Protein synthesizing systems from spores and vegetative cells of Bacillus cereus

A system of polyphenylalanine synthesis was optimized for a comparison of the polymerizing activities of ribosomes from spores and vegetative cells of Bacillus cereus T. Ribosomes of both types react similarly, showing a magnesium optimum of about 6 mM and spermidine optima of about 5 mM and 4 mM for vegetative and spore ribosomes, respectively. These lead to optimum mono- to multivalent cation rations of 9 and 10 respectively at 100 mM ammonium ion. A comparison of the response of these ribosomes to suboptimal concentrations of magnesium and spermidine show that they differ qualitatively from each other, suggesting that they possess different structure, macromolecular or ionic components.Abbreviations DFP diisopropylfluorophosphate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid     

Properties of fructose 1,6-diphosphate aldolases from spores and vegetative cells of Bacillus cereus

Fructose 1,6-diphosphate aldolase from cells of Bacillus cereus appears to be typical Class II aldolase as judged by its functional and physical properties. Spore and vegetative cell aldolase had similar enzymatic, immunochemical, and heat resistance properties in the absence of calcium, but they differed in their thermal stabilities in the presence of calcium, their Stokes' radii, their mobility in acrylamide gel electrophoresis, and their molecular weights. The pH optimum for both enzymes was 8.5, and their K(m) with respect to substrate was 2 x 10(-3)m. Highly purified spore and vegetative cell aldolases were both heat labile with half-lives of 4 min at 53 C and pH 6.4. In the presence of 3 x 10(-2)m solution of calcium ions, the stability of the spore protein increased 12-fold but the vegetative form became more heat labile. The enhanced stability of the spore aldolase was not diminished by dialysis or gel filtration but was lost after chromatography on diethylaminoethyl cellulose at pH 7.4. Aldolase from vegetative cells exists in an equilibrium mixture of two molecular weights, 115,000 and 79,000 in the approximate ratio of 1:4, respectively. The molecular weight of spore aldolase is 44,000. Spore aldolase was more mobile during electrophoresis than its vegetative cell counterpart because of its smaller size.     

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. Methods and RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Transformation of Bacillus cereus vegetative cells by electroporation

Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 (1.8 megadaltons) by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell. Transformation of a 130-megadalton plasmid occurred at a comparable frequency. The method was simple, rapid, and yielded transformant colonies in 14 to 24 h. Transformation was obtained with unpurified total plasmid DNA.     

Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats

The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples from animals receiving vegetative cells, only few B. cereus cells were detected. Spores survived the gastric barrier well, and were in some cases detected up to 2 weeks after ingestion. Selective growing revealed no major changes in the intestinal flora during passage of B. cereus. However, denaturing gradient gel electrophoresis analysis with universal 16S rRNA gene primers revealed significant changes in the intestinal microbiota of animals dosed with spores. Vero cell assays and a commercial kit (BCET-RPLA) did not reveal any enterotoxin production from B. cereus F4433/73R in the intestinal tract.     

Microgermination of Bacillus cereus spores

The biphasic nature of germination curves of individual Bacillus cereus T spores was further characterized by assessing the effects of temperature, concentration of germinants, and some inorganic cations on microgermination. Temperature was shown to affect both phases of microgermination as well as the microlag period, whereas the concentration of l-alanine and supplementation with adenosine exerted a significant effect only on the microlag period. The germination curves of individual spores induced by inosine were also biphasic and resembled those of spores induced by l-alanine. High concentrations (0.1 m or higher) of calcium and other inorganic cations prolonged both phases of microgermination, particularly the second phase, and had a less pronounced effect on the microlag period. The second phase of microgermination was completely inhibited when spores were germinated either in the presence of 0.3 m CaCl(2) or at a temperature of 43 C; this inhibition was reversible. Observations on the germination of spore suspensions (kinetics of the release of dipicolinic acid and mucopeptides, loss of heat resistance, increase in stainability, decrease in turbidity and refractility) were interpreted on the basis of the biphasic nature of microgermination. Dye uptake by individual spores during germination appeared also to be a biphasic process.     

Transformation of Bacillus cereus vegetative cells by electroporation.

Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 (1.8 megadaltons) by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell. Transformation of a 130-megadalton plasmid occurred at a comparable frequency. The method was simple, rapid, and yielded transformant colonies in 14 to 24 h. Transformation was obtained with unpurified total plasmid DNA.     

Analysis of broth-cultured Bacillus atrophaeus and Bacillus cereus spores

Aims: To compare physical properties of spores that were produced in broth sporulation media at greater than 10⁸ spores ml⁻¹. Methods and Results: Bacillus atrophaeus reproducibly sporulated in nutrient broth (NB) and sporulation salts. Microscopy measurements showed that the spores were 0·68 ± 0·11 μm wide and 1·21 ± 0·18 μm long. Coulter Multisizer (CM3) measurements revealed the spore volumes and volume-equivalent spherical diameters, which were 0·48 ± 0·38 μm³ and 0·97 ± 0·07 μm, respectively. Bacillus cereus reproducibly sporulated in NB, sporulation salts, 200 mmol l⁻¹ glutamate and antifoam. Spores were 0·95 ± 0·11 μm wide and 1·31 ± 0·17 μm long. Spore volumes were 0·78 ± 0·61 μm³ and volume-equivalent spherical diameters were 1·14 ± 0·11 μm. Bacillus atrophaeus spores were hydrophilic and B. cereus spores were hydrophobic. However, spore hydrophobicity was significantly altered after treatment with pH-adjusted bleach. Conclusions: The utility of a CM3 for both quantifying Bacillus spores and measuring spore sizes was demonstrated, although the volume between spore exosporium and spore coat was not measured. This study showed fundamental differences between spores from a Bacillus subtilis- and B. cereus-group species. Significance and Impact of the Study: This is useful for developing standard methods for broth spore production and physical characterization of both living and decontaminated spores.     

Isolation, purification and identification of protective substances from a culture broth of germinating Bacillus cereus spores

A fraction increasing the resistance of resting spores to UV-irradiation and high temperature has been isolated from the culture medium at the stage of B. cereus st. 96 spore initiation. Amino acid analysis, gas chromatography, electrophoresis, and TLC of the products of acidic and alkaline hydrolysis of the isolated fraction demonstrated that the active component of the fraction was the lipoteichoic acid.     

Low-pH activation of Bacillus cereus spores

Heat activation of bacterial spores at low pH was investigated in detail. Unlike activation of spores in distilled water at a neutral pH, activation at low pH involves two superimposed processes: enhanced activation and death. Low-pH-activated spores failed to germinate in d-alanine, in contrast to spores activated at neutral pH, owing to the abolition of alanine-racemase activity. Morphological and permeability changes such as release and partial disruption of spores were dipicolinic acid-observed during low-pH activation. The kinetics pattern of low-pH activation, as well as the change in properties of the spores thereafter, suggest that the mechanism of low-pH activation differs from that of other kinds of heat-activation.