Quantitative aspects of exchangeable calcium in spores of Bacillus megaterium

Rode, L. J. (The University of Texas, Austin), and J. W. Foster. Quantitative aspects of exchangeable calcium in spores of Bacillus megaterium. J. Bacteriol. 91:1589-1593. 1966.-More than 90% of the calcium in Ca(45)-labeled native spores was released from the cells during germination. Some 95% of the spore calcium was not exchangeable when ungerminated native spores were titrated to pH 4 with HNO(3). Ca, Mg, Na, Si, and Fe were displaced from the spores by H(+). The adsorption of Ca(45) by H-spores and its subsequent release were studied under a variety of conditions. The isolated "coat fraction" of spores adsorbed substantial amounts of Ca(45). Release of the adsorbed Ca was achieved with various reagents.     

Influence of temperature on the effectiveness of a biogenic carbonate surface treatment for limestone conservation

So far, most studies on microbiologically induced carbonate precipitation for limestone conservation have been performed at temperatures optimal for the activity of the calcinogenic bacteria (i.e., 20–28 °C). Successful application in practice, however, requires adequate performance in a wide range of environmental conditions. Therefore, the aim of this study was to select microorganisms that are most suited for biodeposition at temperatures relevant for practice. In a first step, ureolytic microorganisms were screened for their growth and ureolytic activity at different temperatures (10, 20, 28, and 37 °C). Large differences in calcinogenic activity could be observed between experiments performed on agar plates and those performed in solution and in limestone. In a second step, the influence of temperature on the performance of the biodeposition treatment with different ureolytic microorganisms was evaluated, both on the consolidative and protective effect of the treatment. In contrast with the experiments on agar plates, the Sporosarcina psychrophila strains failed to produce significant amounts of calcium carbonate on limestone in conditions relevant for practice, even at 10 °C. This resulted in a poor performance of the treatment. From experiments performed on limestone prisms, it appeared that the mesophilic Bacillus sphaericus produced the highest amount of carbonate in the shortest amount of time at all temperatures tested. As a result, compared to the untreated specimens, the highest consolidative (64 % lower weight loss upon sonication) and protective effect (46 % decreased sorptivity) were observed for the treatments with this species. From this study, it appears that among all ureolytic strains tested, B. sphaericus is most suited for biodeposition applications in practice.     

Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus

Foerster, Harold F. (The University of Texas, Austin), and J. W. Foster. Endotrophic calcium, strontium, and barium spores of Bacillus megaterium and Bacillus cereus. J. Bacteriol. 91:1333-1345. 1966.-Spores were produced by washed vegetative cells suspended in deionized water supplemented with CaCl(2), SrCl(2), or BaCl(2). Normal, refractile spores were produced in each case; a portion of the barium spores lost refractility and darkened. Thin-section electron micrographs revealed no apparent anatomical differences among the three types of spores. Analyses revealed that the different spore types were enriched specifically in the metal to which they were exposed during sporogenesis. The calcium content of the strontium and the barium spores was very small. From binary equimolar mixtures of the metal salts, endotrophic spores accumulated both metals to nearly the same extent. Viability of the barium spores was considerably less than that of the other two types. Strontium and barium spores were heat-resistant; however, calcium was essential for maximal heat resistance. Significant differences existed in the rates of germination; calcium spores germinated fastest, strontium spores were slower, and barium spores were slowest. Calcium-barium and calcium-strontium spores germinated readily. Endotrophic calcium and strontium spores germinated without the prior heat activation essential for growth spores. Chemical germination of the different metal-type spores with n-dodecylamine took place at the same relative rates as physiological germination. Heat-induced release of dipicolinic acid occurred much faster with barium and strontium spores than with calcium spores. The washed "coat fraction" from disrupted spores contained little of the spore calcium but most of the spore barium. The metal in this fraction was released by dilute acid. The demineralized coats reabsorbed calcium and barium at neutral pH.     

Energy-dependence of calcium accumulation during sporulation of Bacillus megaterium KM.

