Forespore signaling is necessary for pro-sigmaK processing during Bacillus subtilis sporulation despite the loss of SpoIVFA upon translational arrest

The sigmaK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-sigmaK, and the resulting sigmaK RNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-sigmaK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-sigmaK processing began during sporulation. In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-sigmaK was observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent of spoIVB. The inability to process pro-sigmaK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-sigmaK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-sigmaK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.     

SpoIVB-mediated cleavage of SpoIVFA could provide the intercellular signal to activate processing of Pro-sigmaK in Bacillus subtilis

SpoIVB is the critical determinant for intercompartmental signalling of pro-sigmaK processing during sporulation in Bacillus subtilis. We show here that the SpoIVB serine peptidase can cleave the SpoIVFA protein, which is one component of the pro-sigmaK processing complex. SpoIVFA has been shown elsewhere (Rudner, D.Z., and Losick, R., 2002, Genes Dev 16: 1007-1018) to tether BofA and SpoIVFB in a membrane-embedded heteroligomeric complex in which BofA directly inhibits the activity of SpoIVFB. Cleavage of SpoIVFA would provide the necessary signal to dissolve this complex and release BofA-mediated inhibition on the zinc metalloprotease, SpoIVFB, that is responsible for cleaving pro-sigmaK to its mature form. We also show that the SpoIVB PDZ domain is required for self-recognition and trans cleavage of SpoIVB and is probably also used to target an internal motif within the C-terminal region of SpoIVFA exposed in the space between the inner and outer forespore membranes. This work reveals the mechanism of intercompartmental signalling and provides a unified model as to how sigmaK-directed gene expression in the mother cell is co-ordinated with events in the forespore chamber.     

Serine proteases from two cell types target different components of a complex that governs regulated intramembrane proteolysis of pro-sigmaK during Bacillus subtilis development

Upon starvation Bacillus subtilis undergoes a developmental process involving creation of two cell types, the mother cell and forespore. A signal in the form of a serine protease, SpoIVB, is secreted from the forespore and leads to regulated intramembrane proteolysis (RIP) of pro-sigmaK, releasing active sigmaK into the mother cell. RIP of pro-sigmaK is carried out by a membrane-embedded metalloprotease, SpoIVFB, which is inactive when bound by BofA and SpoIVFA. We have investigated the mechanism by which this complex is activated. By expressing components of the signalling pathway in Escherichia coli, we reconstructed complete inhibition of pro-sigmaK RIP by BofA and SpoIVFA, and found that SpoIVB serine protease activity could partially restore RIP, apparently by targeting SpoIVFA. Pulse-chase experiments demonstrated that SpoIVFA synthesized early during B. subtilis sporulation is lost in a SpoIVB-dependent fashion, coincident with the onset of pro-sigmaK RIP, supporting the idea that SpoIVB targets SpoIVFA to trigger RIP of pro-sigmaK. Loss of BofA depended not only on SpoIVB, but also on CtpB, a serine protease secreted from the mother cell. CtpB appeared to cleave BofA near its C-terminus upon coexpression in E. coli, and purified CtpB degraded BofA. We propose that RIP of pro-sigmaK involves a three-step proteolytic cascade in which SpoIVB first cleaves SpoIVFA, CtpB then cleaves BofA and finally SpoIVFB cleaves pro-sigmaK.     

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.     

Substrate requirements for regulated intramembrane proteolysis of Bacillus subtilis pro-sigmaK

During sporulation of Bacillus subtilis, pro-sigmaK is activated by regulated intramembrane proteolysis (RIP) in response to a signal from the forespore. RIP of pro-sigmaK removes its prosequence (amino acids 1 to 20), releasing sigmaK from the outer forespore membrane into the mother cell cytoplasm, in a reaction catalyzed by SpoIVFB, a metalloprotease in the S2P family of intramembrane-cleaving proteases. The requirements for pro-sigmaK to serve as a substrate for RIP were investigated by producing C-terminally truncated pro-sigmaK fused at different points to the green fluorescent protein (GFP) or hexahistidine in sporulating B. subtilis or in Escherichia coli engineered to coexpress SpoIVFB. Nearly half of pro-sigmaK (amino acids 1 to 117), including part of sigma factor region 2.4, was required for RIP of pro-sigmaK-GFP chimeras in sporulating B. subtilis. Likewise, pro-sigmaK-hexahistidine chimeras demonstrated that the N-terminal 117 amino acids of pro-sigma(K) are sufficient for RIP, although the N-terminal 126 amino acids, which includes all of region 2.4, allowed much better accumulation of the chimeric protein in sporulating B. subtilis and more efficient processing by SpoIVFB in E. coli. In contrast to the requirements for RIP, a much smaller N-terminal segment (amino acids 1 to 27) was sufficient for membrane localization of a pro-sigmaK-GFP chimera. Addition or deletion of five amino acids near the N terminus allowed accurate processing of pro-sigmaK, ruling out a mechanism in which SpoIVFB measures the distance from the N terminus to the cleavage site. A charge reversal at position 13 (substituting glutamate for lysine) reduced accumulation of pro-sigmaK and prevented detectable RIP by SpoIVFB. These results elucidate substrate requirements for RIP of pro-sigmaK by SpoIVFB and may have implications for substrate recognition by other S2P family members.     

