BofC negatively regulates SpoIVB-mediated signalling in the Bacillus subtilis sigmaK-checkpoint

The BofC protein acts negatively on intercompartmental signalling of pro-sigma(K) processing in the sigma(K)-checkpoint of Bacillus subtilis. Signalling is brought about by the SpoIVB protein, which is synthesized in the forespore and initiates proteolytic processing of pro-sigmaK to its mature and active form in the opposed mother cell chamber of the developing cell. We have shown here that BofC, like SpoIVB, is secreted across the inner forespore membrane and, from the analysis of a bofC deletion and insertion mutant, is likely to interact with SpoIVB. In the absence of BofC, the amount of SpoIVB found in sporulating cells is substantially reduced, although SpoIVB is still able to activate proteolysis of pro-sigma(K). Conversely, in the absence of SpoIVB, the levels of BofC accumulate suggesting that the fate of each molecule is dependent upon their mutual interaction. Our results suggest that BofC could maintain SpoIVB in a stable but inactive form. Supporting this, we have shown that overproduction of BofC inhibits SpoIVB autoproteolysis and leads to a delay in proteolytic cleavage of pro-sigma(K). Based on our work here, we have proposed a model for BofC's functional role in intercompartmental signalling.     

SpoIVB has two distinct functions during spore formation in Bacillus subtilis

The Bacillus subtilis SpoIVB protein is a critical component of the intercompartmental signal-transduction pathway that activates the sigma factor, σ^K, in the mother cell of the sporulating cell. SpoIVB, synthesized in the forespore chamber, must act across two layers of phospholipid membrane to facilitate proteolytic processing of inactive pro-σ^K to active σ^K. We have used a genetic approach to dissect SpoIVB function and found that this protein has two distinct developmental functions. One function is that of intercompartmental signalling of pro-σ^K processing. The other role is essential to spore formation and is illustrated by mutations of SpoIVB which allow cell–cell signalling of pro-σ^K processing but prevent the formation of viable spores. Using localized and site-specific mutagenesis we have identified a functional domain of SpoIVB that is involved in its non-signalling role.     

Overproducing the Bacillus subtilis mother cell sigma factor precursor, Pro-sigma K, uncouples sigma K-dependent gene expression from dependence on intercompartmental communication.

During sporulation of Bacillus subtilis, proteolytic activation of pro-sigma K and ensuing sigma K-dependent gene expression normally require the activity of many sporulation gene products. We report here that overproducing pro-sigma K at the onset of sporulation substantially uncouples sigma K-dependent gene expression from its normal dependency. Overproducing pro-sigma K in strains with a mutation in spoIIIG, spoIIIA, spoIIIE, or spoIVB partially restored sigma K-dependent gene expression in the mother cell and resulted in accumulation of a small amount of polypeptide that comigrated with sigma K, but these mutants still failed to form spores. In contrast, sporulation of spoIVF mutants was greatly enhanced by pro-sigma K overproduction. The products of the spoIVF operon are made in the mother cell and normally govern pro-sigma K processing, but overproduction of pro-sigma K appears to allow accumulation of a small amount of sigma K, which is sufficient to partially restore mother cell gene expression and spore formation. This spoIVF-independent mechanism for processing pro-sigma K depends on sigma E, an earlier-acting mother cell-specific sigma factor. The spoIIID gene, which encodes a mother cell-specific DNA-binding protein that is normally required for pro-sigma K production, was shown to be required for efficient pro-sigma K processing as well. bof (bypass of forespore) mutations bypassed this requirement for spoIIID, suggesting that SpoIIID is less directly involved in pro-sigma K processing than are spoIVF gene products. However, bof spoIIID double mutants overproducing pro-sigma K still failed to sporulate, indicating that SpoIIID serves another essential role(s) in sporulation in addition to its multiple roles in the production of sigma K.     

The structure of bypass of forespore C, an intercompartmental signaling factor during sporulation in Bacillus

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase sigma-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing sigma(K), (Esigma(K)). sigma(K) is synthesized as an inactive zymogen, pro-sigma(K), which is activated by proteolytic cleavage. Processing of pro-sigma(K) is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-sigma(K) processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-sigma(K) processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded beta-sheet onto one face of which an alpha-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded beta-sheet and three alpha-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the sigma(K) checkpoint are discussed in the light of sequence and structure comparisons.     

