Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant.

A thermosensitive sporulation mutant was used to determine the order of sporulation operonsin the urs region of the Bacillus subtilis chromosome. Data from three-factor transformation crosses and three- and four-factor transduction crosses established the order metC-SPO-96(SpoII)-spo-85(SpoV)-spo-279(SpoII)-furA-ura-cysC-spo-NG1.67(SpoIII). Previously, furA was thought to lie to the right of ura and cysC to the left (Dubnau, 1970; Young and Wilson, 1972).     

Ultrastructural studies of sporulation in Streptomyces.

This is the first study of sporogenesis in Streptomyces carried out on a relatively high number of species (seven) which allows us, using also previously published results, to establish a general picture of this process. In the sporogenesis of Streptomyces two basic stages can be considered: the sporulation septum synthesis and the arthrospore maturation. Our ultrastructural study of the sporulation septum formation suggests the existance within this genus of three basic types. Type I is distinguished because the septum is formed from the beginning by two separate cross walls. Within this type we include Streptomyces erythraeus, Streptomyces albus, and Streptomyces aureofaciens and also include Streptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus. Type II is distinguished because there is a deposit of material previous to the synthesis of the double annulus which completes the septum. This type can be divided into two subtypes. In the first the deposits are wedge-shaped and the double annulus is clearly visible, and to this group belong Streptomyces flaveolus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second the deposits, which have a different shape and are very well developed, constitute almost entirely the sporulation septum in which the double annulus is barely visible; Streptomyces antibioticus and also Streptomyces viridochromogenes belong to this group. Type III, represented by Streptomyces cinnamonensis, is distinguished because the septum is formed by a single cross wall.     

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.     

Motility of Bacillus subtilis during growth and sporulation.

The change of motility and the presence of flagella were followed throughout growth and sporulation in a standard sporulating strain and in 19 cacogenic sporulation mutants of Bacillus subtilis. For the standard strain, the fraction of motile cells decreased during the developmental period to less than 10% at T4. Motility was lost well before the cells lose their flagella. Conditions reducing the decrease of motility also reduced sporulation: motile cells never contained spores. The decrease of motility was not coupled with a decrease in the cellular concentration of adenosine 5'-triphosphate or a decline in oxygen consumption, but an uncoupling agent immediately destroyed motility at any time. Apparently, motility decreased during development because it became increasingly uncoupled from the energy generating systems of the cell. The motility of sporulation mutants decreased after the end of growth at the same time as or earlier than the motility of the standard strain; the early decrease of motility in an aconitase mutant, but not that in an alpha-ketoglurate dehydrogenase mutant, could be avoided by addition of L-glutamate. Sporulation or related events such as extracellular antibiotic or protease production were not needed for the motility decline.     

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.     

Ultrastructural studies of sporulation in Bacillus sphaericus.

Spore septum formation in Bacillus sphaericus 9602 occurs 2 h after the end of exponential growth at one end of the vegetative cell, which retains a uniform diameter. The apparently rigid spore septum contains an inner cell wall layer which disappears when the sporulation septum "bulges" into the mother cell cytoplasm. This process occurs simultaneously with terminal swelling at the end of the cell containing the spore septum. It is suggested that the inner cell wall layer is peptidoglycan and that its dissolution and the terminal swelling are consequences of a localized autolysis. Engulfment of the forespore by membrane proliferation results in the production of a forespore surrounded by two flexible, closely apposed membranes. These membranes appear to become more rigid as a peptidoglycan-like layer appears between them, concomitant with the condensation of the forespore nucleoid into a crescent-shaped structure. After nuclear condensation, visible development of distinct cortex, primordial cell wall, and spore coat layers begin, and the forespore cytoplasm assumes an appearance similar to that of a refractile spore. The spore coats consist of an amorphous inner layer, a lamellar midlayer, and a structured outer layer. As cortex synthesis and spore coat assembly continue, exosporium development commences close to that portion of the mother cell plasma membrane which surrounds the forespore. The exosporium is lamellar and in tangential section is seen to have a hexagonal arrangement of subunits. The timing of these morphological events has the expected correlation with the appearance of unique enzyme activites required for cortex synthesis.     

Biochemical changes during sporulation of Bacillus stearothermophilus.

The process of sporulation was studied in Bacillus stearothermophilus. A medium is described that supports good growth and sporulation of the organism. In this medium, which contains glucose, salts, and amino acids, acetate starts to accumulate before any of the glucose is catabolized. Enzymes of the tricarboxylic acid cycle are present at all times during growth and sporulation and are found in dormant spores. As the glucose in the culture is consumed, acetate rapidly increases and the pH of the medium drops. The acetate rapidly disappears during sporulation and the pH rises. Dipicolinic acid appears during sporulation and several key-enzyme activities fluctuate in a characteristic pattern.     

Alternative medium for Clostridium perfringens sporulation.

A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains. Comparatively this medium gave a greater percentage of sporulation than five other media described previously.