A large number of proteins are known to reside at specific subcellular locations in bacterial cells. However, the molecular mechanisms by which many of these proteins are anchored at these locations remains unclear. During endospore formation in Bacillus subtilis, several integral membrane proteins are located specifically at the interface of the two adjacent cells of the developing sporangium, the mother cell and forespore. The mother cell membrane protein SpoIIIAH recognizes the cell-cell interface through an interaction with the forespore membrane protein SpoIIQ, and then the other proteins are positioned there by the SpoIIIAH-SpoIIQ complex. In this study, we investigated the molecular mechanisms underlying the formation of the SpoIIIAH-SpoIIQ complex. Using gel filtration chromatography and isothermal titration calorimetry, we measured the binding parameters that characterize the SpoIIIAH-SpoIIQ interaction in vitro. We also demonstrated that the interaction of SpoIIIAH and SpoIIQ is governed by their YscJ and degenerate LytM domains, respectively. Therefore, the LytM domain of SpoIIQ provides the positional cue that dictates the localization of mother cell membrane proteins to the mother cell-forespore interface.

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

Proteins SpoIIQ and SpoIIIAH interact through two membranes to connect the forespore and the mother cell during endospore development in the bacterium Bacillus subtilis. SpoIIIAH consists of a transmembrane segment and an extracellular domain with similarity to YscJ proteins. YscJ proteins form large multimeric rings that are the structural scaffolds for the assembly of type III secretion systems in Gram-negative bacteria. The predicted ring-forming motif of SpoIIIAH and other evidence led to the model that SpoIIQ and SpoIIIAH form the core components of a channel or transporter through which the mother cell nurtures forespore development. Therefore, to understand the roles of SpoIIIAH and SpoIIQ in channel formation, it is critical to determine whether SpoIIIAH adopts a ring-forming structural motif, and whether interaction of SpoIIIAH with SpoIIQ would preclude ring formation. We report a 2.8-Å resolution structure of a complex of SpoIIQ and SpoIIIAH. SpoIIIAH folds into the ring-building structural motif, and modeling shows that the structure of the SpoIIQ–SpoIIIAH complex is compatible with forming a symmetrical oligomer that is similar to those in type III systems. The inner diameters of the two most likely ring models are large enough to accommodate several copies of other integral membrane proteins. SpoIIQ contains a LytM domain, which is found in metalloendopeptidases, but lacks residues important for metalloprotease activity. Other LytM domains appear to be involved in protein–protein interactions. We found that the LytM domain of SpoIIQ contains an accessory region that interacts with SpoIIIAH.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a "spotwelding" activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.

Two highly similar RNA polymerase sigma subunits, σ F and σ G, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ F drives synthesis of σ G but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ G while discriminating between σ F and σ G in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ G (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ G in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ G. CsfB is normally produced in the forespore, under σ F control, but sigGN45E mutant cells also expressed csfB and did so in a σ G-dependent manner, autonomously from σ F. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ G activity. N45 is invariant in the homologous position of σ G orthologues, whereas its functional equivalent in σ F proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ F, a E39N substitution in σ F resulted in efficient binding of CsfB to σ F. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ F in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.

During sporulation in Bacillus subtilis, the onset of activity of the late forespore-specific sigma factor σ^G coincides with completion of forespore engulfment by the mother cell. At this stage, the forespore becomes a free protoplast, surrounded by the mother cell cytoplasm and separated from it by two membranes that derive from the asymmetric division septum. Continued gene expression in the forespore, isolated from the surrounding medium, relies on the SpoIIIA-SpoIIQ secretion system assembled from proteins synthesised both in the mother cell and in the forespore. The membrane protein insertase SpoIIIJ, of the YidC/Oxa1/Alb3 family, is involved in the assembly of the SpoIIIA-SpoIIQ complex. Here we show that SpoIIIJ exists as a mixture of monomers and dimers stabilised by a disulphide bond. We show that residue Cys134 within transmembrane segment 2 (TM2) of SpoIIIJ is important to stabilise the protein in the dimeric form. Labelling of Cys134 with a Cys-reactive reagent could only be achieved under stringent conditions, suggesting a tight association at least in part through TM2, between monomers in the membrane. Substitution of Cys134 by an Ala results in accumulation of the monomer, and reduces SpoIIIJ function in vivo. Therefore, SpoIIIJ activity in vivo appears to require dimer formation.

Molecular Microbiology Published by John Wiley & Sons Ltd. During sporulation in Bacillus subtilis, a group of mother cell-specific proteins guides the assembly of the coat, a multiprotein structure that protects the spore and influences many of its environmental interactions. SafA and CotE behave as party hubs, governing assembly of the inner and outer coat layers. Targeting of coat proteins to the developing spore is followed by encasement. Encasement by SafA and CotE requires E, a region of 11 amino acids in the encasement protein SpoVID, with which CotE interacts directly. Here, we identified two single alanine substitutions in E that prevent binding of SafA, but not of CotE, to SpoVID, and block encasement. The substitutions result in the accumulation of SafA, CotE and their dependent proteins at the mother cell proximal spore pole, phenocopying a spoVID null mutant and suggesting that mislocalized SafA acts as an attractor for the rest of the coat. The requirement for E in SafA binding is bypassed by a peptide with the sequence of E provided in trans. We suggest that E allows binding of SafA to a second region in SpoVID, enabling CotE to interact with E and SpoVID to function as a non-competitive hub during spore encasement.