Processing of a Membrane Protein Required for Cell-to-Cell Signaling during Endospore Formation in Bacillus subtilis

Abstract

Activation of the late prespore-specific RNA polymerase sigma factor G during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of G also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of G . We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of G . SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of G following engulfment completion.Two important issues in developmental biology are how adjacent cells communicate so that coordinated responses emerge and how these signal transduction complexes are assembled at appropriate, discrete sites within the cell. The process of spore differentiation by the gram-positive bacterium Bacillus subtilis provides several examples of cell-cell communication pathways that initiate in one cell and influence the behavior of an adjacent sister cell (11,15,39). Sporulation involves the cooperation between two cells formed at the onset of the process by asymmetric division of a sporangial cell. This polar division produces a polar prespore, the future spore, and an adjacent larger mother cell, a terminal cell line, which contributes to spore morphogenesis but that lyses at its completion (11,15,39). The programs of gene expression in the prespore and the mother cell are controlled by the successive activation of cell-type-specific RNA polymerase sigma factors.

Bacillus subtilis SpoIIIJ and YqjG Function in Membrane Protein Biogenesis

“…Both proteins are essential for activation of a forespore-specific transcription factor and could be crosslinked in vivo. However, the level of membrane-inserted SpoIIIAE was not affected in a SpoIIIJ deletion strain (37), possibly suggesting a function in a postinsertional stage of SpoIIIAE biogenesis. Our report now demonstrates that both SpoIIIJ and YqjG mediate membrane insertion of in vitrosynthesized E. coli and B. subtilis F o c and supports the universal hypothesis that Oxa1p-like proteins are required for membrane insertion and biogenesis of subunits of respiratory chain complexes and other energy-transducing complexes.…”

“…Recently, a role for SpoIIIJ in membrane protein insertion was suggested based solely on its sequence similarity to E. coli YidC, although no direct experimental evidence for such a function was provided (4). Another report suggested that SpoIIIJ could be involved in the maturation of SpoIIIAE (37). Both proteins are essential for activation of a forespore-specific transcription factor and could be crosslinked in vivo.…”

A Genomic Signature and the Identification of New Sporulation Genes

“…The isolation of the forespore from the external milieu upon engulfment completion is a hallmark of endosporulation. A novel type of transport system coded for by the E -controlled spoIIIA operon allows the mother cell to feed the forespore, maintaining forespore-specific gene expression, when its engulfment is finalized (66,(104)(105)(106)(107). A central part of the complex is formed by the SpoIIIAH protein and by SpoIIQ, a F -controlled, forespore-specific protein, which interact directly across the intermembrane space, forming a channel that links the cytoplasm of the two cells (104)(105)(106)137).…”

“…Samples (0.6 ml) of DSM cultures were collected during sporulation and resuspended in 0.2 ml of phosphatebuffered saline, and the membrane dye FM4-64 (Molecular Probes) was added at a final concentration of 10 g ml Ϫ1 for visualization of membranes. Microscopy was carried out as described previously (66).…”

“…The signature includes the genes for regulatory proteins Spo0A, E , G , and the ancillary transcription factors SpoIIID and SpoVT, the engulfment genes spoIID, spoIIM, and spoIIP, the gene coding for the SpoIVA ATPase involved in assembly of the spore surface layers, and the spoVA operon, which is required for transport of dipicolonic acid, a compound characteristic of bacterial endospores, into the forespore (reviewed in references 19, 26, 22, 28, and 103). It also includes genes for proteins that function in cell-type-specific activation of the sporulation factors, including the anti-F / G factor SpoIIAB, the SpoIIE phosphatase involved in antagonizing SpoIIAB in the forespore, the F -controlled SpoIIR protein, which is secreted to the intermembrane space and required for the activation of pro-E in the mother cell (reviewed in reference 26), and the E -controlled spoIIIA operon, which codes for a transport system that connects the mother cell to the forespore (66,(104)(105)(106)(107).…”