Date of Award

11-16-2006

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Thomas M. Hill

Abstract

Persistence is the ability of a small fraction of bacteria (one-in-a-million) to survive antibiotic exposure, yet the survivors are as susceptible to antibiotic as the parental cells. "High persistence" in Escherichia coli is linked to mutations in hipA, the toxin entity of the toxin-antitoxin module hipBA. The wild type hipA gene encodes a "toxin" while hipB encodes a DNA-binding protein that autoregulates hip operon expression and binds to HipA to nullify its effects. The hipA7 allele confers persistence to 1:100 cells and serves as a model system to study persistence. Wild-type HipA, when in excess of HipB, causes culture growth to cease and colony forming units (CFU) to decrease sharply, coincident with translation inhibition and development of persistence in up to 20% of cells. In contrast, expression of hipA7 in excess of hipB did not inhibit growth, CFU ability, or protein synthesis, yet conferred persistence to 10% of cells. The bacteriostatic condition conferred by HipA is reversible upon expression of hipB, although the majority of cells remain in a viable but non-culturable condition. The hipA7 allele contains two mutations (G22S and D291A), which are required for the full range of phenotypes associated with hip mutants. Expression of hipA7 in the absence of hipB is sufficient to confer high persistence, indicating that hipB is not required. However, genetic evidence suggests that HipB degradation by the lon protease is required for high persistence. The stringent response, characterized by the production of (p)ppGpp by relA and spoT, plays a key role in persister cell development. hipA7 relA mutants exhibit intermediate persistence frequencies whereas hipA7 relA spoT mutants are altogether incapable of generating persisters. In agreement, all persisters arising in (p)ppGpp0 strains following hipA over-expression were (p)ppGpp revertents, verifying an essential function of (p)ppGpp in persistence. Potentially, persister cells arise stochastically due to fluctuations in gene expression and protein degradation, resulting in the release of bacteriostatic-conferring proteins, shutting down the cell for a period of time sufficient to outlast lethal challenge. Resuscitation of dormant cells is achieved through anti-bacteriostatic protein production, which binds to and neutralizes bacteriostatic proteins, initiating growth and reestablishing the bacterial population.

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