Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate. During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum. We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE. We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

The products of the ftsA and ftsZ genes play a major role in septum formation in Escherichia coli. Their homologues have been found in various bacterial species, such as Bacillus subtilis where they are involved in septation during vegetative growth as well as during sporulation, a developmental process that is initiated by the formation of an asymmetrically positioned septum. Transcription of the B. subtilis ftsAZ operon was studied during exponential growth and sporulation by monitoring beta-galactosidase synthesis in strains harboring fusions of the E. coli lacZ gene with various fragments of the ftsAZ regulatory region. Transcription of the ftsAZ operon was found to be controlled by three promoters which were mapped by primer extension and characterized by their temporal pattern of expression. Two of these promoters, P1 and P3, are dependent on sigma A, the major vegetative sigma factor, and are expressed mainly during growth. The third one, P2, is recognized by sigma H associated RNA polymerase and its activity increases three- to four-fold around the onset of sporulation. The post-exponential enhancement of P2-driven transcription is abolished in a spo0A mutant but partially restored in an abrB spo0A double mutant. After inactivation by oligonucleotide-directed mutagenesis mutated copies of P1 and P2 were introduced into the chromosome upstream from the ftsAZ operon. Transformants could be obtained only when ftsAZ transcription was controlled by a combination of two intact promoters, neither P1, P2 nor P3 being essential for viability. The sporulation efficiency was found to be dependent on the level of transcription of ftsAZ, the absence of P2 still allowing 30% of the normal sporulation rate. Therefore the post-exponential burst of synthesis of the FtsA and FtsZ proteins is not an absolute requirement for the successful completion of the asymmetric septum.

The spo-279(ts) mutation, originally thought to be located in the spoIIG operon of Bacillus subtilis, has been mapped in close proximity but outside of the spoIIG locus. This mutation defines a new gene, spoIIN, located midway between the spoIIG and the spoVE loci, and whose product is required for successful completion of the asymmetric septation step. The spoIIN locus was cloned using a combination of 'walking steps' upstream from the spoIIG region and hybridization screening of a bacteriophage lambda library. Sequencing of DNA fragments able to rescue the spoIIN279(ts) mutation revealed that the spoIIN locus is identical with the B subtilis counterpart of the Escherichia coli ftsA gene. After cloning the ftsA region from a strain containing the spoIIN279(ts) mutation we found that this mutation converts the ninth residue of the FtsA protein from serine to asparagine. The spoIIN279(ts) mutation, which is recessive, leads to filamentation during growth at 42 degrees C and causes defective formation of the sporulation septum at this non-permissive temperature. The FtsA protein is therefore required for proper cell septation, both during vegetative growth and sporulation. Possible additional roles of FtsA during sporulation are discussed.

During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.

Sporulation of Bacillus subtilis is a primitive example of coupling between morphological changes and timing of gene expression during development. A major early control of transcriptional activity is dependent on a new sigma factor, sigma E, which is encoded by the sigE gene and synthesized as an inactive precursor, pro-sigma E. We show that mutations in the spoIIGA gene block the processing of pro-sigma E. Moreover, synthesis of both spoIIGA and sigE products in vegetative cells leads to expression of a sigma E-controlled promoter during growth, suggesting that SpoIIGA has pro-sigma E processing activity. The SpoIIGA polypeptide, which contains five potential transmembrane domains, is synthesized during sporulation 1 hr before processing activity can be detected. We propose that SpoIIGA processing activity is triggered by the presence of the sporulation septum, which is itself dependent on the spoIIAA and spoIIE products. These proteins are normally needed for pro-sigma E processing during sporulation but can be bypassed in vegetative cells. According to this model, a morphological structure would directly control the synthesis of a developmental sigma factor and would modify gene expression.

Summary: The prototrophic clones formed after fusion of mixed protoplasts from two polyauxotrophic strains of Bacillus subtilis have been counted both as L-form colonies and as bacterial colonies, by plating on selective medium in the presence and in the absence of methicillin. On average, one hundred times as many prototrophic colonies were counted when cell wall regeneration was prevented by the antibiotic. Thus genetic inactivation, which occurs regularly in bacterial exfusants, may be dependent on cell wall regeneration.

During the sporulating phase of Bacillus subtilis an extracellular serylprotease (SPE) and an esterase are excreted. The two enzymes follow the same kinetics of appearance in the culture medium and have some properties in common, in particular both enzymes hydrolyze benzoyltyrosine ethylester and are inhibited by phenylmethylsulfonyl fluoride. This raised the question of a possible structural relationship between the two proteins. A specific antiserum against the SPE was prepared. Using a radioimmunological assay, no reaction with the esterase could be detected. Asporogenous mutants (SpoOA and SpoOB) imparied in the production of both enzymes were analysed. Immunological assays with SPE-specific antiserum supported the hypothesis that the regulation of the biosynthesis of both enzymes would be affected by SpoOA and SpoOB mutations.