Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

Sporulation in Bacillus subtilis. Morphological changes

1. When Bacillus subtilis was grown in a medium in which sporulation occurred well-defined morphological changes were seen in thin sections of the cells. 2. Over a period of 7.5hr. beginning 2hr. after the initiation of sporulation the following major stages were observed: axial nuclear-filament formation, spore-septum formation, release of the fore-spore within the cell, development of the cortex around the fore-spore, the laying down of the spore coat and the completion of the corrugated spore coat before release of the spore from the mother cell. 3. The appearance of refractile bodies and 2,6-dipicolinic acid and the development of heat-resistance began between 5 and 6.5hr. after initiation of sporulation. 4. The appearance of 2,6-dipicolinic acid and the onset of refractility appeared to coincide with a diminution of electron density in the spore core and cortex. 5. Heat-resistance was associated with the terminal stage, the completion of the spore coat. 6. The spore coat was composed of an inner and an outer layer, each of which consisted of three or four electron-dense laminae. 7. Serial sections through cells at an early stage of sporulation showed that the membranes of each spore septum were always continuous with the membranes of a mesosome, which was itself in close contact with the bacterial or spore nucleoid. 8. These changes were correlated with biochemical events occurring during sporulation.     

Sporulation in Bacillus subtilis. The role of exoprotease

1. Intracellular turnover of protein was measured in wild-type Bacillus subtilis, which produces exoprotease at stage I in the sporulation process. Protein is degraded at a rate of 8–10%/hr. 2. As a result of this turnover, the proteins of the mother cell are extensively degraded and resynthesized by about 6hr., so that the later stages of spore formation occur in a cytoplasm containing mainly `new' protein. 3. The same protease appears to be responsible both for the intracellular turnover of protein and for extracellular proteolytic activity. In mutants that have lost the exoenzyme the intracellular protein is stable for many hours. In addition, these mutants fail to produce antibiotic and are asporogenous. When the exoprotease is regained as a result of back-mutation all the lost capacities of the cell are restored together. 4. Protease activity also accounts for the change in antigenic pattern of extracts of cells sampled during sporulation. Immunoelectrophoresis shows that, in the wild-type, the antigens characteristic of the vegetative cell have largely disappeared after a few hours; in the proteaseless mutants the vegetative-cell pattern is conserved. Apart from changing the protein pattern of the cell the protease could also have the function of removing protein inhibitors of sporulation. Other possible interpretations of the results are discussed.     

Sporulation of Bacillus subtilis in Continuous Culture

Sporulation of Bacillus subtilis 168 was studied in chemostat cultures. Sporulation occurred at high frequency under limitation of growth by glucose or the nitrogen source in minimal medium, whereas rates of sporulation were low for Mg(2+), phosphate, citrate, or tryptophan limitation. Sporulation was found at all growth rates tested, and the incidence of spores increased with decrease in growth rate of the culture. Within the range of growth rates up to the maximum obtainable with the defined medium, no threshold effect of growth rate on sporulation was observed. By studying transient states, it was possible to determine the time taken for the appearance of a refractile spore after initiation of a cell to sporulation. Under conditions of glucose limitation, cells were found to be committed to sporulation as soon as they were initiated. In nitrogen-limited cultures, however, a partial relief of nitrogen limitation prevented the development of spores during the first hour after initiation. The results of experiments with multistep changes in dilution rate of a chemostat culture indicate that initiation to sporulation is probably restricted to a particular point in the cell division cycle.     

Sporulation in Bacillus subtilis. Antigenic changes during spore formation

1. Antisera, prepared against extracts of cells and spores of Bacillus subtilis, were used in immunoelectrophoretic studies of the changes occurring in cell extracts during the course of spore formation. 2. At least 15 antigens could be detected in vegetative-cell extracts by the antiserum prepared against cell extracts and at least seven could be demonstrated in spore extracts by the homologous antiserum. 3. Cross-absorption studies showed that two of these antigens were probably completely specific for vegetative-cell extracts and that one was probably completely specific for spore extracts. The remainder were probably present in very small quantities in the heterologous extract. 4. In extracts of cells sporulating in an ;exhaustion medium' those antigens characteristic of the spore began to appear about 1hr. after the end of exponential growth. 5. In cells sporulating in a resuspension medium, spore antigens were detected at 4hr., and by 7hr. a decrease in vegetative-cell antigens was observed. 6. In an asporogenous mutant blocked early in sporulation there was neither an increase in spore antigens nor a decrease in vegetative-cell antigens. 7. In an asporogenous mutant blocked later in sporulation, there was an increase in spore antigens similar to that which occurred in the sporogenous strain.     

Ornithine Decarboxylase Activity during Sporulation of Bacillus subtilis

A simple method for overproduction of a target protein by genetic engineering techniques has been established. This method involves rearranging the target gene, which contains a ribosome binding sequence for expression, in plurally repeated form, and inserting it in a 3′ lower part of promoters.

