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Cell cycle-regulated surface structures and mobile DNA in bacteria

English title Cell cycle-regulated surface structures and mobile DNA in bacteria
Applicant Viollier Patrick
Number 182576
Funding scheme Project funding (Div. I-III)
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Experimental Microbiology
Start/End 01.10.2018 - 30.09.2022
Approved amount 1'008'000.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Genetics

Keywords (8)

buoyancy; polarity; capsule; flagellum; glycosylation; phage; pili; protein localisation

Lay Summary (German)

Lead
Unsere Forschung befasst sich mit der Erkundung neuer Kontrollmechanismen und deren Eiweisse in Bakterien. Unser Interesse gilt besonders den Mechanismen, die einer strikten zeitlicher Kontrolle unterliegen, wie zum Beispiel jene, welche bestimmte Vorgänge auf bestimmte Zeitpunkte im Zellzyklus beschränken. Um diese Studien auszuführen, bedienen wir uns des besten Modellsystems für Zellzyklusstudien in Bakterien: dem a-Proteobacterium Caulobacter crescentus. Als nicht-pathogenes Bakterium eignet sich Caulobacter besonders für solche Untersuchungen, da es mit einer simplen Zentrifugationsmethode synchronisiert werden kann, d.h. Zellen von verschiedenen Stadien des Zellzyklus isoliert werden können.
Lay summary

Studien verschiedener Forschungsgruppen haben erwiesen, dass in C. crescentus der Zusammenbau der Geissel, der Fimbrien und der Oberflächenmatrix (Kapsel) in genau vorprogrammierten Phasen des Zellzyklus erfolgt.  Unsere Vorarbeiten zeigten auf, dass die Untereinheiten der Geissel (auch Flagellum genannt) zuerst mit Sialinsäure-Zuckerresten verknüpft werden müssen, damit der Zusammenbau erfolgen kann. Ausserdem wird die Erstellung der Fimbrien und der Geissel durch spezielle gemeinsame Eiweisse koordiniert, die sich am Zellpol befinden und einer Zellzykluskontrolle unterliegt. Die Koordination anderer Oberflächenstrukturen zum Beispiel der Kapsel, die die Bakterien vor Infektion gewisser Bakteriophagen schützt, unterliegt ebenfalls Kontrolle von Eiweissen die am Pol haften. Unsere Studien dieser polaren Eiweisse wird wichtige Erkenntnisse liefern, die als Angriffspunkt zur möglichen Bekämpfung krankheitserregender Bakterien dienen könnten, da die Geissel, Fimbrien und die Kapsel im Allgemeinen als wichtige molekulare Komponenten fungieren die es einige krankheitserregende Bakterien erlaubt Wirtszellen zu befallen. Gelingt es mittels gezielter Inaktivierung jene Mechanismen auszuschalten, sollte es möglich sein, bakterielle Infektionen zu reduzieren oder sogar ganz zu verhindern.

Direct link to Lay Summary Last update: 01.10.2018

Responsible applicant and co-applicants

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Associated projects

Number Title Start Funding scheme
162716 Cell cycle-regulated surface structures in bacteria 01.10.2015 Project funding (Div. I-III)

Abstract

Cell cycle-regulated surface structures and mobile DNA in bacteria Bacterial cell surface structures are important virulence determinants and activators of innate or adaptive immune responses. Many bacteria, especially members of the a-proteobacteria class, feature an overt cell cycle in which they trigger key events at pre-determined phases. In the model a-proteobacterium Caulobacter crescentus the pili and flagellum, two proteinaceous cell surface structures, are known to be cell cycle regulated. We recently also found the polysaccharide capsule and mobile DNA to be constrained during the C. crescentus cell cycle. While the underlying mechanisms are still incompletely understood, the synchronizeability of C. crescentus makes it a superb system in which to interrogate the temporal regulation of such dissimilar cellular components. With mobile DNA being a principal factor in the lateral spread of antibiotic resistance genes, the impact of the cell cycle on DNA mobility has important implications on understanding how resistance genes are mobilized and how to curb their dissemination. Superimposed on the temporal control of piliation and flagellation is the highly localized, but incompletely understood, assembly of these structures at the newborn cell pole. Interestingly, recent evidence suggests that the major subunit of the C. crescentus flagellar filament, FljK, is post-translationally modified (glycosylated) with the “sialic acid-like” nonulosonate sugar, pseudaminic acid, covalently appended by the putative glycosyltransferase FlmG. This indicates that unknown specificity determinants of FlmG exist. With these questions in mind, our new goals are to:1: Resolve the specificity in glycosylation of the flagellum by the FlmG (glycosyltransferase).2: Elucidate how the ZitP polarity factor localizes to the newborn pole to implement pilus assembly.3: Unravel how the HvyA protease homolog prevents capsulation and how its translation is regulated.4: Dissect the cell cycle control of prophage excision and the epigenetic control of FCbK transcription.Collectively our work will illuminate several different molecular and cytological mechanisms of spatio-temporal regulation in bacteria, primarily relying on forward genetic, cytological and genomic approaches we are experienced in. The approaches include live cell microscopy (Aim 2), transposon mutagenesis and comprehensive transposon insertion site sequencing (Tn-Seq, Aim 2, 3), co-immunoprecipitation and immunoblotting (Aim 1, 2, 4), biochemical cell wall purification (Aim 2, 3), binding assays with recombinant proteins (Aim 1, 2) and RNA-Seq (Aim 3 and 4).In the face of ever-increasing spread of antimicrobial resistance genes, it is paramount to advance our understanding of basic mechanisms operating on key virulence determinants (surface structures) and mobile DNA responsive to the cell cycle in bacteria. It is estimated that 30 years from now more people will succumb to untreatable microbial infections than to cancer, warranting the need for research on bacteria, including model systems. By focusing on mechanisms at the level of DNA (epigenetic and recombination), RNA (transcription and translation) and protein (glycosylation and localization), our explorations of regulatory processes are also relevant to understanding how cells function in general and they may set the stage for new applications in biotechnological or medical fields in the future
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