Advisor(s)

Kim Lewis

Contributor(s)

Marin Vulić, Veronica S. Godoy-Carter, Eric J. Stewart, Thomas Bernhardt

Date of Award

2010

Date Accepted

9-2010

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Arts and Sciences. Department of Biology.

Keywords

escherichia coli, genes, antibiotic tolerance

Subject Categories

Genetic engineering - Experiments, Escherichia coli - Genetics - Experiments

Disciplines

Biology, general | Genetics

Abstract

Bacterial populations stochastically produce a small number of non-growing or dormant persister cells that are tolerant to antibiotics. Persisters are phenotypic variants that are genetically identical to the susceptible cells within a clonal population. Persisters contribute to the antibiotic recalcitrance of biofilm infections. A number of recent studies point to the involvement of toxin/antitoxin (TA) systems in persister formation. A strain with two point mutations in hipA, the toxin of the hipBA TA system, produced 10,000 times more persisters than the wild type. Similarly, overexpression of HipA caused a sharp increase in the persister fraction. However, deletion of hipA did not produce a phenotype suggesting that multiple genes/pathways cause persister formation. The antitoxin HipB represses the hipBA operon by cooperative binding to four operator sites and inactivates the toxin HipA. The crystal structure of the HipA-HipB-DNA complex revealed that one HipB dimer is sandwiched by one molecule of HipA on each side and that HipB induces a 70 ° bend in the operator DNA. HipA is a kinase with eukaryotic serine/threonine fold. EF-Tu was identified as a target of HipA suggesting that HipA causes protein synthesis inhibition and dormancy upon phosphorylation of EF-Tu. For HipA to be active, HipB must be removed or degraded. Antitoxins are typically degraded by one of the ATP-dependent proteases. HipB was stabilized in lon- background and also degraded by Lon in vitro demonstrating that Lon is the main protease responsible for HipB proteolysis. The unstructured C-terminus of HipB is critical for rapid proteolysis as a truncated HipB appears to be stabilized. To identify additional genes responsible for persister formation, we performed a screen for mutants with altered antibiotic tolerance using an ordered library of 3,985 Escherichia coli knockout strains. We exposed stationary-state cultures in 96-well plates to ofloxacin at a concentration well above MIC and determined the persister cell level of each culture. 10 mutants with decreased persistence but no difference in growth rate or ofloxacin MIC compared to wild type were identified. Two putative persister genes, yigB and ygfA, encoding a FMN phosphatase and a 5-formyl tetrahydrofolate (THF) cyclo-ligase, respectively, were further validated.

Document Type

Dissertation

Rights Information

Copyright 2010

Rights Holder

Sonja Hansen


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