. Dabei vermittelt RflM nicht-phosphoryliertem RcsB die Spezifität für die flhDC Repression und steigert dessen Affinität. RcsB dient der Stabilisierung von RflM. Da FlhDC selbst die rflM-Transkription kontrolliert, wurde die Hypothese aufgestellt, dass der RcsB/RflM Komplex zur Feinabstimmung der Initiation der Flagellen-Biosynthese dient. Im zweiten Teil dieser Arbeit wurde die Wirkungsweise der HilD-abhängigen Aktivierung der flhDC Transkription untersucht. HilD ist ein DNA-Bindeproteinen und ein Hauptaktivator des Spi-1 Sekretionssystems. In dieser Arbeit wurde gezeigt, dass HilD seine Funktion durch eine Bindung upstream des P5-flhDC Transkriptionsstarts ausübt. Dies trägt zur gegenseitigen Regulation zwischen dem flagellaren und Spi-1 System bei. Im dritten Teil dieser Arbeit wurden die Genregulationsdynamiken der RcsB/RflM- und HilD-vermittelten Regulation der flhDC Transkription untersucht. Einzelzellanalysen zeigten, dass flhDC wachstumsphasenabhängig heterogen exprimiert ist, wodurch zwei Subpopulationen resultierten: flhDC-An und flhDC-Aus. Der negative FlhDC-RflM Rückkopplungskreislauf führt zu einer Herunterregulation der flhDC-An Subpopulation von P1-flhDC während des frühen exponentiellen Wachstums. Der FlhDC-FliZ-HilD Rückkopplungskreislauf dient der flhDC Expression von P5-flhDC während des späten exponentiellen Wachstums. Zusammengenommen ermöglicht dies S. Typhimurium eine Feinabstimmung der flagellaren Motilität und der Spi-1-abhängigen Invasion und somit eine schnelle Anpassung an wechselnde Umweltbedingungen während des Infektionsprozesses.

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4 C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4 C2 -dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post- transcriptional levels. The flagellar master regulator FlhD₄C₂ resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD₄C₂- dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM- dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.