The chloramphenicol acetyltransferase (CAT) structural gene was used to demonstrate the validity of this method. E. coli harboring a CAT expression plasmid, pUS(CAT)₁, which had one inserted CAT gene, was able to produce CAT at the level of only 4% of the total cellular protein according to densitometric scanning on Coomassie-blue-stained SDS-polyacrylamide gel and had the CAT activity of 3.9 × 10³ units/mg protein. However, E. coli harboring a CAT expression plasmid, pUS(CAT)₄, which had inserted four directly repeated copies of the CAT gene, could synthesize CAT up to 16% of the total cellular protein and had the CAT activity of 2.8 × 10⁴ units/mg protein. This suggests that this method should be useful for overproducing many important peptides or proteins in bacteria.     

Fine structure of Bacillus subtilis. I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO(4), OsO(4), or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO(4) in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO(4) fixation appeared to provide much better definition of the boundaries of various structures than did OsO(4). With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO(4) fixation) as two thin lines. In cells fixed first with OsO(4) solution, and then refixed with a mixture of KMnO(4) and OsO(4) solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO(4). One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.     

Sporulation of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A mutant of Bacillus subtilis 168 lacking aconitase (EC 4.2.1.3) was found to be blocked at stage 0 or I of sporulation. Although adenosine triphosphate levels, which normally decrease in tricarboxylic acid cycle mutants at the completion of exponential growth, could be maintained at higher levels by feeding metabolizable carbon sources, this did not permit the cells to progress further into the sporulation sequence. When post-exponential-phase cells of mutants blocked in the first half of the tricarboxylic acid cycle were resuspended with an energy source in culture fluid from post-exponential-phase wild-type B. subtilis or Escherichia coli, good sporulation occurred. The spores produced retained the mutant genotype and were heat stable but lost refractility and heat stability several hours after their production.     

Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potentials inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.     

Selectivity in Protein Degradation during Sporulation of Bacillus subtilis

The breakdown of cellular protein was investigated in Bacillus subtilis labeled with glycine-2-³H or L-phenylalanine-U-¹⁴C at different stages of vegetative growth and sporulation. In cells labeled with l-phenylalanine-U-¹⁴C, multiple protein turnover was observed. However, in cells labeled with glycine-2-³H, the patterns of protein turnover were quite different in the stages of growth and sporulation; proteins which were labeled at the early stationary phase were degraded rapidly, but those labeled at the late sporulation stage were hardly degraded. It was found that glycine incorporated into cells at the late sporulation stage was mainly utilized for biosynthesis of the spore coat protein. These data suggest that the spore coat protein which contains relatively large amounts of glycine is little subject to further degradation.     

Sporulation promoting factor in vegetative cells of Bacillus subtilis

A simple experimental system for detection of sporulation promoting factors was presented. This system showed that there was a sporulation promoting factor in the vegetative cells of Bacillus subtilis cultivated on nutrient agar for 9 hr (at stage T0). The factor was partially purified from the sonicate of vegetative cells by ethanol fractionation, gel filtration, chromatography and preparative gel electrophoresis, and it was identified as manganese-containing protein.     

Metabolism of Sulfur Compounds in Bacillus subtilis during Sporulation

Metabolism of various sulfur compounds in Bacillus subtilis during growth and sporulation was investigated by use of tracer techniques, in an attempt to clarify the mechanism involved in the formation of cystine rich protein of the spore coat.

Methionine, homocysteine, cystathionine, cysteine and some inorganic sulfur compounds (sulfate, sulfite and thiosulfate) were utilized by this organism as sulfur sources for its growth and sporulation. Biosynthesis of methionine from sulfate during growth was more or less inhibited by the addition of cysteine, homocysteine or cystathionine to the culture.

It is suggested from these results that in Bacillus subtilis methionine is synthesized from sulfate through cysteine, cystathionine and homocysteine as is the case in Salmonella or Neurospora. The results also suggest that the metabolism of sulfur-containing amino acids in Bacillus subtilis is strongly regulated by methionine and homocysteine.     

Sporulation in Bacillus subtilis is independent of membrane fatty acid composition.

Growth and sporulation of a Bacillus subtilis mutant deficient in branched fatty acid synthesis (gene symbol bfmB) were examined. The mutant, which produces an acyl-coenzyme A:acyl carrier protein transacylase with reduced affinity for branched fatty acid primers, could grow in media containing any one of a wide range of low-molecular-weight fatty acids having branched, cyclic, saturated, or unsaturated carbon chains. The fatty acid composition of cellular lipids depended on the compound used to support growth. Cultures of the bfmB mutant grown in the presence of 3-methylcrotonate contained an unusually high fraction (73%) of straight-chain fatty acids in the cellular lipids. The mutant sporulated with any one of the precursors of branched fatty acids in the medium; isolated spores contained mainly this branched fatty acid and only 10% or less straight-chain fatty acids regardless of the straight-chain fatty acid content of vegetative cells. Exceptional were spores grown in the presence of cyclobutane-carboxylic acid, which contained 28% straight-chain fatty acids. The branched fatty acid composition of spores could be modified greatly by changing the supply of precursors in the medium.     

Potassium Content During Growth and Sporulation in Bacillus subtilis

Vegetative and sporulating cells of Bacillus subtilis retain a higher level of internal potassium than do nonsporulating stationary-phase cells. The addition of manganese to nonsporulating stationary-phase cells, at concentrations required for sporulation, rapidly stimulates uptake and net accumulation of potassium and induces sporulation.