Coiled coil cytoskeleton in cell architecture and osmotic stress response in Streptomyces

Abstract: Coiled coil proteins emerge as important determinants of bacterial cellular organization. Several such proteins display properties characteristic to metazoan intermediate filament (IF) proteins, and are therefore designated bacterial IF-like proteins. The best studied examples of the latter are crescentin in Caulobacter crescentus that determines the curved cell shape, and FilP (filament forming-protein) in Streptomyces. The aim of this thesis was further characterization of the bacterial IF-like protein FilP in the medically and industrially important model organisms of genus Streptomyces, which are multicellular and mycelial bacteria. Previously, it was shown that FilP spontaneously and without any co-factor polymerizes into filaments in vitro, and that FilP is required for maintaining regular stiffness, elasticity and morphology of the hyphae. In this study we showed that FilP can assemble into tightly interconnected network structure in vitro, that is likely to possess high viscoelasticity, which can explain the role of FilP in hyphal stiffness. By immuno-staining technique FilP was shown to localize to the apical portions of the hyphae in a form of a gradient during active growth. Fluorescence time-lapse microscopy using fluorescently tagged derivatives of FilP showed that the FilP cytoskeleton is dynamically remodeled during growth, and that several different mechanisms including degradation, contribute to the turnover of FilP cytoskeleton and dynamically maintain the apical gradients of the FilP network. Additionally, our data showed that FilP is recruited the tips of the hyphae via an interaction with DivIVA, the determinant of Streptomyces polar growth. We propose a model whereby DivIVA recruits FilP to the apical regions to provide a mechanical support to the intrinsically weak tips of hyphae during a growth. Another avenue of research pursued in this thesis was to study the cell-biological aspects of osmotic stress response. We revealed a so far uncharacterized cellular response of Streptomyces to hyperosmotic stress, which involved complete reprogramming of cell polarity and redistribution of growth sites from hyphal tips to lateral walls, resulting in extensive de novo branch formation. Besides re-arrangement of coiled coil cytoskeletons DivIVA and FilP this response also involved hyper-condensation of nucleoids, slow recovery of turgor, and a change in the fluidity of the cytoplasm. In summary, my study has revealed several new aspects concerning the working principles of the IF-like FilP cytoskeleton, and might even offer insights into the so far poorly understood role of the IF cytoskeleton in metazoan cells. Our cell-biological study of osmotic stress response has shed new light on bacterial stress responses, which have seldom been studied on single-cell level.