A forespore checkpoint for mother cell gene expression during development in B. subtilis

Gene expression in the mother cell compartment of sporulating cells of B. subtilis is partly governed by the mother cell RNA polymerase sigma factor sigma K. Paradoxically, sigma K-directed gene expression also depends on sigma G, the product of the forespore compartment regulatory gene spoIIIG, and on other forespore regulatory proteins. We now identify mutations in the genes bofA and bofB that relieve the dependence of mother cell gene expression on forespore regulatory proteins but not on sigma K. We establish that the dependence of mother cell gene expression on the forespore regulatory proteins is mediated at the level of the conversion of pro-sigma K to its mature, active form. We propose that the bofA and/or bofB proteins govern this conversion in response to a signal generated by the forespore. Activation of pro-sigma K could be a checkpoint for coordinating gene expression between the mother cell and forespore compartments of the developing sporangium.     

Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.

We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.     

A small protein required for the switch from {sigma}F to {sigma}G during sporulation in Bacillus subtilis

A cascade of alternative sigma factors governs the program of developmental gene expression during sporulation in Bacillus subtilis. Little is known, however, about how the early-acting sigma factors are inactivated and replaced by the later-acting factors. Here we identify a small protein, Fin (formerly known as YabK), that is required for efficient switching from σ(F)- to σ(G)-directed gene expression in the forespore compartment of the developing sporangium. The fin gene, which is conserved among Bacillus species and species of related genera, is transcribed in the forespore under the control of both σ(F) and σ(G). Cells mutant for fin are unable to fully deactivate σ(F) and, conversely, are unable to fully activate σ(G). Consistent with their deficiency in σ(G)-directed gene expression, fin cells are arrested in large numbers following the engulfment stage of sporulation, ultimately forming 50-fold fewer heat-resistant spores than the wild type. Based in part on the similarity of Fin to the anti-σ(G) factor CsfB (also called Gin), we speculate that Fin is an anti-σ(F) factor which, by disabling σ(F), promotes the switch to late developmental gene expression in the forespore.     

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.     

A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels

During spore formation in Bacillus subtilis, sigma(E)-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of sigma(G) in the forespore by a pathway of intercellular signalling that is composed of multiple proteins of unknown function. Here, we confirm that the vegetative protein SpoIIIJ, the forespore protein SpoIIQ and eight membrane proteins (SpoIIIAA through SpoIIIAH) produced in the mother cell under the control of sigma(E) are ordinarily required for intercellular signalling. In contrast, an anti-sigma(G) factor previously implicated in the pathway is shown to be dispensable. We also present evidence suggesting that SpoIIIJ is a membrane protein translocase that facilitates the insertion of SpoIIIAE into the membrane. In addition, we report the isolation of a mutation that partially bypasses the requirement for SpoIIIJ and for SpoIIIAA through SpoIIIAG, but not for SpoIIIAH or SpoIIQ, in the activation of sigma(G). We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of sigma(G). Finally, based on the similarity of SpoIIIAH to a component of type III secretion systems, we speculate that signalling is mediated by a channel that links the mother cell to the forespore.