Ca2+ accumulation and endogenous respiration of sporulating Bacillus megaterium are inhibited to the same extent by electron-transport of inhibitors and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that Ca2+ is accumulated by an active transport process. Forespores isolated in stage V of sporulation demonstrated Ca2+-specific carrier-mediated Ca2+ uptake, consistent with downhill transfer [Hogarth & Ellar (1978) Biochem. J. 176, 197-203]. In the present studies forespore Ca2+ uptake was unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by concentrations of respiratory inhibitor that inhibited forespore endogenous respiration by 85%. These data suggest that Ca2+ enters the isolated forespore by facilitated diffusion. Ca2+ uptake into sporulating protoplasts was completely inhibited by concentrations of respiratory inhibitors that had no effect on either Ca2+ uptake or respiration of stage-V forespores, but which resulted in inhibition of mother-cell membrane NADH oxidase. These results indicate that the mother-cell membrane is a site for active transport of Ca2+ into the sporulating cell. The effects of the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide on mother-cell membrane adenosine triphosphatase, NADH oxidase and protoplast Ca2+ uptake were examined.     

Influence of pore structure on the effectiveness of a biogenic carbonate surface treatment for limestone conservation

A ureolytic biodeposition treatment was applied to five types of limestone in order to investigate the effect of pore structure on the protective performance of a biogenic carbonate surface treatment. Protective performance was assessed by means of transport and degradation processes, and the penetration depth of the treatment was visualized by microtomography. Pore size governs bacterial adsorption and hence the location and amount of carbonate precipitated. This study indicated that in macroporous stone, biogenic carbonate formation occurred to a larger extent and at greater depths than in microporous stone. As a consequence, the biodeposition treatment exhibited the greatest protective performance on macroporous stone. While precipitation was limited to the outer surface of microporous stone, biogenic carbonate formation occurred at depths of greater than 2 mm for Savonnières and Euville. For Savonnières, the presence of biogenic carbonate resulted in a 20-fold decreased rate of water absorption, which resulted in increased resistance to sodium sulfate attack and to freezing and thawing. While untreated samples were completely degraded after 15 cycles of salt attack, no damage was observed in biodeposition-treated Savonnières. From this study, it is clear that biodeposition is very effective and more feasible for macroporous stones than for microporous stones.     

Availability of calcium from skim milk, calcium sulfate and calcium carbonate for bone mineralization in pigs

Dairy products provide abundant, accessible calcium for humans, while some calcium sulfate-rich mineral waters could provide appreciable amounts of calcium. But there is little evidence that this calcium is as available as milk calcium for making bone. The availability of calcium was studied by monitoring bone parameters in 2-month-old pigs fed restricted amounts of calcium (70% RDA) for 2.5 months. The 3 main (> or = 50% Ca intake) Ca sources were either CaCO3 or CaSO4 or skim milk powder (29% of the diet). The bones of the pigs fed the milk diet had higher (P < 0.01) ash contents, breaking strength and density (DEXA) than those of the two others groups, in which the bone values were similar. Thus, the calcium provided by a diet containing milk appears to ensure better bone mineralization than do calcium salts included in a non-milk diet. The calcium restriction may have enhanced some milk properties to stimulate calcium absorption in these young, rapidly growing pigs.     

Injury and recovery of Bacillus megaterium from mild chlorhexidine treatment

Treatment of Bacillus megaterium cell suspensions with 12 /μmol/1 chlorhexidine diacetate for 5 min led to an approximate 50%, reduction in viability when plated onto tryptone soya agar (TSA). Fifty percent of the surviving fraction were unable to form colonies on TSA containing 5.5% w/v KCI. Such loss of KCl tolerance is indicative of membrane damage, and was recovered within 30 min of incubation in tryptone soya broth (TSB). Multiplication of the damaged organisms did not recommence in this medium until after 60 min. Inclusion of inhibitors of respiration, and of protein, RNA and DNA synthesis in the TSB recovery medium did not significantly affect either the rate or extent of the recovery of KCl tolerance by damaged organisms.     

Effect of inhibitors on calcium carbonate deposition mediated by freshwater algae

Nine green algae, a diatom and three cyanobacteria were shown to precipitate CaCO₃ in batch culture, when grown in the light in a hard water medium containing 68 mg L⁻¹ soluble calcium. The composition of the medium was based on that found in a natural hardwater marina where precipitation of CaCO₃ within algal biofilms occurred. Deposition occurred as a direct result of photosynthesis which caused an increase in the pH of the medium. Once a critical pH had been reached, typically approximately pH 9.0, precipitation began evidenced by a fall in the concentration of soluble calcium in the medium. Certain characteristics of the precipitation process displayed by the diatom Navicula sp. were different to those of the other algae. All algae produced extracellular crystals of irregular morphology. Using a standardized protocol employing the green algae Chlorococcum sp. and Stigeoclonium variabile, the effects of various inhibitors of CaCO₃ nucleation or growth of crystals were studied. Fifteen compounds were screened and assessed for their performance in this context. Most materials effectively delayed deposition of CaCO₃, many decreased precipitation rates and all had a marked effect on crystal morphology. The most effective compound was HEDP (1-hydroxyethylene 1,1 diphosphonic acid), which inhibited precipitation completely at a concentration of 2.5 mg L⁻¹ The use of such compounds to reduce the precipitation of calcium salts within algal biofilms in natural hard waters is discussed.     

