Role of a SpoVA Protein in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

Abstract

The proteins encoded by the spoVA operon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2, 6-dicarboxylic acid (DPA 2,6 ) and Ca 2؉ into developing spores of the bacterium Bacillus subtilis. The crystal structure of B. subtilis SpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA 2,6 exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA 2,6 and Ca 2؉ -DPA 2,6 with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA 2,6 -binding pocket in SpoVAD essentially abolish its DPA 2,6 -binding capacity. Moreover, replacement of the wild-type spoVAD gene in B. subtilis with any of these spoVAD gene variants effectively eliminated DPA 2,6 uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA 2,6 movement into developing B. subtilis spores.

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

“…At present, the identity of this lower-molecularmass band is not clear, although its intensity was clearly lower than that of the 36-kDa SpoVAD band. The distribution of GerD and SpoVAD was different from that of the GRs, which was perhaps not unexpected, since (i) GerD is relatively hydrophilic and may be held in the IM primarily by its lipid anchor, (ii) significant amounts of GerD were seen in the S fraction of spores previously (26), and (iii) SpoVAD has no obvious hydrophobic segments suitable for membrane anchoring (28). Consequently, SpoVAD and GerD may not be as tightly bound to the IM as are GR subunits and thus may be easily lost from the IM when spores are disrupted and fractionated.…”

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

“…The B. subtilis SpoVA complex is composed of seven different proteins: SpoVAA, SpoVAB, SpoVAC, SpoVAD, SpoVAEa, SpoVAEb, and SpoVAF. Most of these proteins are important for both CaDPA import into the developing spore during sporulation and CaDPA release during germination (e.g., SpoVAD binds DPA [47]). C. difficile does not encode orthologues of many of these proteins.…”

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

“…The resulting PCR products, ⌬spoVAEa and the intact spoVAEa gene, were cloned into a modified pET15b vector containing a His 6 tag and a tobacco etch virus (TEV) protease cleavage site (13). The ⌬SpoVAEa protein (residues 2 [⌬124-135] to 203) and intact SpoVAEa were expressed in Escherichia coli BL21 Star(DE3) (Invitrogen, Grand Island, NY) initially by growth at 37°C in Luria broth (LB) (14) plus ampicillin (100 g/ml) and then by induction with 1 mM isopropyl-␤-D-thiogalactopyranoside at an optical density at 600 nm (OD 600 ) of 0.8 and subsequent growth at 21°C for 16 h. Both SpoVAEa proteins were soluble and were purified by Ni 2ϩ -nitrilotriacetic acid affinity chromatography under native conditions, followed by TEV protease cleavage of the His 6 tag and cation-exchange chromatography (SD200; GE Healthcare, Piscataway, NJ) (6,13).…”

“…These proteins are essential for the uptake of dipicolinic acid (DPA) into the dormant spore (3-7); in turn, DPA is essential for spore stability, as DPA-less B. subtilis spores germinate spontaneously after their release from the sporangium and during spore purification (8). At least some SpoVA proteins are also essential for DPA release in spore germination, and one SpoVA protein, SpoVAD, binds DPA specifically, with this binding essential for DPA uptake in sporulation (6). SpoVAD is a soluble protein and has no obvious signal peptide or membrane-spanning or anchor sequences.…”