Sporulation and Antibiotic Production by Bacillus subtilis Mutants Deficient in Intracellular Proteases

Erythropoietin (Ep) was isolated from the urine of patients with aplastic anemia [Yanagawa et al., J. Biol. Chem., 259, 2707 (1984)] and burst-promoting activity (BPA) was extensively purified from the residue obtained after removal of Ep. These erythropoietic factors were studied for their effcects on erythroid burst-colony formation of human peripheral blood mononuclear cells in methylcellulose cultures. Reddish bursts were formed with the addition of Ep alone. Addition of BPA not only elevated the number of bursts but also greatly reduced the amount of Ep required for burst formation. The presence of BPA alone in cultures did not permit bursts to form but did permit the growth of small colonies that did not contain hemoglobin (Hb). Addition of Ep to these small colonies led to the formation of erythroid bursts. Administration of Ep to the cultures could be delayed for 6 days without decreasing the number of bursts if the cultures were initiated in the presence of BPA; in the absence of BPA, the erythroid precursors (BUF-E) were rapidly lost if Ep was not provided at the start of the cultures. BPA produced larger bursts than those formed in the presence of Ep alone. Microassays of Hb in the bursts indicated that BPA increased the amonut of Hb per burst. This increase could not be entirely explained by the augumentation in cell number per burst but was partly ascribable to the increased amount of Hb per cell.     

Intracellular Proteases of Bacillus subtilis

The purpose of this study was to clarify the relationship between various cultivation conditions and the amounts of the rutin (RT) and protodioscin (PD) in asparagus spears. Green and white spears were grown in open culture and under two different blanching conditions. Although RT was detected only in the green spears, PD was detected mainly in white spears produced by covering with soil. The RT and PD contents of cladophylls grown in an open field and in a closed cultivation system were also investigated, and the closed system resulted in cladophylls with low RT and high PD, unlike the open field.     

Intracellular Proteases of Bacillus stearothermophilus

Cell-free extracts of Bacillus stearothermophilus have been shown to exhibit proteolytic activity toward casein as well as specific activity to catalyze the hydrolysis of furylacryloylglycyl-l-leucine amide, furylacryloylglycine, and carbobenzoxyl-glycine-p-nitrophenyl ester, indicating the presence of a neutral proteinase, a carboxypeptidase-like enzyme, and an alkaline proteinase. The neutral proteinase and carboxypeptidase-like activities were separated by gel filtration over Bio-Gel P-60, and both were reversibly inhibited by 1, 10-phenanthroline. The esterase activity was inhibited by diisopropylfluorophosphate, which did not affect other enzymatic activities and was insensitive to 1, 10-phenanthroline and ethylenediaminetetra-acetic acid.     

Statistical Estimate of the Total Number of Operons Specific for Bacillus subtilis Sporulation

As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23 known operons. From the proportion that do so (10/16), it is calculated that the most probable number of sporulation operons is 37 (68% confidence limits of 31 and 46). If allowance is made for the fact that two of the operons apparently contain mutagenic "hot spots" and the calculation is amended accordingly, the most probable numbers of operons becomes 42 (limits 33 and 59).     

Acrylamide Gel Electrophoresis of Intracellular Proteins During Early Stages of Sporulation in Bacillus subtilis

