Isolation, characterization and biological activities of a toxin from Bacillus megaterium (B-23).

Fungitoxic substance was isolated from the culture filtrate of B. megaterium (B-23). Age of culture and pH of medium influence the fungitoxicity of its culture filtrate. Partially purified toxin was thermolabile, non-dialysable, ethyl acetate soluble, vanillin-sulphuric acid positive and effective within a range of pH 5-9. It exhibited maximum UV absorption at 224 nm. Its melting point was 242 degrees C. The efficacy of this compound was tested on 4 jute parasites namely, C. corchori, C. gloeosporioides, M. roridum and A. citri, of which M. roridum and C. corchori were least and most sensitive to the toxin respectively.

Role of outer coat in resistance of Bacillus megaterium spore.

The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed of galactosamine-6-phosphate (GalN-P) polymers with polypeptides and calcium. OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer of GalN-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experiments revealed that the outer coat limited the entry of compounds with a molecular weight of more than 2,000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of OC-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainly of GalN-P polymers, and the molecular sieving effect.

Characterization and deposition of the proteins in the outermost layer of Bacillus megaterium spore.

It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at t10 (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.

Immunological detection of 22K protein in sporulating cells of Bacillus megaterium ATCC 12872.

The synthesis and deposition of 22,000-dalton (22K) spore coat protein were examined immunochemically on the sporulating cells of Bacillus megaterium ATCC 12872 using the antibody to purified 22K spore coat protein. This antibody cross-reacted with 44K and 25K proteins in immunoblot analysis of dormant spore coat proteins. Immunoblot analysis on the sporulating cells showed that 22K protein was detected from t8 in forespore coat protein fractions. Sandwich enzyme immunoassay revealed that 22K protein in the spore coat protein fraction appeared at t6 and reached a plateau at t9, and 22K protein in the mother cell cytoplasmic fraction was detected at only t7 and t8 at a very low level.

Immunoelectron microscopic studies on spore coat proteins of Bacillus megaterium.

An immunochemical staining technique for the spore coat proteins of Bacillus megaterium ATCC 12872 was developed using colloidal gold as a second antibody. For reducing the non-specific immunogold binding and increasing the specific binding, the affinity-purified IgG was used as a first antibody. In sporulating cells at t10, gold particles were found not only in the spore coat but also in the mother cell cytoplasm, suggesting that some coat proteins were synthesized in the cytoplasm. Use of the specific affinity-purified antibody to 48K-protein demonstrated that this protein was one of the components of the outer coat.

Lipid composition of Bacillus megaterium spores and spore membranes.

Bacillus megaterium QM B1551 spore lipids were extracted by an improved technique, and the phospholipid and fatty acid compositions were determined. Phospholipids accounted for 65% of the total fatty acids; the neutral lipid fraction contained 15% and the remaining fatty acids were in the interphase, aqueous phase and pellet from the lipid extraction. Each phospholipid had similar fatty acid compositions as did the delipidated pellet. However, the aqueous phase and, to some extent, the interphase had unique fatty acid compositions. Also, fatty acids were found acylated to proteins, which was observed by electrophoresis of delipidated proteins from spores grown in [1-14C]palmitate. Therefore, spores contain unique non-phosphatide fatty acid components that can now be analyzed.

The major acid-soluble proteins of Bacillus subtilis spores: partial amino acid sequence and forespore location of their mRNAs.

In Bacillus subtilis the alpha, beta, gamma and delta components comprise 80-90% of the total acid-soluble spore proteins (ASSPs). Sequence analysis demonstrates that alpha and beta share 32 of their first 36 amino acids and are closely related to the A and C ASSPs of Bacillus megaterium spores, confirming the results of analysis of their cloned genes. Despite the difference in apparent size of gamma and delta, they have identical N-terminal sequences (37 residues). Unless gamma and delta derive from very recently duplicated genes, it appears that gamma is derived from delta, either in vivo or during isolation. Although the sequenced regions of gamma and delta have no homology to alpha and beta, outside of the previously recognized pentapeptide recognition sequence for the spore endopeptidase, they share 10 and 15 residue peptides flanking this sequence with ASSP B of B. megaterium, but in reverse order. At least two groups of ASSPs have, therefore, been conserved between B. subtilis and B. megaterium: the multigene AC alpha beta family and the B gamma (delta) group. Sequence conservation in each group implies selection for functions in addition to storage. Both the alpha and beta components of B. subtilis ASSPs and their mRNAs are located in the forespore compartment of cells at t5.5 of sporulation, the time of most rapid ASSP synthesis. The sizes of these transcripts (250-350 bp) and their ability to direct the in vitro synthesis of ASSPs of mature size, indicate that genes for these ASSPs are monocistronic, consistent with dispersed map location. Synthesis of ASSPs is, therefore, coordinately controlled by selective transcription in the forespore.

Isolation and characterization of forespores from Bacillus megaterium.

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.

Luciferin-luciferase assay of adenosine triphosphate from bacteria: a comparison of dimethylsulphoxide (DMSO) and acetone with other solvents.

