Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.     

Microbial biohydrogenation of oleic acid to trans isomers in vitro

Ruminant products are significant sources of dietary trans fatty acids. Trans fatty acids, including various conjugated linoleic acid isomers, have been shown to act as metabolic modifiers of lipid metabolism. Trans fatty acids originate from biohydrogenation of dietary unsaturated fatty acids by gut microbes; however, the exact synthetic pathways are unclear. It was our goal to examine the biohydrogenation pathway for oleic acid, where oleic acid is hydrogenated directly to stearic acid. Our objective in this study was to trace the time course of appearance of 13C in labeled oleic acid to determine if trans monoenes are formed from the 13C-labeled oleic acid or if the 13C appears only in stearic acid as described in reviews of earlier work. Enrichments were calculated from the mass abundance of 13C in major fatty acid fragments and expressed as a percentage of total carbon isotopomers. Significant 13C enrichment was found in stearic acid, oleic acid, trans-6, trans-7, and in all trans C18:1 in positions 9-16. We concluded that the biohydrogenation of oleic acid by mixed ruminal microbes involves the formation of several positional isomers of trans monoenes rather than only direct biohydrogenation to form stearic acid as previously described.     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.     

Induction of phosphoribomutase in Bacillus cereus growing on nucleosides

In this paper we show that phosphoribomutase is induced in Bacillus cereus by the same metabolizable purine and pyrimidine ribonucleosides previously shown to induce the purine nucleoside phosphorylase (Tozzi, M.G., Sgarrella, F. and Ipata, P.L. (1981) Biochim. Biophys. Acta 678, 460-466). The mutase allows ribose 1-phosphate formed from nucleosides to be utilized by the cell through the pentose cycle, upon transformation to ribose 5-phosphate. The equilibrium constant of the mutase reaction is towards ribose-5-phosphate formation. The coordinate induction of the two enzymes completes the picture of the molecular events leading to the utilization of the sugar moiety of purine nucleotides and nucleosides as an energy source (Mura. U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905-7909).     

Induction of pullulanase production in Bacillus cereus FDTA-13

Studies were carried out on the production of pullulanase by a newly isolated strain Bacillus cereus FDTA-13. High titres of the enzyme were obtained in a medium containing branched polysaccharides. To further enhance the yield, induction of pullulanase using conventional inducers were studied. Maltooligosaccharides (maltose to maltotetraose) when added in the medium individually, or in a 1:1 combination of maltotriose and maltotetraose resulted different levels of pullulanase compared to control. Growth under carbon limited conditions (5 g l(-1)) with inducers resulted remarkably enhanced pullulanase activity. Pullulanase activity was severely repressed in presence of glucose. Low levels of pullulanase was observed in nitrogen limited medium, even with combinations of several maltosaccharides.     

Induction of natural competence in Bacillus cereus ATCC14579

Natural competence is the ability of certain microbes to take up exogenous DNA from the environment and integrate it in their genome. Competence development has been described for a variety of bacteria, but has so far not been shown to occur in Bacillus cereus. However, orthologues of most proteins involved in natural DNA uptake in Bacillus subtilis could be identified in B. cereus. Here, we report that B. cereus ATCC14579 can become naturally competent. When expressing the B. subtilis ComK protein using an IPTG‐inducible system in B. cereus ATCC14579, cells grown in minimal medium displayed natural competence, as either genomic DNA or plasmid DNA was shown to be taken up by the cells and integrated into the genome or stably maintained respectively. This work proves that a sufficient structural system for DNA uptake exists in B. cereus. Bacillus cereus can be employed as a model system to investigate the mechanism of DNA uptake in related bacteria such as Bacillus anthracis and Bacillus thuringiensis. Moreover, natural competence provides an important tool for biotechnology, as it will allow more efficient transformation of B. cereus and related organisms, e.g. to knockout genes in a high‐throughput way.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Induction of Systemic Resistance by Bacillus cereus Against Tomato Foliar Diseases Under Field Conditions

