Date of Completion

5-8-2013

Embargo Period

5-8-2013

Keywords

Bacillus subtilis, bacterial spore, spore germination, fluorescence microscopy, recombinant fusion proteins, membrane lipids, membrane proteins

Major Advisor

Peter Setlow

Associate Advisor

Ann Cowan

Associate Advisor

Ji Yu

Associate Advisor

Stephen King

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Bacillus subtilis cells form spores when they are deprived of nutrients. The spores formed are resistant to many insults including: heat, radiation, enzymes and various chemicals. They are also metabolically inactive and can remain in this state indefinitely. Although dormant, spores rapidly return to life via the process of germination when specific nutrients are returned to their environment. The inner membrane of dormant spores is of central importance in spore biology. Previous studies have shown that the components of the inner membrane of dormant spores have the unique properties of being relatively immobile and compressed. The inner membrane is also relatively impermeable and thus plays a major role in spore resistance and dormancy. It remains unclear how the inner membrane acquires these properties. Moreover, the spore inner membrane contains proteins required for initiating germination including the nutrient germinant receptors (GRs), and the GerD and SpoVA proteins. These proteins seem to act cooperatively but the mechanistic details of the initiation of germination are poorly understood. The first aim of this study was to determine whether the inner membrane phospholipids played specific roles in sporulation and spore properties. The results revealed that changing the phospholipid composition of the B. subtilis inner membrane had little effect on sporulation, and only modest effects on spore germination, wet heat and oxidizing agent sensitivity, and inner membrane permeability. The second aim of this study was to determine how germination proteins are organized in the spore inner membrane using fluorescence microscopy. The results show that the GRs and GerD colocalize primarily to a single cluster in dormant spores, pointing to the existence of a specific germination locus or “germinosome” in the dormant spores’ inner membrane. GRs require all their subunits as well as GerD for cluster formation. Diacylglycerol addition to GerD and GRs’ C subunits is also required for clustering. However, the different GRs cluster independently of each other, and GerD forms clusters in the absence of all the GRs. These data suggest that cooperativity in spore germination is achieved at least in part via pre-positioning necessary germination proteins at a single site in the dormant spore.

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