Molecular scar tissue as potential incentive for epigenetic differentiation and memory in clonal bacterial populations

Bacteria typically proliferate by binary fission to form genetically identical and morphologically indistinguishable daughter cells. However, it is becoming clear that these isogenic siblings can have phenotypically distinct behavior irrespective of environmental conditions, which contributes to the development of heterogeneous populations with intricate social behavior. While this phenotypic heterogeneity has typically been attributed to stochastic cellular processes and/or dedicated genetic circuitry, more deterministic drivers of heterogeneity have been found as well. Indeed, recent observations from our group have shown that the emergence of protein aggregates (PAs) after exposure to proteotoxic stresses can serve as an epigenetic incentive for cellular individualization and population heterogeneity. Specifically, it was found that PAs, through their nucleoid-enforced asymmetric segregation during cell division, inherently create subpopulations of PA-bearing and PA-free cells that are phenotypically distinct regarding their survival towards subsequent proteotoxic stresses. As such, this dissertation aims to further elaborate upon these previous observations and explore how molecular marks left after influential previous experiences (i.e. "molecular scar tissue") can shape population heterogeneity and serve as epigenetic memory elements in clonal bacterial populations. In a first part, we elaborated a high-throughput single-cell screening methodology that allows the discovery of intricate promoter expression patterns in the spore forming Gram-positive model organism Bacillus subtilis. As such, this method revealed several novel heterogeneously expressed genes and furthers our understanding of the usage of phenotypic heterogeneity strategies in this organism. Next, we constructed a synthetic PA model system for B. subtilis, in which a fluorescently trackable inert aggregation-prone protein is expressed from an IPTG-inducible promoter. This allowed an in-depth characterization of PA dynamics, specifically in the context of sporulation and germination, which revealed a remarkable robustness of these processes against perturbation by misfolded protein and PAs. Indeed, we could find no detrimental effects of PAs on sporulation, germination or even spore survival against heat or UV stress. Intriguingly, we even noticed protein PAs can be readily incorporated in the endospore and persist throughout the entire sporulation process. In a subsequent part, we reveal how PAs in Escherichia coli confer increased cellular robustness against proteotoxic stresses, such as exposure to heat, peroxide, and the antibiotic streptomycin, both in terms of increased survival frequencies and decreased resuscitation times, but not to a DNA damaging stress such as UV exposure. Our results indicate this may be attributed to the co-inheritance of protein quality control components with asymmetrically dividing PAs, leading to their specific enrichment in PA-bearing cells. Additionally, we explored how PA segregation and disaggregation dynamics can enable lineage-dependent proteomic differences in the PA-free subpopulation. These results thus further highlight that PAs can serve as long-term epigenetic memory elements that also drive phenotypic heterogeneity in stressed clonal populations. Lastly, directed evolution efforts in the E. coli model system allowed us to discover a novel evolutionary route by which bacteria can adaptively tune their heat shock protein levels and corresponding proteostatic robustness. In fact, we found that seemingly random single mutations in the E. coli tnaA gene can confer significant heat resistance through the creation of folding-deficient and aggregation-prone TnaA variants that in turn can endogenously and preemptively trigger HSP expression. More generally, these findings importantly imply that even mutations with disruptive effects on protein structure and functionality can still yield true gain-of-function alleles with a selective advantage in adaptive evolution. In conclusion, this dissertation provides novel insights into how ancestral molecular scar tissue such as PAs can shape population heterogeneity on different levels. Furthermore, modulation of intracellular PA dynamics and management seems to provide the cell with the adaptive potential to fine-tune its cellular robustness such as its heat shock resistance.status: publishe