Influence of substrate mineralogy on bacterial mineralization of calcium carbonate: implications for stone conservation

The influence of mineral substrate composition and structure on bacterial calcium carbonate productivity and polymorph selection was studied. Bacterial calcium carbonate precipitation occurred on calcitic (Iceland spar single crystals, marble, and porous limestone) and silicate (glass coverslips, porous sintered glass, and quartz sandstone) substrates following culturing in liquid medium (M-3P) inoculated with different types of bacteria (Myxococcus xanthus, Brevundimonas diminuta, and a carbonatogenic bacterial community isolated from porous calcarenite stone in a historical building) and direct application of sterile M-3P medium to limestone and sandstone with their own bacterial communities. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), powder X-ray diffraction (XRD), and 2-dimensional XRD (2D-XRD) analyses revealed that abundant highly oriented calcite crystals formed homoepitaxially on the calcitic substrates, irrespective of the bacterial type. Conversely, scattered spheroidal vaterite entombing bacterial cells formed on the silicate substrates. These results show that carbonate phase selection is not strain specific and that under equal culture conditions, the substrate type is the overruling factor for calcium carbonate polymorph selection. Furthermore, carbonate productivity is strongly dependent on the mineralogy of the substrate. Calcitic substrates offer a higher affinity for bacterial attachment than silicate substrates, thereby fostering bacterial growth and metabolic activity, resulting in higher production of calcium carbonate cement. Bacterial calcite grows coherently over the calcitic substrate and is therefore more chemically and mechanically stable than metastable vaterite, which formed incoherently on the silicate substrates. The implications of these results for technological applications of bacterial carbonatogenesis, including building stone conservation, are discussed.     

Bio-deposition of a calcium carbonate layer on degraded limestone by Bacillus species

To obtain a restoring and protective calcite layer on degraded limestone, five different strains of the Bacillus sphaericus group and one strain of Bacillus lentus were tested for their ureolytic driven calcium carbonate precipitation. Although all the Bacillus strains were capable of depositing calcium carbonate, differences occurred in the amount of precipitated calcium carbonate on agar plate colonies. Seven parameters involved in the process were examined: calcite deposition on limestone cubes, pH increase, urea degrading capacity, extracellular polymeric substances (EPS)-production, biofilm formation, ζ-potential and deposition of dense crystal layers. The strain selection for optimal deposition of a dense CaCO₃ layer on limestone, was based on decrease in water absorption rate by treated limestone. Not all of the bacterial strains were effective in the restoration of deteriorated Euville limestone. The best calcite precipitating strains were characterised by high ureolytic efficiency, homogeneous calcite deposition on limestone cubes and a very negative ζ-potential.     

-aminobutyric acid as a required germinant for mutant spores of Bacillus megaterium

Germinated spores of Bacillus megaterium QM B1551 were irradiated with ultraviolet light, and spore-forming survivors were screened for germination requirements. Spore strains which failed to germinate in a variety of defined solutions germinative for spores of the parent strain were obtained. Mutant spores germinated readily in solutions containing yeast extract or one of numerous complex preparations. gamma-Aminobutyric acid, obtained from yeast extract by column chromatography, was shown to be required for germination by the mutant spores. gamma-Aminobutyric acid and l-alanine at final concentrations of 1 mm each, in solutions of KI (40 mm), equaled the potency of yeast extract (1 mg/ml) in the germination of the mutant spores. One of several other amino acids could be substituted, though less effectively, for l-alanine. alpha-Aminobutyric acid, beta-aminobutyric acid, beta-alanine, and 5-aminovaleric acid were ineffective substitutes for gamma-aminobutyric acid in mutant 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.     

Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.     