Acrylamide gel electrophoresis of unfractionated cellular extracts of Bacillus subtilis is shown to be an effective method for characterizing many of the changes in protein composition, when coupled with specific histological-type staining reactions. The results obtained here by using extracts from cells at different stages of growth and sporulation are consistent with observations from other laboratories where extensively purified and highly characterized enzymes have been studied. In several instances, the histochemical reactions can be associated with a specific enzymatic function and appear to indicate the presence of multiple molecular forms. In other instances, the data cannot be evaluated in terms of known enzyme function because the specificity of the histochemical analysis is not certain. However, the assays described permit monitoring of electrophoretic changes at the level of individual proteins within sporulating cultures. The results suggest that B. subtilis may contain two "hexokinase-like" enzymes which cease to function before sporulation is initiated. Aldolase and alanine dehydrogenase are detectable as single bands of enzyme activity during vegetative growth but as multiple molecular forms once sporulation has been initiated. Reduced nicotinamide adenine dinucleotide dehydrogenase activity is represented by an entire family of reactive species in these crude extracts, which undergo multiple changes during the early stages of sporulation. Tricarboxylic acid cycle dehydrogenase enzymes and those bands having esterase activity on alpha-naphthyl acetate show detectable changes in specific activity after cessation of exponential growth. Glucose dehydrogenase is not detectable until the sequence of changes leading to spore formation has progressed for 4 or 5 hr.     

Interaction of Proteinaceous Protease Inhibitor of Bacillus subtilis with Intracellular Proteases from the Same Strain

Intracellular protease inhibitor of B. subtilis IFO 3027 inhibits cytoplasmic serine proteases (IP-I, IP-II and IP-III), but it does not affect membrane-bound serine protease from the same strain. Inhibitory equivalent and binding titration studies revealed that the inhibitor binds and inhibits one mol of IP-I (a major protease in the cells). The inhibitor constant, Ki, against IP-I is estimated to be approximately 10^–9 m. The inhibitor binds and inhibits IP-II and IP-III in the same manner as IP-I, but its association with these enzymes is strongly repressed by the substrate (casein). Incubation of the inhibitor with membrane-bound protease results in the loss of the inhibitory activity.     

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. 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.     

Intracellular Peptidase of Bacillus subtilis

An electrophoretically pure preparation of aminopeptidase was isolated from the cells of a strain of Bacillus subtilis which secreted saccharifying α-amylase. The purified peptidase was active only in the presence of manganese and cobaltous ions. Both the metallic ions were effective to a similar degree for the enzyme on most of the peptides used as the substrate. However, for hydrolysis of leucine amide by the enzyme was effective only cobaltous ion and, in this case, manganese ion showed to act as a competitive inhibitor. On the other hand, for hydrolysis of certain other peptides such as glycyl proline, quite the opposite relationship was observed between those metallic ions. In the present paper is also described a new micro-assay method of amidase activity shown by the peptidase.     

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.     

Intracellular proteinase of Bacillus subtilis.

An intracellular serine proteinase was isolated from Bacillus subtilis, strain A-50. The molecular weight of the enzyme is 30000 +/- 1000, its amino acid composition is enriched in glutamic acid residues, the isoelectric point is 4.3, the N-terminal sequence Glu-Leu-Pro-Glu-Gly-Ile-Gln-Val-Ile-Lys-Ala-Pro-Glu-Leu-Xxx-Ala-Gln-Gly-Phe-Lys Gly-Ser-Asx-Ile-Lys-Ile-Ala-Val-Leu-Asx. The enzyme is structuraly homologous with secretory subtilisins.     

Protease Activities During the Course of Sporulation in Bacillus subtilis

Three proteolytic enzymes have been isolated from sporulating cultures of Bacillus subtilis. These activities were, respectively, a protease inhibited by ethylenediaminetetraacetic acid (EDTA) but not phenylmethylsulfonyl fluoride (PMSF), a protease active on both protein and ester substrates, and an ester-active enzyme with low activity on proteins. The latter two enzymes were inhibited by PMSF but not by EDTA. The specific activity of each was determined both intra- and extra-cellularly during growth and sporulation in a single-defined medium. All three enzymes were shown to exhibit a rapid increase in specific activity at a time coinciding with the appearance of refractile bodies in cells.     

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 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.     

In Vivo Effects of Sporulation Kinases on Mutant Spo0A Proteins in Bacillus subtilis

The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.     

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.     

Mutational analysis of the ribC gene of Bacillus subtilis

The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

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.