A new flavin enzyme catalyzing the reduction of dihydrodipicolinate in sporulating Bacillus subtilis I. Purification and properties.

A dihydrodipicolinate reductase containing flavin was purified from sporulating Bacillus subtilis PCI 219. The purified enzyme appeared homogeneous by dise gel electrophoresis. Its molecular weight was estimated as 74,000 by gel filtration on Sephadex G-200, and as 18,500 by electrophoresis on sodium dodecylsulfate polyacrylamid gel. These results suggest that the enzyme is composed of four subunits. The prosthetic group was identified as FMN, and one mole of the enzyme contained two moles of FMN. Both NADPH and NADH acted as coenzyme, though NADH was less effective. The enzyme also exhibited diaphorase activity. The pH optimum was 6.1. The enzyme was inhibited by dipicolinate but not by lysine or alpha, epsilon-diaminopimelate.     

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.     

Regulation of dihydrodipicolinate synthase and aspartate kinase in Bacillus subtilis.

The regulation of dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4) was studied in Bacillus subtilis 168. Starvation for lysine gave depression of one aspartate kinase isoenzyme but not of dihydrodipicolinate synthase. Strains resistant to growth inhibition by the lysine analogue thiosine exhibited constitutively derepressed synthesis of one aspartate kinase isoenzyme but had normal levels of dihydrodipicolinate synthase. The data provide strong evidence that lysine is not the signal for derepression of dihydrodipicolinate synthase. Nevertheless, dihydrodipicolinate synthase specific activity increased during sporulation, and it is suggested that this increase may result, in part, from resistance to proteolysis of that enzyme.     

Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase.

Dehydroquinate synthase, the enzyme which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) to 5-dehydroquinate, has been purified from Bacillus subtilis in association with chorismate synthase and NADPH-dependent flavin reductase. The enzyme was only active when associated with chorismate synthase, whereas the flavin reductase could be separated from the complex with retention of dehydroquinate synthase activity. The enzyme requires NAD and either Co2+ or Mn2+ for maximal activity. The activity was completely inhibited by EDTA. The Km of the enzyme for DAHP, NAD, and Co2+ were estimated to be 1.3 X 10(-4), 5.5 X 10(-5), and 5.5 X 10(-5) M, respectively. Enzyme activity was completely inhibited by NADH and the inhibition was not reversed by the addition of NAD, NADPH and NADP were not inhibitory. The enzyme was unstable to heat and lost all activity at 55 degrees C. A protein fraction which did not adsorb to phosphocellulose was found to inhibit the enzyme.     

Purification and characterization of NADPH-dependent flavin reductase. An enzyme required for the activation of chorismate synthase in Bacillus subtilis.

NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.     

Transcriptional control of synthesis of acid-soluble proteins in sporulating Bacillus subtilis

The major acid-soluble spore proteins (ASSPs) isolated from mature spores of Bacillus subtilis are designated alpha, beta, and gamma (about 60, 60, and 100 amino acids in length, respectively). Alpha and beta are very similar, and gamma is very similar to a less predominant ASSP called delta (about 115 amino acids). A minor and very basic ASSP called epsilon is the same size as alpha and beta but is unrelated antigenically. These and several minor ASSPs comprise at least three related families of sporulation-specific gene products. Expression of the alpha and beta genes, detectable as functional mRNA in vitro, coincides with the time of synthesis of all of the major ASSPs in vivo. This apparently coordinate expression is dependent on at least the spo0A, spoIIA, and spoIIIA loci, but not on the spoIVA or spoVA loci, consistent with the late stage of this expression (initiating at 3.5 h after the start of sporulation and peaking at 5 h after start of sporulation). A few minor ASSPs may be asynchronously expressed.     

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.     

Effects of antibiotics on synthesis and persistence of sigma E in sporulating Bacillus subtilis.

A potential regulatory link between the activation of a sporulation-specific sigma factor (sigma E) and forespore septum formation was investigated by treating Bacillus subtilis with inhibitors of protein or peptidoglycan synthesis and monitoring the consequences of these treatments on sigma E activation and septation. Western blot (immunoblot) and electron microscopic analyses revealed that both the formation of sigma E and septation were inhibited to a similar degree when either rifampin or chloramphenicol was added at different times before the second hour into sporulation but that penicillin preferentially blocked septation. We interpret these results as indicating that the syntheses of the gene products for both septation and sigma E activation occur at approximately the same time in development but that synthesis of an intact septum is unlikely to be a prerequisite for the formation of sigma E. We observed that penicillin could not only block septation but, depending on the time of its addition, could also inhibit both the activation of sigma E and the synthesis of its precursor. The basis of this effect is unknown, but it is not due to an overall disruption of protein synthesis. The incorporation of [35S] methionine by the sporulating cultures was unaffected by penicillin treatment. A time course study of the effects of rifampin and chloramphenicol treatments on sigma E levels revealed that both the synthesis of sigma E and its disappearance from sporulating cultures is inhibited by these antibiotics. This suggests that ongoing macromolecular synthesis is required for the turnover of sigma E.     

A procedure to remove protease activities from Bacillus subtilis sporulating cells and their crude extracts.

A simple procedure was developed to remove both extracellular and intracellular proteases associated with aporulating Bacillus subtilis cells. Cells are washed four times with 1 m KCl before breakage and their crude extracts are treated with 2 mm diisopropylfluorophosphate before passage through a hemoglobin-Sepharose affinity column. RNA polymerase in crude B. subtilis extracts treated by this procedure was stable, functionally and structurally, for more than 1 month at 4°C. This process for removing all proteases should work essentially with any crude extracts containing proteolytic activities.     

Processing of Bacillus subtilis succinate dehydrogenase and cytochrome b-558 polypeptides. Lack of covalently bound flavin in the Bacillus enzyme expressed in Escherichia coli

The DNA sequence of the Bacillus subtilis sdh operon coding for the two succinate dehydrogenase subunits and cytochrome b-558 (the membrane anchor protein) has recently been established. We have now determined the extent of N-terminal processing of each polypeptide by radiosequence analysis. At the same time, direct evidence for the correctness of the predicted reading frames has been obtained. The cytochrome showed a ragged N-terminus, with forms lacking one residue, and is inserted across the membrane without an N-terminal leader-peptide. Covalently bound flavin was not detectable in B. subtilis succinate dehydrogenase expressed in Escherichia coli despite normal N-terminal processing of the apoprotein. This provides an explanation to why the succinate dehydrogenase synthesized in E. coli is not functional and demonstrates that host-specific factors regulate the coenzyme attachment.     

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.     

A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.     

Genetic suppression analysis of sigma E interaction with three promoters in sporulating Bacillus subtilis.

Genetic evidence suggests that the sigma (sigma) subunit of RNA polymerase determines the specificity of promoter utilization, by making sequence-specific contacts with DNA. We examined the effects of two single amino acid(aa) substitutions in sigma E on the utilization of mutated derivatives of three different promoters in sporulating Bacillus subtilis. We found allele-specific suppression of mutations in all three promoters by each aa substitution in sigma E. These results provide strong evidence that sigma E interacts with each of these promoters in vivo. Moreover, the specificity of suppression of the mutations by the aa substitutions in sigma E lead us to speculate that the Met124 of sigma E closely contacts two adjacent bp in the -10 region of the promoters.