Impact of global gene regulators in E. coli on cellular proteome and metabolic function

  • Almost sixty yearsafter the determination of the DNA helix structure and ten years after the human genome project gene regulation remains still obscure in many aspects. Although many regulatory interactions of regulatory proteins are known as well as their metabolic impact (e.g. lac operon), holistic concepts of regulation are still mainly uncovered or attributed to single effectors. Nevertheless, certain concepts were already revealed showing for example boolean like logics in the organization of the Escherichia coli regulatory network also found in neuronal networks and computer chips supporting the view of an information processing system. According to the last example transcription is mostly regarded as the complex interaction of regulators and targets integrated in the transcriptional regulatory network (TRN). Each regulator to target connection was inferred by deletion of the regulator, complementation in trans e.g. on a plasmid and confirmed by in vitro studies in a minimal factor context. The studies were performed under certain environmental conditions and are therefore in principle only valid under these conditions. The TRN can therefore be considered as the sum of possible regulatory interactions known down to the present day. The architecture of the network is mainly considered as hierarchical. Within the hierarchy, comprehensive regulatory overlaps were found called DORs. These overlaps exhibit more or less specific functions. We investigated the regulatory overlaps of major regulators (σ-factors and NAPs) regulons creating so called couplons. Gene ontology analysis showed an enrichment of specific metabolic function compared to random subsets taken from individual regulons. Moreover, the presence of highly overlapping regulons implies the reciprocal regulation of the involved regulators. These regulators are in turn organized in a heterarchically organized network of major regulators, constructing a decision making organization coordinating downstream regulation. This is supported by noise damping feedforward connections to couplons on the next hierarchy level most permeable for consistent signal sent by the upstream heterarchical network. However the majority of genes is not yet included in the TRN but also show complex transcription patterns. We show indications, that these genes may not be integrated into the TRN in a digital way. Here digital refers to a on-off target behavior dependent on a one to one relation of regulator and target. It has been shown, that supercoilii ing of the DNA is besides the TRN a global regulator of transcription driving growth related metabolic pathways and controlling anabolism/catabolism ratios. However the analysis was based on pure microarray data lacking relevance for the proteome composition. Therefore we performed a proteome analysis based on the experimental design of previous microarray experiments and find a strong correlation of transcriptome and proteome with a temporal delay of 20 minutes. Moreover, we find the same metabolic pathways activated on the proteomics level, proving the functional relevance of supercoiling changes. Analysing different gene sets under altered supercoiling conditions we found a diametrally opposed transcription pattern of TRN regulated genes and the complementary set of isolated genes during altered supercoiling supporting previous observations of two distinct types of information (TRN and gene neighborhood) executing regulatory control. However, we find regulators reacting on supercoiling similarly to isolated genes connecting supercoiling sensitive isolated genes with TRN regulators and in fact the TRN. Moreover, major TRN regulators also constituting major structural proteins like HU, FIS or H-NS show non-random distribution of binding sites. These proteins are also involved in buffering supercoiling dynamics and therefore coordinate gene regulation and chromosome topology. Although protein binding sites may play a structural role, like matS sites for the terminus macrodomain formation, or HU for transcription foci formation, the chromosome compaction itself has major effects on regulatory control. HU mutants have shown to create the chromosomal replication origin/terminus dependent transcription differences. The genes in the origin region were more active than in the wild type and the opposite was found for the terminus. In accordance with the HU transcription pattern also the binding sites of σ-factors show clear spatial gradients. Especially the antagonists σ70 and σ38 show a diametral gradient from origin to terminus indicating a spatial coding of temporal gene expression patterns. In this study we describe a spatio-temporal transcription program based on gradients of different DNA features monitored throughout the growth cycle of Escherichia coli. We show that gene order is conserved in the γ-Proteobacteria implementing a temporal program in space starting from origin of replication in exponential phase to the terminus in stationary phase. We finally propose that supercoiling, GC-content, gene orientation and binding site distributions coordinate of the spatio-temporal gene expression program.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Patrick Sobetzko
Referee:Georgi Muskhelishvili, Georgi Muskhelishvili, Marcelo Fernandez Lahore, Andrew Travers
Advisor:Georgi Muskhelishvili
Persistent Identifier (URN):urn:nbn:de:101:1-201305294899
Document Type:PhD Thesis
Date of Successful Oral Defense:2012/10/05
Date of First Publication:2012/10/29
PhD Degree:Bioinformatics
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QH Natural history - Biology / QH301-705.5 Biology (General) / QH324 Methods of research. Technique. Experimental biology / QH324.2 Data processing. Bioinformatics
Call No:Thesis 2012/33

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