Zöllner, Robert (2019). Linking intercellular forces to colony dynamics and fitness in bacterial populations. PhD thesis, Universität zu Köln.

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Abstract

Biofilm formation is a collective phenomenon of prokaryotic systems. Within biofilms cells show enhanced resistance against environmental stresses. Bacteria can use type 4 pili (T4P) for active force generation, adhesion and formation of biofilms. The first part of this work focusses on T4P-mediated single cell interactions. These interactions are quantified by means of laser tweezers and connected to dynamics of early biofilms. The second part of this work focusses on the interplay of T4P-mediated single cell interactions, evolution and structure of macroscopic biofilms. Genome sequencing in combination with phenotypic analysis is used to connect mutations to interbacterial interaction and collective phenomena. Using a dual laser trap, we showed that the waiting time distribution of cell-cell interaction events is comparable to the waiting time distribution of cell-bead interaction events. Monomers of T4P are post-translationally modified. We observe that cell-cell rupture forces and binding kinetics are fine-tuned by pilin glycosylation and phosphoform modification pathways. In addition, intercellular binding kinetics change with varying T4P-motor activity. These microscopic findings are correlated with results from mesoscopic multicellular systems. Our data indicate that diversification of rupture forces and binding probabilities induces cell sorting. Fusion dynamics of multicellular aggregates shows liquid-like behavior. T4P-mediated interaction kinetics regulate these dynamics and strongly affect the material properties surface tension and viscosity. For a long time T4P mediated force generation was thought to be coupled to the retraction ATPase PilT, which is essential for twitching motility and multicellular dynamics. However, we observed that force generation by T4P retraction occurs without retraction ATPase PilT. We demonstrate that retraction in the absence of PilT is independent of proton motive force and of the pilT-paralogues pilU and pilT2. Retraction is two orders of magnitude slower than pilT-driven T4P retraction and stalls frequently at opposing forces smaller than 10 pN. Furthermore, the velocity of pilT-independent T4P retraction depends on the opposing force and the periplasmatic PilE-concentration. Previous studies show that positioning of individual cells within biofilms governs dispersal, growth, nutrient consumption and collective protection against environmental stresses. We tested whether tuning of T4P-mediated interaction forces governs positioning and affects population dynamics of expanding biofilms. Our data shows that weakly interacting cells without T4P segregate to the periphery of growing populations and show a strong selective advantage. Spatially resolved sequencing demonstrates that pilin antigenic- and phase-variation are natural mechanisms that generate a standing diversity of pilin sequences within the populations and cause segregation of subpopulations. Populations evolving in liquid medium favor dispersive arrangements over dense aggregation while maintaining their ability to use T4P for active force-generation. Whole genome sequencing data suggests, that mutations in pilC and pilE are likely to be involved in changing intercellular T4P-mediated interactions as to enable disperse arrangements. In conclusion, different pilin post-translational modifications and variations in motor activity fine-tune attractive T4P-mediated interactions. Altered single-cell interactions affect cell communities and have drastic effects on material properties of multicellular systems. Investigation of T4P retraction in the absence of pilT indicates that PilT-independent T4P retraction is a universal T4P phenomenon. Our results suggest that T4P-mediated interactions are subject to rapid evolution, which enables cells to change and adjust aggregation, and react to environmental conditions.

Item Type: Thesis (PhD thesis)
Creators:
CreatorsEmailORCID
Zöllner, Robertrzoellne@uni-koeln.deUNSPECIFIED
URN: urn:nbn:de:hbz:38-94479
Subjects: Natural sciences and mathematics
Physics
Life sciences
Uncontrolled Keywords:
KeywordsLanguage
Biophysics;Laser tweezers;Biofilms;Intercellular interaction;Molecular motors;Neisseria gonorrhoeae;English
Faculty: Faculty of Mathematics and Natural Sciences
Divisions: Faculty of Mathematics and Natural Sciences
Language: English
Date: 2019
Date of oral exam: 21 February 2019
Referee:
NameAcademic Title
Maier, BerenikeProf. Dr.
Bollenbach, TobiasProf. Dr.
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/9447

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