The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

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.     

Acid-Soluble Nucleotides in an Asporogenous Mutant of Bacillus subtilis

An asporogenous mutant of Bacillus subtilis Sp(-)H12-3, which is considered to have a block at stage 0, showed general growth characteristics similar to those of sporulating cultures. However, a sudden increase in the total amount of acid-soluble nucleotides observed at t(2) in sporulating bacteria was completely absent in this mutant. In sporulating cells, a marked increase in two nucleotides which were identified to be uridine diphosphate (UDP)-galactose and UDP-N-acetylglucosamine was noted, whereas UDP-glucose appeared to be accumulated in the mutant cells at t(2). No unusual nucleotides were found in the strains of B. subtilis examined. The possible role of these UDP derivatives in early stages of sporulation in B. subtilis is discussed.     

Analysis of Isoaccepting Transfer Ribonucleic Acid Species of Bacillus subtilis: Changes in Chromatography of Transfer Ribonucleic Acids Associated with Stage of Development

Changes in chromatographic profiles of tyrosyl-, leucyl-, tryptophanyl-, and lysyl-transfer ribonucleic acids (tRNAs) are presented as a function of the growth stage in Bacillus subtilis. All of the tRNA groups investigated expressed different temporal patterns of change in isoaccepting species. Tyrosyl-tRNAs were the earliest to change and were followed by changes in leucyl- and then tryptophanyl-tRNAs. Lysyl-tRNAs were unique in having two times of change: one early and one very late. As an aid in understanding the temporal aspect of tRNA alterations during sporulation, the chromatographic profiles of aminoacyl tRNAs from an early blocked asporogenous mutant were studied. The asporogenous mutant used was blocked at the axial filament stage, stage 0 of sporulation. Nevertheless, those tRNAs which showed differences between the spore and cells in exponential growth exhibited similar changes in the asporogenous mutant after 24 h of growth. The data suggest that several tRNA changes occur during development in B. subtilis but that the events leading to these changes are either independent of, or occur before, stage 0 of sporulation, except in the case of lysyl-tRNA.     

Sporulation in Bacillus subtilis. The appearance of sulpholactic acid as a marker event for sporulation

1. The synthesis of sulpholactic acid in sporulating cultures of Bacillus subtilis was studied. 2. Sulpholactic acid was first detected about 4h after the initiation of sporulation and 1h before refractility. The rate of synthesis paralleled that of the other events of sporulation examined. 3. Sulpholactic acid accounted for 1.7% of the material of the spore. 4. Because the addition of chloramphenicol in the earlier stages of sporulation inhibited formation of the compound, it is likely that the enzymes concerned are synthesized de novo during sporulation. 5. In asporogenous mutants only those blocked at a late stage and showing partial refractility were able to produce sulpholactic acid. This correlation makes sulpholactic acid a useful marker event in sporulation.     

Characterization of aBacillus subtilis germination mutant with pleiotropic alterations in spore coat structure

One class of spore germination mutants ofBacillus subtilis produces lysozymesensitive spores with altered surface structure. These mutations were pleiotropic in that the pattern of soluble and insoluble spore coat proteins was extensively changed with the virtual absence of a major 12kd polypeptide. Reversion to the lysozyme-resistant phenotype (and wild-type spore coat profile) at or near the site of the original mutation occurred at a frequency consistent with an initial point mutation.The 12kd protein was also absent from extracts of sporulating cells of the mutant although antigens of 14kd and 32kd protein cross-reacting with antibody to the 12kd polypeptide were detected. The 32kd antigen was also present in extracts of sporulating cells but not in the extracts of the spore coat of the wild type and is probably a precursor. Improper processing of such a precursor could account for the extensive alterations of coat structure.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

Functional modifications of the translational system in Bacillus subtilis during sporulation.

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.     

Appearance of a ribonucleic acid polymerase-binding protein in asporogenous mutants of Bacillus subtilis

A 70,000-dalton protein that is found in sporulating Bacillus subtilis and that binds to ribonucleic acid polymerase is present in asporogenous mutants that proceed to or beyond stage II of sporulation, but is absent from mutants blocked at stage zero.     

Membrane fractions from the outer layers of spores of Bacillus thuringiensis with toxicity to lepidopterous larvae.

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.     

Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.     

Synthesis of a spore-specific surface antigen during sporulation of Saccharomyces cerevisiae

Antisera raised against purified yeast ascospores caused agglutination of both ascospores and vegetative cells. A spore-specific activity was obtained by absorbing out anti-vegetative activity with vegetative cells. The anti-vegetative cell activity was directed against mannan, and was probably due to exposure of some spore coat mannan at the spore surface since concanavalin A and lentil lectin also caused agglutination of ascospores. The spore-specific activity was probably determined by a protein or proteins, since extraction of spores with a mixture of sodium dodecyl sulphate and dithiothreitol markedly affected their agglutination by the spore-specific serum. The spore-specific antigen was synthesized in a soluble form during sporulation several hours before the appearance of the spore surface and the pool of soluble antigen declined as the spore was assembled. Synthesis of the soluble antigen was inhibited by adding cycloheximide at all times up to its first appearance in the sporulating cell.     

Purification and characterization of a new glucose dehydrogenase from vegetative cells of Bacillus megaterium

A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (M_r 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d-glucose and 2-deoxy-d-glucose), and the affinity for glucose (a K_m value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The K_m values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d-glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase.