Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

Degradation of ornithine transcarbamylase in sporulating Bacillus subtilis cells.

When Bacillus subtilis cells grew and sporulated on glucose-nutrient broth, ornithine transcarbamylase (OTCase) was synthesized in the early stationary phase and then inactivated. The loss of OTCase activity was much slower in a mutant that was deficient in a major intracellular serine protease (ISP). Immunochemical analysis showed that synthesis of OTCase decreased to a low, but detectable, level during its inactivation and that loss of activity was paralleled by loss of cross-reactive protein. Because the antibodies were capable of detecting denatured and fragmented forms of OTCase, we conclude that inactivation involved or was rapidly followed by degradation in vivo. Native OTCase was not degraded in crude extracts or when purified ISP and OTCase were incubated together under a variety of conditions. Synthesis of OTCase was not shut off normally in the ISP-deficient mutant. When the effects of continued synthesis were minimized, OTCase was degraded only slightly slower in the mutant than in its parent. Thus, the mutant had unanticipated pleiotropic characteristics, and it was unlikely that ISP played a major role in the degradation of OTCase in vivo.     

Characterization of an intracellular serine protease from sporulating cells of Bacillus brevis.

Sporulating cells of Bacillus brevis ATCC 9999 produced a high level of an intracellular serine protease when grown in nutrient medium. The protease activity in the crude extracts of this strain appeared at hour 5 (t5) after the end of exponential growth and increased gradually during sporulation, reaching a maximum at t12 to t13. The enzyme isolated in a partially purified state showed a pH optimum between 7.3 and 9.0 and had an apparent molecular weight of about 60,000. The activity was completely inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, EDTA, and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The protease possessed a high activity for azocoll and low activities for azocasein and 14C-labeled hemoglobin. It cleaved the cyclic decapeptide gramicidin S specifically at the peptide linkage between valine and ornithine and hydrolyzed the oxidized insulin B-chain mainly at peptide bonds 4-5 (Glu-His), 6-7 (Leu-CysSO3H), and 15-16 (Leu-Tyr). No catalysis of bond cleavage by the enzyme on a variety of small peptides or esters was detected. Unlike other Bacillus species, B. brevis ATCC 9999 grown in nutrient medium excreted no extracellular proteases.     

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.     

Sporulation in Bacillus subtilis 168. Comparison of alkaline phosphatase from sporulating and vegetative cells

1. The purification of the `vegetative' alkaline phosphatase of Bacillus subtilis 168 was simplified by ionic elution of the enzyme from intact cells. 2. The enzyme has a molecular weight of about 70000 and treatment of the enzyme with 10mm-hydrochloric acid or 6.0m-guanidine hydrochloride, β-mercaptoethanol (0.1m) gives rise to enzymically inactive subunits. 3. The amino acid composition of the enzyme was determined. The N-terminal residue determined by the DNS chloride method is glycine. 4. The properties of this enzyme were compared with the `sporulation' alkaline phosphatase of the same strain. 5. Although the `sporulation' enzyme differs from the `vegetative' enzyme in its physiology of appearance and apparent mRNA stability, an examination of properties of the enzymes revealed no differences. 6. The enzyme from both cell forms is bound to the particulate fraction of cell extracts, but can be solubilized by high concentrations of magnesium chloride; removal of the magnesium chloride, by dialysis, results in precipitation of both enzymes. Both enzymes can be removed from intact cells by ionic elution. 7. The `vegetative' and `sporulation' enzymes have identical pH optima, K_m and K_i values and electrophoretic mobilities in cellulose acetate. 8. Their half-life is 28min at 65°C and their Q₁₀ is 1.25. 9. The molecular size determined by gel filtration on Sephadex G-100 is about 69000. 10. `Vegetative' and `sporulation' forms gave precipitin lines that were continuous and non-spurred when tested against antiserum prepared against the `vegetative' enzyme. 11. The `sporulation' alkaline phosphatase appears to be associated with stage II of sporulation and appears to be induced by something specifically concerned in sporulation and not by phosphate starvation.     

Specific extraction of bacterial cardiolipin from sporulating Bacillus subtilis

A method for rapid purification of bacterial cardiolipin is presented. The cardiolipin level was first increased by suspending Bacillus subtilis cells in a buffer containing an uncoupling agent. At least 90% of the phosphatidylglycerol molecules were rapidly converted into cardiolipin. In sporulating strains, the accumulated cardiolipin appeared to be unextractable by conventional phospholipid extraction procedures. Sporulating bacteria were therefore treated first by a classical technique in order to eliminate lipids other than cardiolipin; a second extraction in a highly acidic medium then allowed us to quantitatively extract the remaining cardiolipin. Besides simplicity and rapidity, this method has the advantage of yielding cardiolipin in a nearly pure form from a relatively low number of bacteria.     

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.     

Purification and properties of RNA polymerases from mother cells and forespores of sporulating cells of Bacillus subtilis

Bacillus subtilis sporulating cells at stage III were fractionated into mother cell and forespore fractions by means of a lysozyme-detergent method. Three forms of DNA-dependent RNA polymerase enzymes, termed M sigma, F sigma, and F delta, in addition to core enzyme (alpha 2, beta', and beta) have been purified from the cell fractions. Enzymes M sigma and F sigma are present in the mother cell and forespore, respectively, and contain sigma factor of 55,000 daltons in addition to the core subunits. On the other hand, enzyme F delta is present specifically in the forespore and contains delta 1 factor of 28,000 daltons instead of the sigma factor. The amount of RNA polymerase in the forespore is about twice that in the mother cell. The enzymes M sigma and F sigma also differed in their elution profiled from DEAE-cellulose columns and in their heat stabilities indicating that the two sigma-containing holoenzyme forms may be different in their structural properties. The enzyme F delta transcribed B. subtilis DNA about 1.6 times more actively than enzyme F sigma, and the enzymes M sigma and F sigma transcribed the DNA about 2.2 times more actively than did core enzyme.     

Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis.

Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.     

A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis

The cis-acting sequences required for chromosome segregation are poorly understood in most organisms, including bacteria. Sporulating cells of Bacillus subtilis undergo an unusual asymmetric cell division during which the origin of DNA replication (oriC) region of the chromosome migrates to an extreme polar position. We have now characterized the sequences required for this migration. We show that the previously characterized soj-spo0J chromosome segregation system is not essential for chromosome movement to the cell pole, so this must be driven by an additional segregation mechanism. Observations on a large set of precisely engineered chromosomal inversions and translocations have identified a polar localization region (PLR), which lies approximately 150-300 kbp to the left of oriC. Surprisingly, oriC itself has no involvement in this chromosome segregation system. Dissection of the PLR showed that it has internal functional redundancy, reminiscent of the large diffuse centromeres of most eukaryotic cells.     

Thermostability of Bacillus subtilis neutral protease.

The thermostability of the B. subtilis neutral protease was studied under various conditions. At elevated temperatures the enzyme was inactivated as a result of autolysis. The rate of inactivation did not depend on the enzyme concentration and the enzyme was most stable near its pH optimum. The rate of inactivation was unaffected by the presence of a second protease during the incubation at high temperatures. The results indicate that the rate of thermal inactivation of the neutral protease is determined by the kinetics of local unfolding processes that precede autolysis rather than by the catalytic rate of the autodigestion reaction or an irreversible unfolding step.     

In vitro translation of messenger ribonucleic acid from sporulating and nonsporulating strains of Bacillus subtilis.

An Escherichia coli translation system supplemented with ribonucleic acid from sporulating Bacillus subtilis produces unique polypeptides which are missing among translation products of ribonucleic acid from six early sporulation mutants.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.