Date of Completion

11-13-2013

Embargo Period

5-11-2014

Keywords

Bacillus subtilis, spore germination, mechanism, superdormancy, gerD

Major Advisor

Dr. Peter Setlow

Associate Advisor

Dr. Ann Cowan

Associate Advisor

Dr. Lawrence Klobutcher

Associate Advisor

Dr. Bing Hao

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Members of Bacillus and Clostridium species sporulate under adverse conditions and the resultant spores can remain metabolically dormant and resistant for years. However, when conditions are favorable, primarily when specific nutrients become available, spores can resume vegetative growth via the process of germination. The overall goal of the work outlined in this thesis is to achieve a better understanding of the process of germination in bacterial spores, since spores are a major concern in food spoilage and food borne diseases as well as in bioterrorism due to Bacillus anthracis spores.

There are two types of germination: nutrient and non-nutrient germination. Specific nutrient germinants that trigger germination are recognized by germinant receptors (GRs), with different GRs having different nutrient germinant specificities and this interaction somehow causes the release of Ca-DPA from the spore core via SpoVA proteins. Spores can also be germinated by non-nutrient germinants by bypassing the GRs and causing spore cortex peptidoglycan hydrolysis leading to DPA release. Interestingly, there is tremendous heterogeneity in the germination of individual spores in populations, and the reasons for this heterogeneity have been unclear. In this study, spores of Bacillus species that germinated extremely poorly with saturating levels of nutrient and non-nutrient germinants, termed superdormant (SD) spores, were isolated from spore populations and the causes for superdormancy of these spores were determined. The results indicate that a low level of a specific GR or GRs is a major cause of spore superdormancy using nutrient germinants, while in case of non-nutrient germinants a spore coat defect seems to be the most likely cause.

Another goal of this thesis was to identify a protein that is involved in the coupling reaction between binding of nutrients germinants to the GRs and the release of DPA from the spore core via SpoVA proteins. Hence a genetic screen was performed to identify such genes encoding additional proteins essential for spore germination with nutrient germinants but this work did not identify any new protein. However, the fact that we identified a dominant negative mutation in gerD suggests that perhaps GerD is the signal integrator that we were looking for.

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