Universität zu Köln

Welker, Anton (2021). Spatio-temporal analysis of architecture and growth in bacterial colonies. PhD thesis, Universität zu Köln.

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Abstract

Most microorganisms prefer to live in surface associated communities called biofilms, where their lifestyle differs considerably compared to their planktonic counterpart. The cell shape, as well as physical interactions determine the structure of bacterial biofilms. Due to cell growth, the size of the biofilm increases with time and the structure changes during biofilm maturation. This work aims at characterizing the structure and growth dynamics of dense colonies formed by gonococci. The first part of this thesis focuses on the characterization of the spatial structure of gonococcal colonies. Image analysis tools were developed that allowed determin- ing the positions of single cells within the spherical colonies. Using the position data, the radial distribution function (RDF) was calculated. The RDF showed short-ranged order but not long range order, reminiscent of liquids. Neisseria gonorrhoeae interact via their type 4 pilus (T4P) with surfaces and cells. T4P- T4P binding between adjacent cells generates attractive force that controls colony formation. We investigated the effect of T4P retraction of the local and meso- scopic structure of gonococcal colonies using strains with varying T4P retraction phenotypes. Reducing speed and frequency of T4P retraction reduced cell density and increased order in 6 h old colonies. Deleting T4P retraction results in loss of local order. After 24 h, density and local order increase for all strains, and larger holes inside the structures of gonococcal colonies emerged, which were independent of T4P motor activity. In conclusion, we show that gonococcal T4P active force generation is not necessary for development of local order, but it accelerates the process of achieving higher densities and local ordering inside spherical colonies. 1 1 Abstract In the second part of this thesis, we developed methods for measuring growth rates of colony-bound bacteria with spatial and temporal resolution. Growing gonococci generated a radial velocity field inside colonies that pointed from the centre of mass (COM) of colonies to its periphery. Close to the colony centres, velocities were minimal and increased towards the periphery of colonies. We showed that by characterizing the velocity field within the colony, the local growth rates can be measured. Independently, growth rates were determined by counting the offspring of single fluorescent cells that were distributed homogeneously inside the colonies. Both methods complement each other, because they have different advantages and disadvantages. Unexpectedly, heterogeneous growth profiles inside small gonococcal colonies emerged after 2 h of growth. To assess the hypothesis that nutrient limitation causes growth heterogeneity, we optimized the nutrient supply with a higher flow rate. Even though gonococcal growth improved slightly, growth profiles were still heterogeneous, indicating different limitations like mechanical constraints. Surprisingly, colonies that could not activate the stringent response developed heterogeneity in spatial and temporal growth even earlier. We suggest that stringent response is important for gonococcal biofilm maturation. Finally, the effect of azithromycin treatment on colony growth dynamics was investigated. We observed that after two generations times, growth rates dropped to low values throughout the colony indicating that azithromycin diffuses quickly through the whole colony and effects the majority of cells. In summary, we established tools for characterizing growth and death within dense spherical colonies at spatial and temporal resolution. This method will be useful to study the mechanisms of development of heterogeneity inside gonococcal colonies and their response to environmental changes like antimicrobial treatment.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Welker, Anton antonwelker1@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-537593
DOI:	10.1006/jmbi.1998.2354
Journal or Publication Title:	Journal of Molecular Biology
Volume:	285
Number:	1
Page Range:	pp. 1-32
Date:	7 August 2021
ISSN:	00222836
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Physics > Institut für Biologische Physik
Subjects:	Data processing Computer science Physics
Uncontrolled Keywords:	Keywords Language Bacterial biofilm English Growth Dynamic English
Date of oral exam:	31 August 2021
Referee:	Name Academic Title Maier, Berenike Prof Bollenbach, Tobias Prof Neundorf, Ines Prof Cronenberg, Tom Dr
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/53759