Universität zu Köln

Müller, Janina ORCID: 0009-0007-0828-334X (2025). Quantification of microbial fitness: costs of protein overexpression and phage infection. PhD thesis, Universität zu Köln.

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Abstract

Fitness is the most fundamental variable in quantitative biology. It is used to understand and predict evolutionary dynamics, analyze the effects of gene expression, and evaluate how organisms interact with each other and their environment. Scalable, quantitative fitness measurements are therefore essential tools for microbial systems biology. The absence of practical, standardized methods for such measurements hampers progress and results in less comparable data across studies. In this work, we developed and applied two high-throughput techniques to address these challenges—one for bacterial fitness and the other for phage fitness. These methods significantly improve accessibility to fitness measurements and enabled us to tackle questions that were previously beyond scope due to infeasible experimental demands. Protein overexpression is linked to many diseases and plays a central role in antibiotic resistance, particularly through drug targets or resistance genes like membrane-localized efflux pumps. Using a high-throughput colony-imaging technique, we performed a genome-wide analysis of overexpression fitness costs in Escherichia coli at finely resolved expression levels. Our analysis revealed that most membrane proteins impose steep fitness costs, with bacterial growth collapsing abruptly once a critical expression threshold is exceeded. The prevailing hypothesis for the high fitness costs of membrane proteins is the supposed saturation of the Sec translocon, the cornerstone of the primary membrane translocation pathway. Through the use of synthetic membrane proteins targeting different translocation pathways, we excluded Sec translocon saturation as the origin of the fitness costs. We used single-cell time-lapse imaging with fluorescently tagged membrane proteins to observe competition between membrane proteins during overexpression. These experiments showed that the overexpression costs stem from the displacement of endogenous membrane proteins. This displacement of the endogenous membrane proteome can abruptly diminish growth during membrane protein overexpression. Displacing 10% of the endogenous membrane proteins traps bacteria in a non-functional membrane proteome state. Compared to bacterial fitness measurements, techniques for quantifying phage fitness remain significantly underdeveloped and often rely on century-old methods like the plaque assay. This severely limits throughput in phage fitness measurements and therefore systematic comparisons of phage phenotypes, such as their amplification rates in bacterial populations and their bactericidal effects under varying environmental conditions, are rare. To address this gap, we developed a novel high-throughput approach termed PHORCE (Phage-Host Observation for Rate Estimation from Collapse Events). PHORCE uses a minimal mathematical model to analyze bacterial population growth and collapse dynamics under phage predation, enabling accurate quantification of lytic phage amplification rates. Our findings demonstrate that the amplification rate quantified through PHORCE reliably captures the bactericidal effect of phages, independent of the initial bacterial and phage population sizes and for different growth conditions. Using this approach, we observed amplification rate differences of more than three orders of magnitude across E. coli phages. Moreover, PHORCE revealed that phage-antibiotic interactions are primarily influenced by the antibiotic rather than the phage. For instance, the ribosome-inhibiting antibiotic doxycycline exhibited antagonistic interactions with phage amplification, whereas the DNA-damaging antibiotic nitrofurantoin showed synergistic effects. By enabling quantitative, high-throughput characterization of phage phenotypes, PHORCE provides a robust framework for systematic phage studies and facilitates screens to identify phage candidates for antibacterial therapeutics. The results of this thesis highlight the importance of developing high-throughput methods and show that their application leads to a comprehensive knowledge gain and can have an impact on various areas of microbiology, biomedicine and biotechnology.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Müller, Janina janina_mueller@yahoo.de orcid.org/0009-0007-0828-334X UNSPECIFIED
URN:	urn:nbn:de:hbz:38-755011
Date:	17 March 2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Ehemalige Fakultäten, Institute, Seminare > Faculty of Mathematics and Natural Sciences
Subjects:	Physics Life sciences
Uncontrolled Keywords:	Keywords Language protein overexpression English membrane proteins English high-throughput screening English colony imaging English Sec pathway English bacteriophage English phage infection English phage-antibiotic interactions English mathematical modeling English
Date of oral exam:	14 March 2025
Referee:	Name Academic Title Bollenbach, Tobias Prof. Maier, Berenike Prof.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/75501