Chromosomal Architecture and its Influence on Gene Expression in Native and Engineered Bacteria
Research in recent years has yielded new insights into the influence of chromosomal architecture at different levels on bacterial gene regulation and expression. On the topological level, chromosome compaction can bring distant genes or regions in spatially proximity, suggesting a regulatory concept...
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|Research in recent years has yielded new insights into the influence of chromosomal architecture at different levels on bacterial gene regulation and expression. On the topological level, chromosome compaction can bring distant genes or regions in spatially proximity, suggesting a regulatory concept of co-expression of distant genes. DNA supercoiling, which is highly dynamic and one of the major factors in nucleoid formation, can have a significant influence on gene expression by modulatory effects on transcription. Furthermore, the replication-induced copy number effect increases the expression of genes by transiently increasing the number of gene copies during replication. However, how this impacts the organism on a systemic level (global gene expression) has not been shown yet. In this work, the influence of the replication-induced copy number effect on gene expression in Escherichia coli has been investigated. It was previously shown, that genes closer to the replication origin (oriC) are higher expressed during the exponential phase compared to the stationary phase. This effect decreases with increasing distance to the oriC. In the course of this work, it was demonstrated that this expression pattern is due to the copy number effect instead of the strategic positioning of genes regulated by global transcription factors. Furthermore, the regulatory impact of the replication-induced copy number effect was determined for individual genes. It could be shown that around 40% of the genes are predominantly copy number regulated, suggesting an important role of the copy number effect for gene regulation and expression in E. coli. In addition, the influence of the copy number effect on the chromosome organization was investigated. The conservation of the position of genes relative to oriC indicates a strong influence of the copy number effect on bacterial chromosome evolution, especially in fast-growing bacteria. Moreover, a genome editing tool based on CRISPR/Cas9 (CRISPR SWAPnDROP) was established for the chromosomal modifications required for these investigations. Beyond its initial purpose, this tool was designed to facilitate large chromosomal rearrangements and the transfer of chromosomal regions between bacterial species. As a proof of principle, a 151kb chromosomal region was transferred from one E. coli strain to another as well as to the biotechnology relevant Vibrio natriegens. In addition, the RP4 conjugation system of E. coli was transferred to both V. natriegens and the plant pathogen Dickeya dadantii and its functionality was demonstrated in these organisms. Furthermore, the transfer of the E. coli lac operon to V. natriegens and the transfer of the GanB ORF from D. dadantii to E. coli demonstrate successful gain of function genome edits using CRISPR SWAPnDROP.