9.B.70 The Multicomponent Putative SpoIIIAE Exporter (SpoIIIA-E) Family
The SpoIIIAA-AH operon encodes 8 proteins, all of which are required for activation of sigma G during spore development in Bacillus subtilis. Mutations in any one of these genes blocks sporulation. This operon is transcribed from a sigma E promoter in the mother cell after asymmetric cell division. SpoIIIAH is known to have a single N-terminal TMS with the remainder of the proteins facing outward. It interacts with SpoIIQ which is present on the forespore cell surface. These two proteins possibly function as a 'zipper' to facilitate engulfment (Blaylock et al., 2004). The other proteins may serve as a protein export system with SpoIIIAA serving as the ATPase and SpoIIIAE serving as the substrate protein. SpoIIIAA is an ATPase with Walker A and B motifs, both of which are essential for sporulation. Residues 97-246 show weak similarity to residues 252-404 of FtsY (TC #3.A.5.1.1), the bacterial equivalent of the SRP receptor subunit. It also shows similarity in the Walker A (GX4GKT) and B (Hy4DE) motifs with the ABC proteins of the ABC superfamily (TC #3.A.1.14.2 and #3.A.1.4.1, respectively). SpoIIIAA is a member of the triple A or VirB11 family.
SpoIIIAE is a 405 aa protein with 7 putative TMSs. It appears to have an N-terminal Sec-type signal peptide followed by TMSs 2-7. Residues 200-350 appear to show very limited sequence similarity with many transport proteins from different families. These include: 2.A.23.1.4 (CPA1); 9.A.15.1.1(YhaG); 3.A.7.1.1. (VirB6); 2.A.31.3.2 (AE); 3.A.1.1.14 (AraH in ABC); 2.A.52.1.4 (NiCoT); 2.A.6.2.15 (RND) and 4.A.1.2.4 (Tre(Glc-PTS)). The significance of these observations is not known. These regions in SpoIIIAE and the other transporters generally show 2 TMSs, a hydrophilic loop of ~50 aas, and 2 more TMSs. SpoIIIAE may possibly be exported from the mother cell via the SpoIIIA exporter and inserted into the forespore membrane where it might function as a small molecule permease to promote sigma G-activation. All other constituents of the system have 1-3 TMS (see below) and may form part of the export system. Based on the similarity of SpoIIIAH to a component of type III secretion systems, Camp and Losick (2008) have suggested that signalling is mediated by a channel that links the mother cell to the forespore.
At an early stage during Bacillus subtilis endospore development, the bacterium divides asymmetrically to produce two daughter cells. The smaller cell (forespore) differentiates into the endospore, while the larger cell (mother cell) becomes a terminally differentiated cell that nurtures the developing forespore. During development the mother cell engulfs the forespore to produce a protoplast, surrounded by two bilayer membranes, which separate it from the cytoplasm of the mother cell. The activation of σG, which drives late gene expression in the forespore, follows forespore engulfment and requires expression of the spoIIIA locus in the mother cell. One of the spoIIIA-encoded proteins SpoIIIAH is targeted specifically to the membrane surrounding the forespore, through an interaction of its C-terminal extracellular domain with the C-terminal extracellular domain of the forespore membrane protein SpoIIQ (P71044). Meisner et al., 2008 identified a homologous relationship between the C-terminal domain of SpoIIIAH and the YscJ/FliF protein family, members of which form multimeric rings involved in type III secretion systems and flagella. If SpoIIIAH forms a similar ring structure, it may also form a channel between the mother cell and forespore membranes. To test this hypothesis Meisner et al., 2008 developed a compartmentalized biotinylation assay, which they used to show that the C-terminal extracellular domain of SpoIIIAH is accessible to enzymatic modification from the forespore cytoplasm. These and other results led them to suggest that SpoIIIAH forms part of a channel between the forespore and mother cell that is required for the activation of σG.
Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation (Rodrigues et al. 2016). An early step in the assembly of this transenvelope complex (called the A-Q complex) is an interaction between the extracellular domains of the forespore membrane protein SpoIIQ and the mother cell membrane protein SpoIIIAH. This interaction provides a platform onto which the remaining components of the complex assemble and also functions as an anchor for cell-cell signalling and morphogenetic proteins involved in spore development. SpoIIQ is required to recruit SpoIIIAH to the sporulation septum on the mother cell side. Rodrigues et al. 2016 identified GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. GerM and SpoIIIAH, derived from the mother cell, and SpoIIQ, from the forespore, have reciprocal localization dependencies suggesting they constitute a tripartite platform for the assembly of the A-Q complex and a hub for the localization of mother cell and forespore proteins.
During engulfment, an essential channel, the so-called feeding tube apparatus, crosses both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Zeytuni et al. 2017 presented the near-atomic resolution structure of SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM), common to the architecture of various dual membrane secretion systems of distinct function. The structure shows that SpoIIIAG exhibits a dramatic adaptation of the RBM fold with a β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro (Zeytuni et al. 2017).
The reactions thought to be catalyzed by the SpoIIIA exporter is:
Small molecules(mother cell), SpoIIIAE(mother cell) and other proteins(mother cell) → small molecules(forespore), SpoIIIAE(forespore membrane) and other proteins(forespore)