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.     

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.     

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.     

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.     

Proteolysis of SpolVB is a critical determinant in signalling of Pro-sigmaK processing in Bacillus subtilis

SpoIVB is essential for intercompartmental signalling in the sigma(K)-checkpoint of Bacillus subtilis. SpoIVB is synthesized in the spore chamber and is the signal which activates proteolytic processing of pro-sigma(K) to its mature and active form sigma(K). We show here that SpoIVB is a serine peptidase of the SA clan. Expression of SpoIVB in Escherichia coli has shown that SpoIVB is able to self-cleave into at least three discrete products, and in vitro studies have shown cleavage in trans. Autoproteolysis of SpoIVB is tightly linked to the initiation of the two developmental functions of this protein, signalling of pro-sigma(K) processing and a yet, uncharacterized, second function which is essential for the formation of heat-resistant spores. In B. subtilis, SpoIVB is synthesized as a zymogen and is subject to two levels of proteolysis. First, autoproteolysis generating intermediate products, at least one of which is proposed to be the active form, followed by processing by one or more enzymes to smaller species. This could provide a mechanism for switching off the active SpoIVB intermediate(s) and suggests a similarity to other proteolytic cascades such as those found in blood coagulation.     

Analysis of the signal for attachment of a glycophospholipid membrane anchor

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH-terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.     

E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells

Although it is generally recognized that cystic fibrosis transmembrane conductance regulator (CFTR) contains a PSD-95/Disc-large/ZO-1 (PDZ)-binding motif at its COOH terminus, the identity of the PDZ domain protein(s) that interact with CFTR is uncertain, and the functional impact of this interaction is not fully understood. By using human airway epithelial cells, we show that CFTR associates with Na(+)/H(+) exchanger (NHE) type 3 kinase A regulatory protein (E3KARP), an EBP50/NHE regulatory factor (NHERF)-related PDZ domain protein. The PDZ binding motif located at the COOH terminus of CFTR interacts preferentially with the second PDZ domain of E3KARP, with nanomolar affinity. In contrast to EBP50/NHERF, E3KARP is predominantly localized (>95%) in the membrane fractions of Calu-3 and T84 cells, where CFTR is located. Moreover, confocal immunofluorescence microscopy of polarized Calu-3 monolayers shows that E3KARP and CFTR are co-localized at the apical membrane domain. We also found that ezrin associates with E3KARP in vivo. Co-expression of CFTR with E3KARP and ezrin in Xenopus oocytes potentiated cAMP-stimulated CFTR Cl(-) currents. These results support the concept that E3KARP functions as a scaffold protein that links CFTR to ezrin. Since ezrin has been shown previously to function as a protein kinase A anchoring protein, we suggest that one function served by the interaction of E3KARP with both ezrin and CFTR is to localize protein kinase A in the vicinity of the R-domain of CFTR. Since ezrin is also an actin-binding protein, the formation of a CFTR.E3KARP.ezrin complex may be important also in stabilizing CFTR at the apical membrane domain of airway cells.     

GerM is required to assemble the basal platform of the SpoIIIA–SpoIIQ transenvelope complex during sporulation in Bacillus subtilis

Sporulating Bacillus subtilis cells assemble a multimeric membrane complex connecting the mother cell and developing spore that is required to maintain forespore differentiation. 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; however, the mechanism by which SpoIIQ specifically localizes to the septal membranes on the forespore side has remained enigmatic. Here, we identify GerM, a lipoprotein previously implicated in spore germination, as the missing factor required for SpoIIQ localization. Our data indicate that 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.     

Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10

We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis.