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

Spores of Bacillus subtilis are extremely resistant to heat, radiation, desiccation, pH extremes, and toxic chemicals (31). Like many endospore formers, B. subtilis is primarily a soil organism and may be subject to large fluctuations in environmental conditions, in particular nutrient availability. Consequently, spore formation in times of low nutrient availability can be advantageous, as the dormant spore can ride out bad times and, when conditions are favorable, return to growth through spore germination and outgrowth. Germination of B. subtilis spores normally begins with the binding of specific nutrient germinants, either l-alanine or a mixture of l-asparagine, d-glucose, d-fructose, and potassium ions (AGFK), to specific receptors located in the spore's inner membrane (7, 16, 19, 23). Three functional nutrient receptors are present in B. subtilis spores, each encoded by the homologous tricistronic gerA, gerB, and gerK operons. The GerA nutrient receptor responds to l-alanine, while the GerB and GerK nutrient receptors cooperate in some fashion to respond to AGFK. The GerD protein also plays a role in germination, as spores of strains with both point and deletion mutations in gerD germinate poorly with both l-alanine and AGFK and are blocked very early in the spore germination pathway (18, 19, 25). The gerD gene encodes an ∼20-kDa protein with a putative 11-amino-acid signal sequence as well as a likely recognition sequence for diacylglycerol addition to a specific cysteine residue near the protein's amino terminus (Fig. ​(Fig.1)1) (33, 38). GerD homologs are present throughout the bacilli (Fig. ​(Fig.1),1), although there is no obvious GerD homolog in Clostridium species. The GerD sequence also does not resemble that of any of the nutrient receptors. Transcription of gerD takes place only in the developing forespore compartment of the sporulating cell and is directed by the forespore-specific RNA polymerase sigma factor, σG, as is transcription of the gerA, gerB, and gerK operons (9, 10, 15, 30). Although components of the GerA and GerB receptors have been localized to the spore's inner membrane, the location of GerD is not yet known (7, 23). One report suggests that GerD is in the spore's integument fraction, i.e., the coat plus the cortex (17). However, the putative lipobox in GerD that appears to be essential for GerD function (25) suggests that this protein likely resides in a membrane. FIG. 1. Alignment of amino acid sequences of the amino-terminal regions of GerDs from various species. The sequences shown are from B. subtilis, B. licheniformis, Geobacillus stearothermophilis, and B. anthracis. Gray shading indicates similar residues, and black ... As noted above, GerD is most likely a lipoprotein that has a diacylglycerol linked to a specific cysteine residue (25, 33, 38) (Fig. ​(Fig.1).1). This type of lipoprotein has been characterized in the following four ways: (i) by sequence data indicating the presence of a conserved signal sequence and lipobox; (ii) by mutation of the invariant cysteine, the site for diacylglycerol addition, within the lipobox region, leading to abrogation of function; (iii) by globomycin treatment, which inhibits processing by signal peptidase II (Lsp); and (iv) by radiolabeling with fatty acids, usually palmitic acid (33). Typically, these lipoproteins contain a signal peptide sequence followed by a lipobox (28, 29, 32, 33). The signal peptide sequence is characterized by an n domain, consisting of the basic amino acids lysine and arginine, followed by a central hydrophobic h domain and, finally, a cleavage (c) domain. The cleavage domain contains a lipobox characterized by the consensus amino acid sequence Leu-Ala/Ser-Gly/Ala-Cys, with cleavage just prior to the cysteine residue (32, 33). GerD contains all three domains, as well as a similar lipobox in its cleavage domain (Fig. ​(Fig.1),1), and changing the putative diacylglycerylated cysteine to alanine results in a gerD spore germination phenotype (25). During the maturation of these types of lipoproteins, the diacylglycerol moiety from phosphatidylglycerol is initially transferred to the sulfydryl group of cysteine by a prelipoprotein diacylglycerol transferase (Lgt). The modified lipoprotein precursor is then cleaved by signal peptidase II, Lsp, and after cleavage of the signal peptide, the N-terminal cysteine may be N-acylated (28, 29, 33, 34, 35). Both lgt and lsp genes have been identified in B. subtilis; however, no gene encoding an N-acyltransferase (Lnt) has been found (11, 13, 27). Since GerD appears to be a lipoprotein and is synthesized in the same compartment and at approximately the same time as the nutrient germinant receptors, it seems most likely that GerD is located in the spore's inner membrane. Indeed, in this work we demonstrate that GerD is a lipoprotein that is associated with the dormant spore's inner membrane. This localization of GerD in the same membrane as the spore's nutrient germinant receptors may assist in our understanding of the role of GerD in the nutrient receptor-mediated spore germination pathway.