Members of the Bacillus genus, when faced with unfavorable environmental conditions such as depletion of nutrients, undergo an asymmetric division process ultimately leading to the formation of an endospore. In some instances, the spore serves as the infectious agent of an associated disease; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of unfavorable environmental conditions including traditional decontamination techniques. Spore resistance is due to the formation of specialized structures that contribute to spore dormancy through several mechanisms, including maintenance of the dehydrated state of the spore core. Spore germination is a rapid process resulting in the irrevocable transformation of the non-metabolizing dehydrated spore into a vegetative outgrowing bacterium. The exact mechanism by which individual proteins function in the germination pathway remains unknown. In this study, we have focused on the roles of putative ion transporters and other germination-active proteins in affecting spore formation and germination.
Metal ions can activate enzymes during the sporulation process and/or be factors in spore resistance properties. In B. subtilis, six proteins within the spore membrane proteome (ChaA, YcnL,YflS, YloB, YugS, ZnuA) are similar to components of known cation transport systems. These proteins may play roles in the accumulation of ions during sporulation and/or the release of ions during germination. Multiple mutants altered in the putative ion transporter genes were generated, and the effects of these mutations were analyzed. All strains containing a yloB deletion showed a decrease in heat resistant cfu/ml, and >40% of the spores appeared phase dark during microscopy, indicating the formation of unstable spores. Studies were conducted to quantify the amounts of individual ions in phase-bright spores using atomic emission spectroscopy and to analyze the rate at which ions are released from germinating spores. The transport of Ca2+ from mother cell to forespore during sporulation seems to be affected in the yloB deletion mutant. This Ca2+ deficit apparently renders the spores unstable, heat sensitive, and partially germination defective, suggesting that a high-affinity transporter for Ca2+ is nonfunctional.
To better understand the underlying mechanisms of germination, a high-throughput genetic screening method called transposon sequencing was used. This analysis identified genes that had not been previously implicated in germination. To investigate their functions, a number of functional assays of all the Ger mutant strains were performed that indicated a delay in stage I of germination. The mutant strains showed significant reduction in germination efficiency with L-valine: about 50% of the population failed to initiate germination suggesting a defect in the GerA-mediated response. The expression of gerA was studied using a lacZ transcriptional fusion followed by quantitative western blot analyses to determine abundance of GerA in mutant strains. The mutants were classified based upon normal or decreased gerA transcription and normal or reduced GerA protein. Further work involves understanding the functions of the identified genes and their correlation to the GerA receptor.
Insight into ion transporters of spore-forming bacteria and understanding the germination apparatus may lead to promising new applications, detection methods, or therapeutics for spores, and may allow the development of better spore decontamination procedures. / Doctor of Philosophy / Bacillus subtilis is an ubiquitous bacterium that is capable of forming endospores when faced with unfavorable environmental conditions. Spores are highly resistant to heat, radiation, lack of nutrients, desiccation and oxygen deprivation. They lack metabolism, which effectively keeps them in a state of suspended animation until germinated. They may remain stable and viable in this state for extremely long periods of time. Several important pathogenic bacteria are spore formers. This leads to difficulty in their environmental eradication and the treatment of patients. Germination allows spores to resume metabolism and reestablish a vegetative state. Certain key molecules activate the germination process. Each species of spore-forming bacteria has a specific set of these molecules called germinants that will enable the spore to exit its dormant state. The work presented focuses on the understanding of the germination apparatus of Bacillus subtilis, which may provide a model to understand the germination of other spore formers and help to improve methods of decontamination.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/98541 |
| Date | 22 May 2020 |
| Creators | Barat, Bidisha |
| Contributors | Biological Sciences, Popham, David L., Caswell, Clayton C., Stevens, Ann M., Scharf, Birgit E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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