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Title: Dynamics of E. coli genome and cytosol under antibiotics
Author: Wlodarski, Michal
ISNI:       0000 0004 7228 7506
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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In light of an urgent need for improved antimicrobial diagnostics and therapeutics, understanding bacterial behaviour, and bacterial responses to treatments in particular, is one of the key objectives of modern medical research. While the molecular mode of action of antibiotics is usually well known, their effect on the cell at a "systems" level (on the regulatory networks, metabolism, etc.) is only beginning to be quantitatively understood. We address some of these response phenotypes in Escherichia coli testing different antibiotic classes and growth conditions. We study the short (< 15 s) time-scale fluctuation dynamics of fluorescently-tagged chromosomal loci and cytosolic aggregates, which report for the state of locus ”compaction” and the levels of macromolecular crowding of the cytosol, respectively. We improve the precision of those measurements developing a novel data treatment procedure and discover that sub-lethal doses of ciprofloxacin, rifampicin, and vancomycin as well as hyperosmotic shock conditions cause small but consistent changes (unique to each treatment agent) to the physical organisation of chromosomal Ori2 and Ter3 loci and the cytosol. We reveal, among other findings, strong correlations between the effects in different parts of the chromosome and between the chromosome and cytosol. In addition, we complement the marker dynamics work with single-cell level gene expression measurements during sub-lethal translation inhibition. Specifically, we compare responses to tetracycline and chloramphenicol from constitutive and ribosomal promoters in Ori3 and Ter3 chromosomal positions over long (7 h) treatment times in exponentially growing bacteria. We reveal, for the first time, the kinetics of cellular resource allocation and provide novel insights on globally regulated transcription, relevant to the three-component proteome partitioning model, gene-length dependent effects of the processivity of translation, and ”reversibility” of ribosome-binding antibiotics. In addition, we discover a strong correlation between the timing of responses from promoters in the Ori3 and Ter3 positions, and a small but consistent difference in the response magnitude between the two positions.
Supervisor: Cicuta, Pietro Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: chromosomal dynamics ; cytosol dynamics ; antibiotics ; E.coli