Characterization, purification and synthesis of spore-coat protein in Bacillus megaterium KM.

The spore-coat fraction from Bacillus megaterium KM, when prepared by extraction of lysozyme-digested integuments with SDS (sodium dodecyl sulphate) and urea, contains three N-terminal residues and a major component of apparent mol.wt. 17500. Electron microscopy of this fraction shows it to consist of an ordered multilamellar structure similar to that which forms the coat region of intact spores. The 17500-dalton protein, which has been purified to homogeneity, has an N-terminal methionine residue, has high contents of glycine, proline, cysteine and acidic amino acids and readily polymerized even in the presence of thiol-reducing agents. It is first synthesized between late Stage IV and early Stage V, which correlates with the morphological appearance of spore coat. Before Stage VI the 17500-dalton protein is extractable from sporangia by SDS in the absence of thiol-reducing reagents. Between Stage VI and release of mature spores the protein becomes resistant to extraction by SDS unless it is supplemented by a thiol-reducing reagent. In addition to that of the spore-coat protein, the timing of synthesis of all the integument proteins was analysed by SDS/polyacrylamide-gel electrophoresis and non-equilibrium pH-gradient electrophoresis. Several integument proteins are conservatively synthesized from as early as 1h after the end of exponential growth (t1), which may reflect protein incorporation into the spore outer membrane.

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.

Location of elements in ashed spores of Bacillus megaterium.

The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore. These reuslts suggest strongly that the core is the site of Ca associated with dipicolinic acid.

Phospholipid metabolism and membrane synthesis during sporulation in Bacillus megaterium.

In view of previously published reports of localized membrane growth in exponentially growing Bacillus megaterium and in sporulating Bacillus cereus, an attempt was made to describe phospholipid metabolism and the topology of membrane synthesis during sporulation in B. megaterium. The cells were pulsed with radioactive glycerol or acetate at the time of septum formation, and the specific activity of the lipid fraction was measured at various times through the free spore stage. The bulk of the material labeled during septation could not be recovered in the spore. Rather, it was found that the labeled lipid fraction underwent considerable turnover during spore development. Additionally, other experiments revealed that the lipid made before the initiation of sporulation was also subject to extensive turnover. In order to minimize both the confounding effects of lipid turnover and the possible presence of lateral diffusion of labeled lipid in the membrane, autoradiography of cells pulse labeled with radioactive glycerol at the time of septation was performed; a symmetrical grain distribution resulted. Thus, despite previously published suggestions to the contrary, the current experimental techniques could not demonstrate the existence of localized membrane synthesis in B. megaterium during sporulation.

Utilization of collagenous by-products from the meat packing industry: production of single-cell protein by the continuous cultivation of Bacillus megaterium.

The conditions for continuous cultivation of Bacillus megaterium on a collagen-derived substrate (SP-100) were determined. The optimum conditions of temperature, pH, and dilution rate were 34 C, pH 7.0, and 0.25/hr, respectively. Increasing the substrate concentration in plain tap water resulted in proportional increases in the productivity of cell mass from 0.6 g per liter per hr at 1% substrate to 1.8 g per liter per hr at 10% substrate; however, the protein content of the biomass decreased from 60 to 36%, and the protein yield decreased from 91 to 50% at substrate concentrations of 1 and 10%, respectively. These effects (decreases) were reversed up to 7.5% substrate by mineral supplementation of the medium. The productivity of biomass increased from 0.6 to 1.9 per liter per hr; the protein content of the biomass, from 43 to 54%; and the protein yield, from 60 to 93%, respectively, as the substrate concentration (with mineral supplementation of the medium) was increased from 1 to 7.5%. Spent medium could be refortified and recycled as often as five times. The amino acids in the substrate protein appeared to be utilized for growth and metabolism more or less uniformly. Analysis of the B. megaterium biomass indicated considerable enrichment of the essential amino acids and reduction of proline, glycine, and hydroxyproline as compared to the collagen-derived substrate. The Protein Efficiency Ratios obtained on the collagen-derived substrate (SP-100) and on the B. megaterium biomass, expressed as percentages of the casein reference protein, were 14 and 74%, respectively. Thus, considerable improvement in nutritional value was effected by bacterial conversion of the collagen-derived substrate into single-cell protein.

Chemical and physical characteristics of the deoxycholate-soluble and magnesium-reaggregated membrane nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.

The inactive components of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase present in the 0.4% deoxycholate-soluble fraction obtained from Bacillus megaterium KM membranes were reaggregated into active NADH oxidase by dilution in the presence of Mg(2+). The reaggregated oxidase was different from the original membrane with respect to sedimentation behavior in a sucrose gradient and morphological appearance. The deoxycholate-insoluble portion of the membrane had membrane-like structure whereas the reaggregated oxidase appeared to be a filamentous aggregate of small particles. The reaggregated oxidase and the deoxycholate-insoluble membrane residue were similar to the original membrane with respect to total protein and total lipid content. The inactive components of the NADH oxidase system exist in deoxycholate as two molecular species which were separable by sucrose density gradient centrifugation or gel filtration in deoxycholate-containing solutions. Both components and dilution in the presence of Mg(2+) were necessary for restoration of oxidase activity. The smaller-molecular-weight component contained all of the NADH-2,6-dichlorophenolindophenol oxidoreductase activity of the original membrane.

Isomers of glucosaminylphospatiylglycerol in Bacillus megaterium.

1. The lipids of Bacillus megaterium were extracted and three lipids containing glucosamine were identified. One of these is not a phospholipid, but the other two, which differ in their chromatographic behaviour, contain phosphorus, glycerol, fatty acid and d-glucosamine in the molar proportions 1:2:2:1. 2. In both phosphoglycolipids, the fatty acids are bound in ester linkage, and both yield 2,5-anhydromannose and 3-sn-phosphatidyl-1'-sn-glycerol on treatment with sodium nitrite. 3. Both phosphoglycolipids were N-acetylated and, after removal of fatty acids by mild alkaline hydrolysis, in both cases N-acetylglucosamine was quantitatively released by beta-N-acetylhexosaminidase. 4. The glucosaminylglycerols derived from the two phosphoglycolipids by partial acid hydrolysis differ in their behaviour towards periodate. In one case 1 mole of periodate is rapidly consumed/mole of glucosaminylglycerol, but in the other case under identical conditions the consumption of periodate is negligible. 5. The phosphoglycolipids were identified as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-3'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol and as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-2'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol. 6. Both phosphoglycolipids are good substrates for phospholipase A: neither is a substrate for phospholipase C from Clostridium perfringens, and only the 3'-glucosaminide is a substrate for phospholipase D.