Title: Single-cell expression and structure-function analysis of a conserved bacterial GTPase in protection against lethal stress
Other Titles: Single-cell expressie en structuur-functie analyse van een geconserveerd bacterieel GTPase in bescherming tegen lethale stress
Authors: Kint, Cyrielle
Issue Date: 7-Jan-2013
Abstract: Persistence is the phenomenon whereby a small fraction of cells within a bacterial population displays tolerance to treatment with high doses of bactericidal antibiotics. In contrast to antibiotic resistant cells, persisters are genetically identical to susceptible bacteria. Although the mechanistic basis for persister formation is currently poorly understood, variation in gene expression between single cells is likely to play an important role. A general hypothesis on persistence states that cells in which the expression level of a key persister protein exceeds a certain threshold switch to the persistent state. Previous research in our group led to the identification of a novel persistence regulator in E. coli. This PhD project further explored the function of this protein, named PerA, in E. coli persistence. By using single-cell techniques, we showed that natural variation in PerA expression between single cells, greatly contributes to the formation of persister cells in E. coli populations. In addition, an artificial system was developed to experimentally control gene expression noise. This system will allow to determine the threshold level of perA expression required for transition to the persistent state. A second approach to examine the role of PerA in E. coli persistence consisted of a detailed structure-function analysis. We showed that the persistence function of PerA is dependent on its GTP-binding domain. Furthermore, five amino acid substitutions within this domain were identified that specifically abolish the PerA persistence function. Biochemical analysis of these mutant proteins suggests that the GTP-bound form of the wild-type protein mediates persistence. The residues identified in this work will facilitate elucidation of the mechanism of PerA-mediated persistence, which could in turn lead to the development of a PerA-targeted anti-persister therapy.In addition to the function of PerA in persistence, we investigated its role in the cellular response to replication fork stress following treatment with a replication inhibitor. Using site-directed as well as random mutagenesis combined with mutant background screening, we were able to provide mechanistic insights into the PerA-mediated effects under these conditions. More specifically, our results suggest that PerA protects the cell from replication fork stress by preventing the formation of toxic hydroxyl radicals. The identification of PerA variants mutated in amino acid residues crucial for resistance to DNA replication stress will facilitate the identification of direct and relevant PerA interaction partners in future work.Finally, we describe the cellular effects of expression of a mutant PerA protein carrying a single amino acid substitution within its GTP-binding domain. This mutant protein displays strong bactericidal activity against certain E. coli strains, seemingly as a consequence of induction of severe DNA damage. Although the mechanism of action of this mutant protein remains to be elucidated, the severe effects on cellviability add to the relevance of PerA as a potential therapeutic target for the development of alternative antibacterial agents.In summary, this PhD research contributes to the understanding of the complex role of the GTPase PerA in stress responses, particularly in tolerance towards bactericidal antibiotics, in the response to replication fork stress and in cell viability in E. coli. The mutants generated in this work provide excellent starting points for the elucidation of the molecular mechanisms of these PerA-mediated processes. Eventually, the knowledge on PerA function gained in this thesis could be applied in the development of PerA-based anti-persister compounds or of novel antibacterial agents.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics
Molecular Imaging and Photonics

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