An Experimental Framework to Examine the Influence of Promoter Architecture and Genomic Context on Gene Expression

Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol. In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work

The role of replication-induced chromosomal copy numbers in spatio-temporal gene regulation and evolutionary chromosome plasticity

For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers

Pristionchus.org: a genome-centric database of the nematode satellite species Pristionchus pacificus

Comparative studies have been of invaluable importance to the understanding of evolutionary biology. The evolution of developmental programs can be studied in nematodes at a single cell resolution given their fixed cell lineage. We have established Pristionchus pacificus as a major satellite organism for evolutionary developmental biology relative to Caenorhabditis elegans, the model nematode. Online genomic information to support studies in this satellite system can be accessed at . Our web resource offers diverse content covering genome browsing, genetic and physical maps, similarity searches, a community platform and assembly details. Content will be continuously improved as we annotate the P.pacificus genome, and will be an indispensable resource for P.pacificus genomics

Supercoiling of an excised genomic island represses effector gene expression to prevent activation of host resistance

The plant pathogen Pseudomonas syringae pv. phaseolicola, which causes halo blight disease of beans, contains a 106kb genomic island PPHGI-1. PPHGI-1 carries a gene, avrPphB, which encodes an effector protein that triggers a resistance response in certain bean cultivars. Previous studies have shown that when PPHGI-1 is excised from the bacterial chromosome avrPphB is down regulated and therefore the pathogen avoids triggering the host’s defence mechanism. Here we investigate whether the down regulation of avrPphB is caused by supercoiling of PPHGI-1. We also investigate the effect of a PPHGI-1 encoded type 1A topoisomerase, TopB3, on island stability and the bacterial pathogenicity in the plant. Supercoiling inhibitors significantly increased the expression of avrPphB but did not affect the excision of PPHGI-1. An insertional mutant of topB3 displayed an increase in avrPphB expression and an increase in PPHGI-1 excision as well as reduced population growth in resistant and susceptible cultivars of bean. These results suggest an important role for topoisomerases in the maintenance and stability of a bacterial encoded genomic island and demonstrate that supercoiling is involved in the down regulation of an effector gene once the island has been excised, allowing the pathogen to prevent further activation of the host defence response

Interference between Triplex and Protein Binding to Distal Sites on Supercoiled DNA

We have explored the interdependence of the binding of a DNA triplex and a repressor protein to distal recognition sites on supercoiled DNA minicircles using MD simulations. We observe that the interaction between the two ligands through their influence on their DNA template is determined by a subtle interplay of DNA mechanics and electrostatics, that the changes in flexibility induced by ligand binding play an important role and that supercoiling can instigate additional ligand-DNA contacts that would not be possible in simple linear DNA sequences

DNA Supercoiling: an Ancestral Regulator of Gene Expression in Pathogenic Bacteria?

International audienceDNA supercoiling acts as a global and ancestral regulator of bacterial gene expression. In this review, we advocate that it plays a pivotal role in host-pathogen interactions by transducing environmental signals to the bacterial chromosome and coordinating its transcriptional response. We present available evidence that DNA supercoiling is modulated by environmental stress conditions relevant to the infection process according to ancestral mechanisms , in zoopathogens as well as phytopathogens. We review the results of transcriptomics studies obtained in widely distant bacterial species, showing that such structural transitions of the chromosome are associated to a complex transcriptional response affecting a large fraction of the genome. Mechanisms and computational models of the transcriptional regulation by DNA supercoiling are then discussed, involving both basal interactions of RNA Polymerase with promoter DNA, and more specific interactions with regulatory proteins. A final part is specifically focused on the regulation of virulence genes within pathogenicity islands of several pathogenic bacterial species

Protein/DNA interactions in complex DNA topologies: expect the unexpected

DNA supercoiling results in compacted DNA structures that can bring distal sites into close proximity. It also changes the local structure of the DNA, which can in turn influence the way it is recognised by drugs, other nucleic acids and proteins. Here, we discuss how DNA supercoiling and the formation of complex DNA topologies can affect the thermodynamics of DNA recognition. We then speculate on the implications for transcriptional control and the three-dimensional organisation of the genetic material, using examples from our own simulations and from the literature. We introduce and discuss the concept of coupling between the multiple length-scales associated with hierarchical nuclear structural organisation through DNA supercoiling and topology

Chromosomal replication dynamics and interaction with the β sliding clamp determine orientation of bacterial transposable elements

Insertion sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here, we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to nine different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. Although we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, because in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phylogenetically distant species