Spore control (Sco) mutations in Bacillus subtilis

Summary Spore control (Sco) mutants were isolated after nitrosoguanidine-induced mutagenesis of germinated spores. They were recognized as colonies showing high proteolytic activity on protein-agar (generally elastin-agar) test plates. Fourteen such mutants were isolated. The Sco mutations were transferred by transformation into an isogenic collection of genetically marked strains. Most of them appeared to be single mutations. Transduction experiments permitted the localisation of six Sco mutants in three loci, all in the argC-metC region. ScoA is located between argC and metC, ScoB is to the right of metC and ScoC is to the left of argC. ScoC and the previously described catA mutation are probably placed in the same gene.Two ScoC strains also appear to carry a second mutation, ScoD, probably localised in the same locus as ScoB or in a locus close to it. Eight other Sco mutations, apparently unlinked to the argC-metC region, were not localised. The results indicate complex regulation of sporulation-associated products such as the proteases, dependent on several genes.     

Spore control (Sco) mutations in Bacillus subtilis

Summary Morphogenesis of spore control (Sco) mutants of Bacillus subtilis was followed by quantitative electron microscopy. In wild type cultures the morphological stages II, III, IV and V attain their peaks of frequency at 1.5, 3.5, 4.5 and 5 h after t ₀, respectively. Stages II and IV are short, stages III and V occupy the main part of the process. Morphogenesis is slowed down in Sco A1, Sco B2 and Sco12 strains: all the stages are delayed and prolonged. Stage III cells are predominant for a long time, until t ₉ or later. It is suggested that the mechanism which switches off sporulation genes is affected and this leads to overproduction of sporulation-associated products. In other Sco mutants, such as Sco C3, part of the population sporulates normally but a fraction of cells persists for a long time at stages III and V. The pleiotropy of the spore control mutations is discussed.     

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.     

Biochemical genetics of bacterial sporulation. IV. Sequential development of resistances to chemical and physical agents during sporulation of Bacillus subtilis

Summary Resistances to various chemical agents appear sequentially during the sporulation of B. subtilis, with the following order: xylene-toluene-benzene-octanol-butanol-methanol, ethanol, chloroform, acetone, dioxane-pyridine-TCA, phenol. Heat-resistance increases gradually: resistance to 80°C for 10 min appears simultaneously with that to TCA and phenol, but spore maturation, as detected by heating at 90°C for two hours, continues for another 120 minutes. Various solvents and temperatures can be used as specific markers for the later stages of sporulation. Such markers cover more than a third of the entire process. Both chemical and temperature resistance markers are useful tools in the study of late sporulation events in wild type and in sporulation mutants.     

Biochemical genetics of bacterial sporulation

Summary The morphological and biochemical characters of twenty nine sporulation mutants were compared. Some of the predictions made on the basis of unidirectional pleïotropic interactions were confirmed, namely that the latest proteolytic enzymes, like elastase, are related to late morphological stages. From the cytological point of view, mutants blocked at various stages were described. Among the late mutants, both coatless mutants with normal but incomplete cortex and cortexless mutants with flexible spore coats were found. Particularly interesting is the class of abnormal late sporulation mutants which form normal mature heat-resistant spores at high frequencies, but, in addition, present various anomalies in the structure of the spore coats and various sporangial inclusions such as a spongy fibrous material, resembling the cortex, and either onion-like or rod-shaped inclusions, probably formed by spore coat components. The presence of these structures is related to the derepression of elastase activity and may reflect overproduction of spore components. Several mutants also contain abnormal, large, dark, membrane-bound mesosomes, either compact or loose, whose presence is related to the lack of oxidation of tetrazolium dyes. The morphological heterogeneity of mutant populations is also noted. These findings are discussed in relation to the theory of sequential gene activation.     

A Bacillus subtilis mutant requiring dipicolinic acid for the development of heat-resistant spores.

A Bacillus subtilis mutant is described which forms heat-resistant spores only in the presence of external dipicolinic acid (DPA). The mutation, dpa-1, is localized in a new sporulation locus, linked to pyrA. The dpa-1 strain is unable to synthesize DPA but can incorporate external DPA. The amount of DPA incorporated, the frequency of heat-resistant spores and their degree of resistance are all dependent on the concentration of external DPA. Spores of dpa- 1 strains exhibit normal resistance to most chemicals, including octanol and chloroform, but not to ethanol, pyridine, phenol and trichloroacetic acid. Complete resistance to the latter group depends on DPA. DPA incorporation is slow and apparently requires an energy supply but not protein synthesis. Direct involvement of DPA in the heat-resistance of the spores is suggested. Thin sections of DPA-less spores exhibit clearly visible cytoplasmic membranes and ribosomes. These structures are absent or less visible in the core of spores obtained with added DPA.