Advisor(s)

Slava S. Epstein

Contributor(s)

A. L. (Arnold L.) Demain (1927-), Veronica S. Godoy-Carter, Edward L. Jarroll, Eric J. Stewart

Date of Award

2010

Date Accepted

11-2010

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

microbiology, bacterial survival strategies, dormancy, latent infections

Subject Categories

Microbial ecology, Microorganisms - Dormancy

Disciplines

Microbiology

Abstract

It has been frequently observed that microorganisms from different sources exist in a non-growing state. We suggest that this state represents a population level strategy utilized by non-sporeforming bacteria to maximize survival under unfavorable conditions. Individual members of the population enter a state of reversible dormancy from which they exit in a seemingly random fashion due to noise in gene networks. We term these stochastically awakened cells scouts, and further hypothesize that scouts may produce signaling compounds that cue the remaining dormant population to initiate growth (Epstein 2009a). To test this model, we employed single cell incubations from a mixed marine sediment sample, a model organism for the pathogen Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli.

Single cells of environmental samples from the top oxic layer of the marine sediment incubated over extended times under static conditions showed initiation of growth well beyond typical growth times, ranging up to 18 months. Identification of isolates via sequencing of the 16S rRNA gene revealed that the majority of late growing isolates were typical marine species with known growth rates well below what was evidenced in our experiments. Furthermore, upon subculture the majority of the isolates (>60%) grew in under 1 month regardless of their initial time required to visualize growth. Escherchia coli cells treated in various ways (antibiotic challenge, exposure to environmental conditions, etc) resulted in a stark increase in the non-growing population relative to total cell counts. Isolation of single cells showed that individual cells were able to initiate growth at seemingly random intervals stretching up to 2 months. This indicates that E. coli exhibits recovery in a manner consistent with the scout model. Further development of a model utilizing E. coli would be extremely useful for downstream studies into the mechanism of dormancy. Dormant M. smegmatis cells, incubated at various concentrations, exhibited visible growth at time points ranging from the typical 48-72 hours, up to over 2 months, in a linear fashion. An ethyl acetate extract of spent medium from actively growing mid-exponential phase M. smegmatis cultures applied to dormant cells resulted in an increase of cells exhibiting visible growth after 5 days of incubation. Methanol fractionation of the extract has allowed us to close in on the potential molecule causing this activity.

These experiments indicate that reversible dormancy consistent with the proposed scout model is present in bacteria from diverse sources, giving us insight into bacterial behavior, potentially leading to the cultivation of previously uncultivated species from the environment, as well as offering a different approach to developing treatments for recalcitrant infections.

Document Type

Dissertation

Rights Holder

Sandra M. Buerger


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