Spore formation by Bacillus subtilis takes place in a sporangium consisting of two chambers, the forespore and the mother cell, which are linked by pathways of intercellular communication. One pathway, which couples the activation of the forespore transcription factor s G to the action of s E in the mother cell, has remained mysterious. Traditional models hold that s E initiates a signal transduction pathway that specifically activates s G in the forespore. Recent experiments indicating that the mother cell and forespore are joined by a channel have led to the suggestion that a specific regulator of s G is transported from the mother cell into the forespore. As we report here, however, the requirement for the channel is not limited to s G . Rather, it is also required for the persistent activity of the early-acting forespore transcription factor s F as well as that of a heterologous RNA polymerase (that of phage T7). We infer that macromolecular synthesis in the forespore becomes dependent on the channel at intermediate stages of development. We propose that the channel is a gap junction-like feeding tube through which the mother cell nurtures the developing spore by providing small molecules needed for biosynthetic activity, including s G -directed gene activation. 2005). Examples of direct intercellular transport of molecules can also be found for bacteria. In these cases, a specialized apparatus, the type III or type IV secretion channel, transfers specific DNA and/or protein molecules from a donor bacterium to the cytoplasm of a recipient bacterium or a eukaryotic host cell (Christie et al. 2005;Galan and Wolf-Watz 2006). However, no examples of contiguous cytoplasmic bridges analogous to gap junctions or plasmodesmata have been described in bacteria.Here we provide evidence for a gap-junction-like ''feeding tube'' that links the developing spore to its mother cell during differentiation in the bacterium Bacillus subtilis. Spore formation takes place in a two-chamber sporangium that consists of a forespore, which will become the spore, and a mother cell (Fig. 1A). Initially, the forespore and the mother cell, which arise by asymmetric division, lie side by side. Next, at intermediate stages of spore formation, the forespore is engulfed by the mother cell to create a free protoplast within the mother cell cytoplasm.

SummaryDuring spore formation in Bacillus subtilis, s E -directed gene expression in the mother-cell compartment of the sporangium triggers the activation of s 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 s E are ordinarily required for intercellular signalling. In contrast, an anti-s 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 s G . We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of s 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.

Using an oligonucleotide microarray, we searched for previously unrecognized transcription units in intergenic regions in the genome of Bacillus subtilis, with an emphasis on identifying small genes activated during spore formation. Nineteen transcription units were identified, 11 of which were shown to depend on one or more sporulation-regulatory proteins for their expression. A high proportion of the transcription units contained small, functional open reading frames (ORFs). One such newly identified ORF is a member of a family of six structurally similar genes that are transcribed under the control of sporulation transcription factor E or K . A multiple mutant lacking all six genes was found to sporulate with slightly higher efficiency than the wild type, suggesting that under standard laboratory conditions the expression of these genes imposes a small cost on the production of heat-resistant spores. Finally, three of the transcription units specified small, noncoding RNAs; one of these was under the control of the sporulation transcription factor E , and another was under the control of the motility sigma factor D .

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Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by singleparticle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure. secretion system | SpoIIIAG | ring-building motif | sporulation