Production of extracellular polysaccharide by Bacillus megaterium RB-05 using jute as substrate

Bacillus megaterium RB-05 was grown on glucose and on “tossa-daisee” (Corchorus olitorius)-derived jute, and production and composition of extracellular polysaccharide (EPS) were monitored. An EPS yield of 0.065 ± 0.013 and of 0.297 g ± 0.054 g⁻¹ substrate after 72 h was obtained for glucose and jute, respectively. EPS production in the presence of jute paralleled bacterial cellulase activity. High performance liquid chromatography (HPLC), matrix assisted LASER desorption/ionization-time of flight (MALDI-ToF) mass spectroscopy, and fourier transform infrared (FT-IR) spectroscopy demonstrated that the EPS synthesized in jute culture (JC) differed from that synthesized in glucose mineral salts medium (GMSM). While fucose was only a minor constituent (4.9 wt.%) of EPS from GMSM, it a major component (41.9 wt.%) of EPS synthesized in JC. This study establishes jute as an effective fermentation substrate for EPS production by a cellulase-producing bacterium.     

Formation of transient amorphous calcium carbonate precursor in quail eggshell mineralization: an in vitro study

To understand the mechanism of quail eggshell biomineralization, we have performed two CaCO(3) precipitation experiments. In the reprecipitation experiments, supersaturated Ca(HCO(3))(2) was prepared by bubbling CO(2) through a slurry of biogenic CaCO(3) obtained from bleach-treated eggshell followed by filtration to obtain a clear solution for crystallization experiments. The nucleated crystals were collected at various time intervals and analyzed. In the second experiment, the extracted SOM from the bleach-treated eggshell was added to the supersaturated clear solution of Ca(HCO(3))(2) solution obtained by bubbling CO(2) gas through a slurry of synthetic CaCO(3) followed by filtration. The crystals/precipitates collected at various time intervals were analyzed. Both experiments showed that amorphous CaCO(3) (ACC) was precipitated in the early stages, which then transformed to the most stable crystalline calcite phase. Amino acid analysis of the soluble organic matrixes (SOM) indicated the presence of high amounts of Glx and Asx amino acids. Ovomucoid--an acidic glycoprotein, and lysozyme--a basic protein, are the two major components along with a few low molecular weight peptides present in the SOM of quail eggshell matrix. Both ovomucoid and lysozyme did not induce precipitation of the ACC phase in in vitro conditions, while the fraction containing low molecular weight peptides induced the precipitation of ACC, suggesting that the latter play an important role in the eggshell biomineralization. Thus, organisms can produce inorganic minerals which assume nonequilibrium morphologies and intricate architecture by precipitating transient ACC, which then transformed into the crystalline phase. Altogether, these observations further demonstrate that this strategy may be common in both vertebrate and invertebrate mineralized structures.     

Identification of penicillin-binding protein 5a of Bacillus megaterium KM as a DD-carboxypeptidase.

Measurement of the stabilities of DD-carboxypeptidase activity and the penicillin-binding activity of proteins 5 and 5a in membranes isolated from vegetative cells and stage-V forespores suggests that the unique sporulation-specific protein 5a may be a penicillin-sensitive DD-carboxypeptidase.     

CYP105A1 mediated 3-hydroxylation of glimepiride and glibenclamide using a recombinant Bacillus megaterium whole-cell catalyst

CYP105A1 from Streptomyces griseolus belongs to a widespread family of soluble prokaryotic cytochromes P450. For in vitro studies we established an electron transfer system, consisting of the ferredoxin Etp1(fd) and the ferredoxin reductase Arh1 from the fission yeast Schizosaccharomyces pombe. We investigated the metabolism of glibenclamide and glimepiride, hypoglycemic drugs of sulfonylurea type, and determined corresponding in vitro kinetic parameters. The resulting 3-cyclohexyl-hydroxylation activity towards glibenclamide and glimepiride was demonstrated by NMR analysis. Furthermore, the main product of glibenclamide, cis-3-hydroxy-glibenclamide is identical with the phase-1-metabolite of this drug in human. The orientation of glimepiride and glibenclamide in the active site of the enzyme is shown by a computational docking model. For high scale production of sulfonylurea derivatives, we designed whole-cell biocatalysts based on Bacillus megaterium MS941. Surprisingly, the system expressing only CYP105A1 showed a similar activity towards hydroxylation of glimepiride and glibenclamide compared to the system expressing additionally the redox partners, Arh1 and Etp1(fd)(516-618), indicating that the host strain provides a functional endogenous electron transfer system.