Date of Completion

6-4-2015

Embargo Period

6-3-2015

Keywords

Bacillus, spores, germinosome, GerD, germination

Major Advisor

Dr. Peter Setlow

Associate Advisor

Dr. Ann E. Cowan

Associate Advisor

Dr. Ji Yu

Associate Advisor

Dr. Lawrence Klobutcher

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Fluorescent fusions to germination proteins of Bacillus subtilis were shown to colocalize into a single focus termed the germinosome in sporulating and germinating spores, but not in outgrowing spores. In sporulating cultures, fluorescent foci are seen immediately upon fluorescent signal detection, ~3 hours before dipicolinic acid uptake, and in fluorescent strains with sspA-lacZ fusions, nearly in parallel with the accumulation of β- galactosidase activity. Furthermore, GerD-GFP self-associated in germinant receptor (GR) null strains, as well as in wild-type Bacillus subtilis, and facilitated germinosome formation in live Escherichia coli when expressed with gerAA-mCherry. These findings suggest the germinosome is made in parallel with GerD and GR synthesis, and that GerD acts as a scaffold that facilitates the colocalization of the GRs in the germinosome. Conversely, we found that after initiation of spore germination, the germinosome foci changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins changed minimally during this period, although GerD levels decreased ∼ 50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼ 2-fold decreases in GerD levels, or other factors such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds. All of these data suggest that GerD, while important for facilitating the formation of the germinosome in sporulation, may not be as significant in maintaining this protein complex in actively germinating spore populations.

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