The ability of a rhizobacterium to protect tomato plants against naturally occurring diseases as well as to improve crop yield under field conditions was studied. The rhizobacterium was introduced to the plants through seed microbiolization. Treatments consisted of different frequencies of fungicide (Chlorothalonyl) sprayings (5, 10 or 20 applications) of tomato plants grown from either microbiolized or non‐microbiolized seeds over a 90‐day evaluation period. Treatment of non‐microbiolized seeds without fungicide application was included as a control. The progress of the following three naturally occurring diseases was evaluated in the field and quantified: early blight (Alternaria solani), late blight (Phytophthora infestans), and septoria leaf spot (Septoria lycopersici). All treatments resulted in reduced disease severity when compared with the control treatment. Highest final fruit yields were found after treatment of plants grown from non‐microbiolized seeds and sprayed with fungicide 20 times over 90 days, and for treatment of plants from microbiolized seeds that received 10 fungicide spray applications, although all treatments increased yield over that obtained in the control treatment. The results demonstrate that combined rhizobacterial and chemical treatments in the field may permit reducing fungicidal spraying frequency while at the same time increasing crop yields.     

Two omega-amino acid transaminases from Bacillus cereus.

Bacillus cereus strain K-22 produced two distinct omega-amino acid transaminases, one catalyzing the transamination between beta-alanine and pyruvic acid and the other that between gamma-aminobutyric acid and alpha-ketoglutaric aic. The two enzymes were partially purified and separated from each other by various chromatographies. beta-Alanine:pyruvic acid transaminase and gamma-aminobutyric acid:alpha-ketoglutaric acid transaminase were induced by the addition of beta-alanine and gamma-aminobutyric acid, respectively, to the growth medium. beta-Alanine transaminase showed an optimum pH of 10.0 and optimum temperature of 35 degrees C, and its Km values for beta-alanine and pyruvic acid were both 1.1 mM. gamma-Aminobutyric acid, epsilon-aminocaproic acid, 2-aminoethylphosphonic acid, and propylamine showed about 30-40% of the activity of beta-alanine as amino donors, and oxalacetic acid was as good an amino acceptor as pyruvic acid. The optimum pH and temperature of gamma-aminobutyric acid transaminase were 9.0 and 50 degrees C, respectively, and its Km value for gamma-aminobutyric acid was 2.8 mM, while that for alpha-ketoglutaric acid was 2.3 mM. gamma-Aminobutyric acid and delta-aminovaleric acid were good amino donors but other omega-amino acids were virtually inactive with gamma-aminobutyric acid transaminase; alpha-ketoglutaric acid, and to a lesser extent glyoxylic acid, were active amino acceptors. Sulfhydryl reagents specifically activated gamma-aminobutyric acid transaminase.     

Dynamics of accumulation of extracellular lipoteichoic acid in Bacillus cereus

The dynamic of accumulation of extracellular lipoteichoic acid (LTA) was studied depending on the growth stage of Bacillus cereus st. 96. A maximum amount of extracellular LTA was detected in the middle of the exponential growth. The quantity of the biopolymer present in the culture medium at the beginning of the stationary growth under conditions of catabolite repression of sporulation and without repression was found to be different. Experimentally increased concentrations of LTA inhibited B. cereus sporulation. Besides, dormant spores of B. cereus st. 96 were found to contain LTA.     

Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid: kinetics

The kinetics of the inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopenicillanic acid are described. Loss of beta-lactamase activity is accompanied by a decrease in protein fluorescence, by the appearance of a protein-bound chromophore at 326 nm, and by loss of tritium from 6 alpha-[3H]-6 beta-bromopenicillanic acid. It is shown that all of the above changes probably have the same rate-determining step. The inactivation reaction is competitively inhibited by cephalosporin C, a competitive inhibitor of this enzyme, and by covalently bound clavulanic acid, suggesting that 6 beta-bromopenicillanic acid reacts directly with the beta-lactamase active site. It is proposed that this inhibitor reacts initially as a normal substrate and that the rate-determining step of the inactivation is acylation of the enzyme. A rapid irreversible inactivation reaction rather than normal hydrolysis of the acyl-enzyme then follows acylation; 6 beta-bromopenicillanic acid is thus a suicide substrate.     

Ribonucleic acid polymerase of germinating Bacillus cereus T.

It appears that a de novo synthesis of the deoxyribonucleic acid-dependent ribonucleic acid-polymerase in Bacillus cereus T takes place fairly late in outgrowth, at the onset of the vegetative cycle. Therefore, the ribonucleic acid-polymerase used by germinating spores is the one carried on from sporulating cells. However, the sporal enzyme is less soluble that the vegetative one, and its "core" is bound to two extra peptides. This complexing to other molecules could play a role in the regulation of gene expression during germination.     

Microbial production of 4-vinylguaiacol from ferulic acid by Bacillus cereus SAS-3006

Abstract

Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.