Topology and Accessibility of Germination Proteins in the Bacillus subtilis Spore Inner Membrane

Abstract

Access to a membrane-impermeant biotinylation reagent as well as protease sensitivity was used to determine germination proteins' topology in the inner membrane (IM) of decoated dormant spores and intact germinated Bacillus subtilis spores. The proteins examined were four nutrient germinant receptor (GR) subunits, the GerD protein, essential for normal GR-dependent spore germination, the SpoVAD protein, essential for dipicolinic acid movement across the IM, the SleB cortex-lytic enzyme, and the YpeB protein, essential for SleB assembly in spores, as well as green fluorescent protein (GFP) in the spore core. GerD and SpoVAD as well as GFP in the spore were not biotinylated in decoated dormant spores. However, GR subunits, SleB, and YpeB were biotinylated 4 to 36% in decoated dormant spores, although these levels were not increased by higher biotinylation reagent concentrations or longer reaction times. In contrast, the germination proteins were largely biotinylated in germinated spores, although GFP was not. All of the germination proteins in the germinated spore's IM, but not spore core GFP, were largely sensitive to an exogenous protease. These results, coupled with predicted or experimentally determined structural data, indicate that (i) these germination proteins are at least partially and in some cases completely on the outer surface of the spore's IM and (ii) there is significant reorganization of these germination proteins' structure or environment in the IM during spore germination. Spores of Bacillus species can remain dormant for years, yet can return to life within minutes in the process of spore germination followed by outgrowth (1, 2). A number of proteins are specifically involved in spore germination (1-5) including (i) the multiple-nutrient germinant receptors (GRs), each of which recognizes different nutrient germinants and has A, B, and C subunits; (ii) the GerD protein required for rapid GR-dependent germination; (iii) the SpoVA proteins needed for uptake of the spore core's large depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in sporulation and its release in spore germination; and (iv) cortex-lytic enzymes (CLEs) needed to hydrolyze spores' large peptidoglycan (PG) cortex. Many of these proteins, in particular the GRs and the GerD and SpoVA proteins, as well as one CLE (SleB) and the YpeB protein essential for SleB assembly in spores, are in the inner membrane (IM) that surrounds the spore core (6-12), and the GRs and GerD appear to be associated in a small cluster or focus in the IM (7). However, there is no definitive knowledge of the precise topology of any of these proteins in the spore IM other than predicted topology based on the following: (i) knowledge that GRs and GerD and SpoVA proteins are synthesized in the developing spore (1, 2, 4); (ii) the presence or absence of a likely N-terminal signal sequence; (iii) in some cases, a recognition signal for diacylglycerol addition to a cysteine residue near the protein's N terminus (13); and (iv) the facts that the sleB ge...

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

“…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). However, how GRs recognize specific nutrients, how nutrient recognition triggers the downstream events in spore germination, and how GerD influences GR function are largely unknown.…”

“…In addition, at least GerBA remains in the spores' IM after germination and on into outgrowth (5). GR A and B subunits are predicted to have multiple transmembrane domains (13,15), and GR C subunits have an N-terminal cysteine with a diacylglycerol moiety (14); these structural features further suggest that GR proteins are present in the IM. In addition, the localization by fluorescence microscopy of fluorescent fusions of a number of GR proteins as well as GerD and a SpoVA protein in the IM region just outside the spore core (12) is further evidence for the location of these proteins in the IM.…”

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…The GR's C subunits are largely on the IM's outer surface, where they are held by a diacylglycerol anchor and, presumably, interaction with their cognate A and B proteins (4,54,55). The structure of the C protein of the B. subtilis GerB GR has been determined to 2.3-Å resolution (56).…”

“…While the GRs are essential for nutrient germination, the normal function of GRs requires the GerD protein (4,58). This small protein is also on the outer sur-face of the IM and is held there, at least in part, by a lipid anchor (54,59). The sequence of GerD is well conserved among bacilli, but GerD is not present in spores of clostridia (6, 8; Li Y, Jin K, Ghosh S, Devarakonda P, Carlson K, Davis A, Stewart K-AV, Cammett E, Pelczar-Rossi P, Setlow B, Lu M, Setlow P, Hao B, unpublished data).…”

“…Unfortunately, this novel structure gives no hints as to this protein's function, although predictive programs suggest that all GR C proteins have a similar structure. The GR B proteins are comprised primarily of TMDs with 10 to 12 helices spanning the IM, while the A proteins have 6 to 8 TMDs but also a small C-terminal globular domain and a large, likely N-terminal soluble domain that is probably on the IM's outer surface (54,55). The amino acid sequences of the putative GR D subunits suggest that these proteins are composed primarily of 2 TMDs, but these proteins' sequences are minimally conserved both within and across species.…”