The extraction of adenosine triphosphate (ATP) from six strains of bacteria, chosen for differences in cell-wall composition and habitat, was performed. The solvents were two in common use, Tris-ethylenediaminetetraacetate (Tris-EDTA) and trichloroacetic acid (TCA), and two promising, though less utilized solvents, dimethylsulphoxide (DMSO) and acetone. ATP was determined by the luciferin-luciferase reaction. Of the solvents used, DMSO and acetone were the most effective considering the different kinds of bacteria tested and, of these two, DMSO was the most convenient to use. Tris-EDTA was not as effective as the other solvents tested and TCA, which was effective with most strains, gave low yields when used with cultures grown in artificial seawater broth. Internal standards were used to determine if there were substances present that could inhibit the reaction of released ATP with the luciferin-luciferase reagent. Extracts of ATP, stored at -20 degrees C, were stable for up to 3 weeks.

Structural studies on N-acetylmannosaminuronic acid-containing and glucuronic acid-containing teichuronic acids in the cell wall of Bacillus megaterium AHU 1375.

Structural studies were carried out on two kinds of teichuronic acid-glycopeptide complexes (designated as TU-GP-I and TU-GP-II) isolated from lysozyme digest of N-acetylated cell walls of Bacillus megaterium AHU 1375 by ion-exchange chromatography and gel chromatography. TU-GP-I, accounting for about 25% of the cell walls, contained N-acetylmannosaminuronic acid, N-acetylglucosamine, glucose, galactose, glycerol, and phosphorus in an approximate molar ratio of 1:1:2:1:0.5:0.5, together with small amounts of glycopeptide components. TU-GP-II, accounting for about 9% of the cell walls, contained glucuronic acid, glucose, and fucose in a molar ratio of about 2:1.5:1, together with small amounts of glycopeptide components. The results of analyses involving Smith degradation, chromium oxidation, methylation, acetolysis, and H-NMR measurement led to the conclusion that the polysaccharide chain of TU-GP-I comprised repeating units,----6) Glc(alpha 1----3)-ManNAcUA(beta 1----4)[Gal(alpha 1----3)][Glc(beta 1----6)]GlcNAc(beta 1----. About half of the repeating units were substituted by glycerophosphoryl residues at C-6 of the beta-glucosyl residues linked to the N-acetylglucosamine residues. By means of a similar procedure, the polysaccharide chain of TU-GP-II was shown to comprise repeating units,----4)GlcUA(alpha 1----3)GlcUA(alpha 1----3)Glc(alpha 1----3)Fuc(alpha 1----, of which about half were substituted by alpha-glucosyl residues at C-3 of the 4-substituted glucuronosyl residues.

Growth of Bacillus megaterium in phosphate-limited medium.

Batch cultures of Bacillus megaterium grown in phosphate-limited media were compared with control cultures grown in phosphate-sufficient media. The effects of phosphate limitation on growth were determined by viable cells counts. Intracellular levels of protein, RNA, poly-3-hydroxybutyrate, carbohydrate and oxygen uptake were significantly affected by phosphate limitation. Electron micrographs of sectioned cells revealed differences in the structure; in particular the thick, rigid cell wall was absent from cells grown in phosphate-limited media, and such cells were larger, pleomorphic, and after 2 d were insensitive to lysozyme.

Isolation and characterization of two distinct fractions from the inner membrane of dormant Bacillus megaterium spores.

Two distinct membrane bands were obtained after sucrose velocity gradient centrifugation of crude inner membranes from dormant Bacillus megaterium spores disrupted under conditions which minimized endogenous enzyme action. These two inner membrane fractions (termed LD and HD) contained similar amounts of total and individual phospholipid species. However, LD and HD differed significantly in phospholipid/protein ratios (4.3 and 0.47 mg/mg, respectively), equilibrium densities (1.12 and 1.18 g/cm3), NADH oxidase specific activity (less than 0.01 and 0.13 mumol/min X mg), and content of specific proteins. In contrast, crude membranes prepared in identical fashion from germinated spores gave only a single inner membrane band (termed G) on sucrose velocity gradients. G had a phospholipid/protein ratio of 0.98 mg/mg, an equilibrium density of 1.16 g/cm3, and an NADH oxidase specific activity of 2.1 mumol/min X mg. Essentially all of the proteins present in LD or HD or both were found in G, consistent with the latter membrane being derived from a mixture of LD and HD. No evidence was found suggesting that there is significant degradation of dormant spore inner membrane protein upon spore germination.

Occurrence of galactosamine-6-phosphate as a constituent of Bacillus megaterium spore coat.

Galactosamine-6-phosphate was identified as a component of the coat of the Bacillus megaterium QM B1551 spore. It was one of the main constituents of the outermost layer of the spore coat, but it was absent from the other integuments including the cortex. These findings suggest that galactosamine-6-phosphate comprises the phosphorus-containing skeleton structure of the spore coat.

Bacteriocin from Bacillus megaterium ATCC 19213: comparative studies with megacin A-216.

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton alpha subunit and a 15,000-dalton beta subunit. Megacin A-19213 is composed of an alpha subunit 18,000 daltons in mass and a beta subunit about 7,500 daltons in mass. No sequence similarities between alpha and beta subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses.

Dielectric characterization of forespores isolated from Bacillus megaterium ATCC 19213.

Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium.

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.

Total and cell wall phosphorus content in Bacillus megaterium in phosphate-limited media.

A comparative study of the amount of total and cell wall phosphorus in Bacillus megaterium ATCC 33085, grown in media with or without phosphate limitation was carried out. The phosphorus levels were investigated during six successive subcultures. A progressive decrease in total phosphorus was found in cells cultivated in a phosphate-limited medium. A decline in the cell wall phosphorus level was observed starting only from the third subculture in phosphate-limited medium, and no phosphorus was detected in the walls of cells in the fifth subculture.