During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA. We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.In response to nutrient depletion, Bacillus subtilis can undergo a complex differentiation process culminating in the formation of a dormant endospore (34). Sporulation begins with an asymmetric cell division that partitions the sporangium into a large mother cell and a smaller prespore, each of which contains one copy of the bacterial chromosome. Hydrolysis of the septal peptidoglycan (PG) allows the mother cell to engulf the prespore, a process that yields a cellular compartment, the forespore, completely surrounded by the mother cell cytoplasm. Maturation of the spore proceeds by the coordinated synthesis of the necessary gene products from both the forespore and the mother cell sporangial compartments. At the end of the developmental process, the spore is released into the environment upon lysis of the mother cell (20,34).The endospore is highly resistant to a variety of insults, including dehydration, organic solvents, lysozyme treatment, and extreme temperatures. However, in spite of its dormant nature, the spore can sense its environment and initiate germination within minutes of exposure to germinants. These spore properties are attributable to the physical and chemical composition of the structures which encase the spore (6,12,20). Thin-section electron microscopy reveals two main layers surrounding the core of mature spores: a thick electron-transparent layer of a modified PG known as the cortex (reviewed in reference 4) and a multila...

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.Proteins are targeted to specific subcellular locations during the assembly of a variety of bacterial structures. The structures assembled by prokaryotes include, for example, the cell division septum (3), surface protein layers (S-layers) (36), and a number of surface appendages such as flagella (24) and pili (17). Here we are concerned with the assembly of the Bacillus subtilis spore coat, a proteinaceous structure that encases spores (reviewed in references 7 and 16). The B. subtilis spore is a metabolically dormant cell type that is formed as an adaptive response to nutrient depletion (9, 30, 37). The spore coat provides protection against physical and chemical insults and can sense and respond to nutrients that trigger germination (9, 26).Spore morphogenesis commences by the placement of an asymmetric septum that divides the rod-shaped cell into a larger mother cell and a smaller prespore, each containing a copy of the chromosome. The septal membranes migrate around the prespore, in a process known as engulfment, which eventually results in the formation of a free-floating protoplast (the forespore) separated from the mother cell cytoplasm by a double-membrane system. Cell wall material (the cortex) is deposited between the membranes of the forespore (9, 30, 37). A thick coat composed of more than two dozen proteins is assembled around the outer forespore membrane. The majority of the proteins that make up the coat are synthesized in the mother cell under the direction of mother cell-specific RNA polymerase sigma factors (7,16,30).

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.

Assembly of each Salmonella typhimurium flagellum filament requires export and polymerisation of ca. 30 000 flagellin (FliC) subunits. This is facilitated by the cytosolic chaperone FliS, which binds to the 494 residue FliC and inhibits its polymerisation. Yeast two-hybrid assays, co-purification and affinity blotting showed that FliS binds specifically to the C-terminal 40 amino acid component of the disordered D0 domain central to polymerisation. Without FliS binding, the C-terminus is degraded. Our data provide further support for the view that FliS is a domain-specific bodyguard preventing premature monomer interaction.

SummaryDuring Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon (cotJA and cotJC) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA. Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.