Localization of the Germination Protein GerD to the Inner Membrane in Bacillus subtilis Spores

Abstract

GerD of Bacillus subtilis is a protein essential for normal spore germination with either L-alanine or a mixture of L-asparagine, D-glucose, D-fructose, and potassium ions. GerD's amino acid sequence suggests that it may be a lipoprotein, indicating a likely location in a membrane. Location in the spore's outer membrane seems unlikely, since removal of this membrane does not result in a gerD spore germination phenotype, suggesting that GerD is likely in the spore's inner membrane. In order to localize GerD within spores, FLAG-tagged GerD constructs were made, found to be functional in spore germination, and detected in immunoblots of spore extracts as not only monomers but also dimers and trimers. Upon fractionation of spore extracts, GerD-FLAG was found in the inner membrane fraction from dormant spores and was present at ϳ2,000 molecules/spore. GerD-FLAG in the inner membrane fraction was solubilized by Triton X-100, suggesting that GerD is a lipoprotein, and the protein was also solubilized 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. However, unprocessed GerD-FLAG still gave bands corresponding to monomers and dimers of slightly higher molecular weight than that of GerD-FLAG from a strain with Lgt, further suggesting that GerD is a lipoprotein. Upon spore germination, much GerD became soluble and then appeared to be degraded as the germinated spores outgrew and initiated vegetative growth. All of these results suggest that GerD is a lipoprotein associated with the dormant spore's inner membrane that may be released in some fashion from this membrane upon spore germination.

Numbers of Individual Nutrient Germinant Receptors and Other Germination Proteins in Spores of Bacillus subtilis

“…2 and Table 1) and PS3478 (data not shown) spores as well as wild-type spores disrupted with lysozyme with or without sonication or by mechanical breakage alone (data not shown). The high levels of GerD and SpoVAD in the S fraction might be considered surprising, since there are data indicating that both GerD and at least one SpoVA protein are in the IMs of intact spores (8,9,12,26). However, previous work has also found significant amounts of these proteins in the S fraction of disrupted spores (8,9,26).…”

“…Disruption of any cistron encoding a GR's A, B, or C subunit eliminates germination with that GR's corresponding germinant or germinant mixture, indicating that all three GR subunits are essential for that GR's function. The GerD protein is also essential for rapid GR-dependent spore germination and for germinosome assembly, and this protein is also present in the spore's IM, where it is presumably anchored by a covalently attached diacylglycerol moiety that is also present in GR C subunits (9,12,13). Loss of this diacylglycerol anchor eliminates both GerD and GR C protein function, consistent with these proteins' inner membrane location, and almost certainly on the outer leaflet of the inner membrane (13,15,16).…”

“…Spore breakage was Ͼ80%, as determined by phase-contrast microscopy, and the extract was fractionated as described above. In yet another experiment, ϳ20 mg (dry weight) of intact wild-type spores was broken by dry rupture with glass beads, and the powder was extracted and fractionated as described above and previously (9).…”

“…Based on previous results, GerD and SpoVAD are more abundant than GerBA in wild-type spores (5,8,9). Therefore, detection of these proteins should not be as challenging in spores where only GRs are overexpressed, because GerD and SpoVAD levels remain at wild-type levels in such strains (7).…”

“…One such layer is the inner membrane (IM), a lipid bilayer with extremely low permeability in which lipid probes and presumably proteins are immobile prior to germination (3). The IM also contains a number of proteins crucial for spore germination, including the nutrient germinant receptors (GRs) that trigger spore germination in response to specific nutrient germinants (4)(5)(6)(7)(8)(9).…”