Exchange of precursor-specific elements between Pro-sigma E and Pro-sigma K of Bacillus subtilis.

sigma E and sigma K are sporulation-specific sigma factors of Bacillus subtilis that are synthesized as inactive proproteins. Pro-sigma E and pro-sigma K are activated by the removal of 27 and 20 amino acids, respectively, from their amino termini. To explore the properties of the precursor-specific sequences, we exchanged the coding elements for these domains in the sigma E and sigma K structural genes and determined the properties of the resulting chimeric proteins in B. subtilis. The pro-sigma E-sigma K chimera accumulated and was cleaved into active sigma K, while the pro-sigma K-sigma E fusion protein failed to accumulate and is likely unstable in B. subtilis. A fusion of the sigE "pro" sequence to an unrelated protein (bovine rhodanese) also formed a protein that was cleaved by the pro-sigma E processing apparatus. The data suggest that the sigma E pro sequence contains sufficient information for pro-sigma E processing as well as a unique quality needed for sigma E accumulation.     

Sporulation protein SpoIVFB from Bacillus subtilis enhances processing of the sigma factor precursor Pro-sigma K in the absence of other sporulation gene products.

Processing of inactive pro-sigma K to active sigma K in the mother cell compartment of sporulating Bacillus subtilis is governed by a signal transduction pathway emanating from the forespore and involving SpoIVFB in the mother cell. Coexpression of spoIVFB and sigK (encoding pro-sigma K) genes in growing B. subtilis or Escherichia coli enhanced pro-sigma K processing in the absence of other sporulation-specific gene products. The simplest explanation of these results is that SpoIVFB is a protease that processes pro-sigma K.     

Mutational analysis of the precursor-specific region of Bacillus subtilis sigma E.

sigma E is a sporulation-specific sigma factor of Bacillus subtilis that is formed from an inactive precursor protein (pro-sigma E) by the removal of 27 to 29 amino acids from the pro-sigma E amino terminus. By using oligonucleotide-directed mutagenesis, sequential deletions were constructed in the precursor-specific region of sigE and analyzed for their effect on the gene product's activity, ability to accumulate, and susceptibility to conversion into mature sigma E. The results demonstrated that the first 17 residues of the pro sequence contribute to silencing the sigma-like activity of pro-sigma E and that the amino acids between positions 12 and 17 are also important for its conversion into sigma E. Deletions that remove 21 or more codons from sigE reduce sigma E activity in cells which carry it, presumably by affecting pro-sigma E stability. A 26-codon deletion results in a gene whose product is not detectable in B. subtilis by either reporter gene activity or Western blot (immunoblot) assay. The primary structure as well as the size of the pro region of sigma E contributes to the protein's stability. The placement of additional amino acids into the pro region reduces the cell's ability to accumulate pro-sigma E. Additional sigE mutations revealed that the amino acids normally found at the putative processing site(s) of pro-sigma E are not essential to the processing reaction; however, a Glu residue upstream of these sites (position 25) was found to be important for processing. These last results suggest that the pro-sigma E processing apparatus does not recognize the actual site within pro-sigma E at which cleavage occurs but rater sequence elements that are upstream of this site.     

Perturbations to engulfment trigger a degradative response that prevents cell-cell signalling during sporulation in Bacillus subtilis

During sporulation in Bacillus subtilis, the mother cell membranes migrate around the forespore in a phagocytic-like process called engulfment. Developmental gene expression requires the successful completion of this key morphological event. Here we show that perturbations to engulfment block the accumulation of proteins secreted into the space between the mother cell and forespore membranes. Our data support a model in which engulfment defects cause the proteolytic clearance of these secreted proteins. Importantly, we show that this degradative response is reversible; once proper engulfment is restored, secreted proteins again accumulate. In particular, we have found that the forespore signalling protein SpoIVB fails to accumulate when engulfment is impaired and, as a result, late mother cell gene expression under the control of sigma(K) is blocked. If engulfment is restored, SpoIVB accumulates and cell-cell signalling resumes. Thus, this degradative pathway functions like a developmental checkpoint ensuring that mother cell gene expression does not commence unless morphogenesis proceeds normally.     

The Bacillus subtilis Signaling Protein SpoIVB Defines a New Family of Serine Peptidases

The protein SpoIVB plays a key role in signaling in the final sigma(K) checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-final sigma(K) to its active form, final sigma(K), in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the final sigma(K) checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily.