Swimming motility of Rhodobacteraceae – Phylogenomic analysis and functional characterization of the archetypal flagellar system in Phaeobacter inhibens DSM 17395

Abstract

Many roseobacters exhibit a biphasic ‘swim-or-stick’ lifestyle and need two crucial capacities swimming motility and biofilm formation. Recently, three distinct flagellar geneclusters (FGC) have been identified within the family Rhodobacteraceae (fla1-fla3), of which one was horizontally transferred. Fla1 is the most abundant flagellar system, but the function of four universally conserved proteins (CP1-CP4) remained unclear. The model organism Phaeobacter inhibens DSM 17395 harbors the most abundant fla1-type flagellum and was ideal for an extensive investigation of its FGC. In this thesis a comprehensive phylogeny regarding the flagellar systems of more than 300 completely sequenced Rhodobacteraceae and a phylogenomic reference tree was established. The analyses clearly showed that fla1 is the archetypical FGC of the Rhodobacteraceae. Furthermore, motility assays of 120 Rhodobacteraceae strains revealed functionality of all three FGCs and unraveled a previously unknown type of dendritic motility on MB soft agar plates. Extensive screening of more than 12,000 P. inhibens transposon mutants on soft agar plates resulted in the identification of genes essential for fla1 motility, thereunder CP1 to CP4 and all three genes of the CtrA-phosphorelay. Genetic complementation of these mutants validated their essential function for swimming motility. An exoproteomic detection of flagellar proteins and electron microscopy suggested that CP1 and CP4 still synthesize a complete but static flagellum. CP2 and CP3 lack secreted flagellar proteins, pointing either towards a structural or a regulatory function of the proteins. Analyses of high throughput transcriptome data of the CtrA phosphorelay mutants showed a strong downregulation of chemotaxis and flagellar genes. The comparison of these data with the model organism Dinoroseobacter shibae revealed a core regulon of only 30 genes composed of flagellar genes. These data clearly show the crucial function of the archetypical fla1-type flagellum for Rhodobacteraceae. The most conspicuous motility phenotype among the transposon mutants was developed by a mutant of a novel ECF sigma factor, swimming in concentric rings. Further experiments documented an involvement of the ECF in TDA-biosynthesis and chemotaxis. Overall, this thesis provides the most comprehensive analysis on flagellar motility in Rhodobacteraceae to date and thus essentially contributes towards a deeper understanding of this important bacterial family.