The Impact of Genome Organization on the Regulation of Gene Expression in Escherichia coli

Bacteria shape the world around us. They are an integral part of various ecosystems, live in animals’ microbiomes for mutual benefit, they can be pathogens, and they are production workhorses in our industries. Bacterial genomes are a subject of study to increase our understanding of how biological information is stored and processed. In this thesis, we investigated the interplay of genome structure and gene regulation from two different angles: We explored the relative spatial structuring of transcription factors and their targets as well as the role of DNA supercoiling in the regulation of carbon metabolism and the response to growth arrest in the stationary phase.

The majority of all transcriptional networks in Escherichia coli are organized in clusters of operons and are distributed around the genome. We rearranged the spatial architecture of the MalT regulon by relocating malT to different locations around the genome. The MalT regulon consist of the set of genes whose expression is activated by the transcriptional regulator MalT and is responsible for maltose and maltodextrin degradation. Upon malT relocation we observed different growth phenotypes. However, a strong fitness disadvantage was common to all of the engineered strains in conditions where switching between different carbon sources was required. Our results highlight the relevance of the spatial organization of genetic designs for efficient cellular information processing.

DNA supercoiling is involved in shaping DNA topology both as a means for effective genome condensation but also in connection with transcriptional regulation. In order to better understand the regulatory effect that DNA supercoiling has on specific genes in their native context, we need to be able to determine local differences in DNA supercoiling across the bacterial genome. Here, we propose a new approach based on two sensor promoters, which generate time-resolved, localized data of DNA supercoiling in different positions in the E. coli genome. We used this system as well as classical plasmidbased detection of DNA supercoiling to determine changes in DNA supercoiling during the consumption of different carbon sources and in the stress response connected to the stationary phase. The temporal resolution of DNA supercoiling dynamics adds a new dimension to our understanding of chromosome structure over the entire growth cycle of E. coli.

The results presented in this thesis contribute to our understanding of the spatial structure of bacterial genomes. These results are not only relevant in a general microbiology context, but can also be used to drive advanced synthetic biology applications forward. Both project lines were supported by the development of a standardized genome engineering platform (SEGA), enabling facilitated construction and
integration of genome-based expression cassettes in E. coli.