Identification and characterization of the Bacillus subtilis spoIIP locus

Abstract

We have identified an additional sporulation gene, named spoIIP, in the region of the Bacillus subtilis chromosome located immediately downstream of the gpr gene (227؇ on the genetic map). A null mutation of spoIIP arrests sporulation at an early stage of engulfment (stage II ii ), a phenotype similar to that already described for spoIID and spoIIM mutants. This gene encodes a 401-residue polypeptide, which is predicted to be anchored in the membrane, most of the protein being localized outside the cytoplasm. The spoIIP gene is transcribed from a promoter located in the interval between the gpr and the spoIIP reading frames. This promoter has the structural and genetic characteristics of a E -dependent promoter. Transcription of spoIIP is abolished by a mutation in spoIIGB, the gene encoding E , and can be induced during exponential growth in cells engineered to produce an active form of E . Plasmid integration-excision experiments leading to the formation of genetic mosaics during sporulation indicate that as with SpoIID and SpoIIM, SpoIIP is required only in the mother cell. Disruption of spoIIP had little or no effect on the expression of F -and E -controlled regulons but inhibited transcription from G -dependent promoters and abolished transcription from promoters under the control of K . We propose that, together with SpoIID and SpoIIM, the SpoIIP protein is involved in the dissolution of the peptidoglycan located in the sporulation septum.Sporulation in Bacillus subtilis involves the production of two cell types, the forespore and the mother cell. These two cells are generated by the formation of a septum in an asymmetric position, followed by engulfment of the smaller forespore by the mother cell (7). Differential transcription occurs in the two cells shortly after septation because of the selective activation of F in the forespore and E in the mother cell (37). Mutations in spoIIAC (the gene encoding F ) and in spoIIGB (the gene encoding E ) completely block engulfment (12). Mutations in spoIID and spoIIM, two genes under the control of E , allow partial degradation of the cell wall in the central part of the septum but prevent further progression of the engulfing membrane (3, 30). Some missense mutations in spoIIAC lead to a more advanced phenotype, although engulfment is not complete, which suggests the existence of some gene(s) under the control of F required in the late stages of the engulfment process (12). Only three genes have been identified as belonging to the F regulon, spoIIIG (38), gpr (41), and dacF (28), and only mutations in spoIIIG (encoding G , the late-acting forespore sigma factor) block sporulation (15). The dacF gene (which encodes a penicillin-binding protein) is located immediately upstream of the spoIIA operon (45), and transcription from the dacF promoter reads into the spoIIA locus (28). The gpr gene (which encodes a protease degrading some spore proteins at germination) does not appear to be followed by a rho-independent transcription termination signal (41). As part of our eff...

Identification of sporulation genes by genome-wide analysis of the σ E regulon of Bacillus subtilis

“…Candidates were prioritized on the basis of two criteria: the presence of predicted membrane-spanning domains in the protein, or recognition of the promoter by s E663 . The products of several known sporulation genes dependent on s E (spoIIB, spoIID, spoIIM, spoIIP) are involved in prespore engulfment and are membrane proteins (Abanes-De Mello et al, 2002;Frandsen & Stragier, 1995;Perez et al, 2000;Smith et al, 1993). It seems likely that other s E -dependent genes involved in engulfment would have domains capable of inserting into the membrane.…”

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…Given that E -directed genes are required to cause septal wall autolysis during engulfment (1,37,57,85,228,232,246,271,272), it seemed plausible that similar activity might enlarge a breach in the septum resulting from loss of SpoIIIE. Indeed, E -directed transcription of spoIID and spoIIP is required to disrupt compartmentalization in spoIIIE class II mutants (110).…”

“…Members of the E regulon that are important for sporulation include spoIID, spoIIM, and spoIIP, which are required for engulfment and to prevent a second asymmetric division from occurring in the mother cell (1,37,57,85,228,232,246,271,272); spoIVA, cotE, and spoVID, which encode scaffold proteins for spore coat assembly (18,245,273,317,318); the spoIIIA operon, which is required for activation of the lateprespore specific sigma factor G (125, 146); sigK, the composite gene for the late mother cell-specific transcription factor K (155,280); and spoIVCA, the gene for the recombinase that generates sigK via a chromosomal rearrangement (155,235,256). In addition, E activates transcription of spoIIID, which encodes a regulator of some E -dependent genes (102).…”

Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis

“…s E plays a decisive role in spore morphogenesis. The spoIID, spoIIM and spoIIP genes are essential for the engulfment process, and also act to prevent a second polar division of the sporulating cell (Abanes-De Mello et al, 2002;Eichenberger et al, 2001;Frandsen & Stragier, 1995;Lopez-Diaz et al, 1986;Pogliano et al, 1999;Rong et al, 1986;. The initial stages in assembly of the spore coat require expression of the spoIVA, cotE, spoVID and safA genes, whose products are morphogenetic proteins which guide the assembly of the several coat structural components (Beall et al, 1993;Driks et al, 1994;Ozin et al, 2000;Takamatsu et al, 1999;Zheng et al, 1988), and several genes which encode spore coat components with no obvious morphogenetic functions are also under the control of s E (e.g.…”