Title

Characterization, analysis, and localization of the GerD protein in spores of Bacillus subtilis

Date of Completion

January 2008

Keywords

Biology, Microbiology

Degree

Ph.D.

Abstract

Spores of a B. subtilis strain with a gerD deletion mutation responded much slower than wild-type spores to nutrient germinants, although they did ultimately germinate, outgrow and form colonies. The germination defect of gerD spores was not suppressed by any changes in the sporulation or germination conditions. Ectopic expression of gerD suppressed the slow germination of gerD spores with nutrients, but overexpression of GerD did not increase rates of spore germination. Loss of GerD had no effect on spore germination induced by agents that do not act through nutrient receptors. Deletion of GerD's putative signal peptide or change of its likely diacylglycerylated cysteine residue to alanine reduced GerD function. The latter findings suggest that GerD is located in a spore membrane, most likely the inner membrane where the nutrient receptors are located. Further experiments using a GerD-FLAG construct to localize GerD within the spore confirmed this hypothesis. GerD-FLAG in the inner membrane fraction was solublized by Triton X-100 suggesting that GerD is a lipoprotein, and the protein was also solublized by 0.5 M NaCl. GerD-FLAG, was not processed proteolytically in a B. subtilis strain lacking gerF (lgt), which encodes prelipoprotein diacylglycerol transferase (Lgt), indicating that when GerD does not have a diacylglycerol moiety, signal sequence processing does not occur. Upon spore germination, much GerD became soluble and then appeared to be degraded as the germinated spores outgrew and initiated vegetative growth. All these data suggest that while GerD is not essential for nutrient germination, this protein has an important role in spores' rapid response to nutrient germinants, by either direct interaction with nutrient receptors or some signal transduction essential for germination. ^