Structural and functional studies of proteins involved in spore formation in Bacillus subtilis

Bacillus subtilis and Clostridioides difficile are Gram-positive bacteria that may adapt to unfavourable conditions by forming a dormant spore. Sporulation involves an asymmetric cell division resulting in a larger mother cell and a smaller forespore. The forespore is later engulfed by the mother cell in a phagocytosis like process.

Activation of the sporulation pathway is tightly regulated through complex sensor systems, crucial to which are peptides imported into the cell through peptide transport systems belonging to the ATP-binding cassette (ABC) family. Peptides have an important nutritional role as well as the signalling role and B. subtilis has three ABC peptide transporters with overlapping specificity, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App). Here the crystal structures of the receptors for two of these transporters OppA and DppE are described revealing unexpectedly that DppE is a murein tripeptide binding protein whilst the OppA structure reveals a tetrapeptide bound whose average electron density closely resembles Ser-Asn-Ser-Ser.

Crystallographic and peptide binding studies of OppA and AppA from C. difficile have led us to conclude that neither of these proteins is a receptor for peptide transport as previously reported. OppA is more likely to serve as a receptor for the uptake of nickel ions.

Upon completion of engulfment, a channel is formed between mother cell and forespore at the heart of which is an intercellular interaction between SpoIIIAH and SpoIIQ that has been described structurally. The stoichiometry of this complex in vivo is unknown though models of rings containing 15-24 molecules have been proposed. Here experiments are described to fuse fluorescent reporters to SpoIIQ and SpoIIIAH in order to perform super resolution microscopy to determine the stoichiometry of the complex in live cells for the first time.