Swarming Behavior of and Hemolysin BL Secretion by Bacillus cereus

An association between swarming and hemolysin BL secretion was observed in a collection of 42 Bacillus cereus isolates (P = 0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation (P = 0.021), suggesting that swarming B. cereus strains may have a higher virulence potential than nonswarming strains.     

Intracellular Proteases of Bacillus stearothermophilus

Cell-free extracts of Bacillus stearothermophilus have been shown to exhibit proteolytic activity toward casein as well as specific activity to catalyze the hydrolysis of furylacryloylglycyl-l-leucine amide, furylacryloylglycine, and carbobenzoxyl-glycine-p-nitrophenyl ester, indicating the presence of a neutral proteinase, a carboxypeptidase-like enzyme, and an alkaline proteinase. The neutral proteinase and carboxypeptidase-like activities were separated by gel filtration over Bio-Gel P-60, and both were reversibly inhibited by 1, 10-phenanthroline. The esterase activity was inhibited by diisopropylfluorophosphate, which did not affect other enzymatic activities and was insensitive to 1, 10-phenanthroline and ethylenediaminetetra-acetic acid.     

Intracellular Proteases of Bacillus subtilis

The purpose of this study was to clarify the relationship between various cultivation conditions and the amounts of the rutin (RT) and protodioscin (PD) in asparagus spears. Green and white spears were grown in open culture and under two different blanching conditions. Although RT was detected only in the green spears, PD was detected mainly in white spears produced by covering with soil. The RT and PD contents of cladophylls grown in an open field and in a closed cultivation system were also investigated, and the closed system resulted in cladophylls with low RT and high PD, unlike the open field.     

Bacillus cereus enterotoxins

Data about Bacillus cereus different enterotoxins including hemolysin (HBL), nonhemolytic enterotoxin (NHE), enterotoxin (T), and emesis-inducing thermostable enterotoxin (ETE) are summarized in the article. Information about synthesis of different diarrhea-inducing and emesis-inducing enterotoxins, methods of their purification, structure, functions, and mechanisms of action are presented. Commercial kits for identification of B. cereus enterotoxins causing food-associated diarrhea are listed.     

Protoplast fusion in Bacillus cereus

Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.     

Degradation of Organomercurials in Bacillus cereus

Degradation of phenylmercuric acetate (PMA) and p-chloromercuribenzoic acid (PCMB) by cells of a mercury resistant strain of Bacillus cereus was demonstrated. Degradation of PMA was also demonstrated with the crude extract of the B. cereus cells grown in nutrient broth containing 1 μm PMA or 10 μm HgCl₂. The crude extract seems to split the carbon-mercury bond of PMA and form benzene and the mercuric ion as degradation products. The splitting enzyme was separated from mercuric reductase which catalyzes the reduction of the mercuric ion by gel filtration on Sephadex G 100.     

Enumeration of Bacillus cereus in Foods

For the enumeration of vegetative cells and spores of Bacillus cereus in foods, a mannitol-egg yolk-phenol red-agar has been developed which exploits the failure of B. cereus to dissimilate mannitol, and the ability of most strains to produce phospholipase C. When a high degree of selectivity was required, polymyxin B sulfate in a concentration of 10 ppm appeared to be the most effective selective additive. Useful characteristics for the identification of presumptive isolates of B. cereus were found to be: morphology, dissimilation of glucose mostly to acetyl methyl carbinol under anaerobic conditions, hydrolysis of starch and gelatin, reduction of nitrate, and growth on 0.25% chloral hydrate agar.     

Membrane-Bound Forms of Serine Proteases in Bacillus intermedius

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.     

Penicillin-Binding Component of Bacillus cereus

(14)C-penicillin is irreversibly bound by Bacillus cereus 569. Incubation of penicillin-treated cells in a cell wall digestion medium results in solubilization of approximately 60% of the irreversibly bound lable. The extent of the solubilization is the same when cells are prepared by either a cold or 37 C treatment procedure. However, spheroplasts prepared by the cold treatment are leaky. When the resulting spheroplasts are incubated in supplemented medium, reduced rates and levels of penicillinase synthesis, relative to induced whole-cell controls, are observed. Spheroplasts from both cold and 37 C prepared cells exhibit this phenomena, although the spheroplasts from 37 C prepared cells synthesized approximately sixfold higher levels of penicillinase. The size distribution of the label solubilized during the preparation of spheroplasts was examined by using Bio Gel P-150 columns. Although no label appeared in the exclusion volume fractions when the cell wall digest of the 37 C treated cells was chromatographed, approximately 10% of the label from cold-treated cells did appear. These results suggest that the presence of irreversibly bound penicillin is required for the synthesis of induced levels of penicillinase and that the irreversibly bound penicillin can be solubilized as a labile complex with material which is excluded from BioGel P-150. It may be concluded that the penicillin-binding lipoprotein complex which has been previously observed is the penicillin-specific binding site. However, the location of this complex in relation to the cell membrane could not be determined.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

Thermostability of Bacillus cereus penicillinase

Williams, Daniel H., III (Hahnemann Medical College, Philadelphia, Pa.), A. Bondi, A. G. Moat, and F. Ahmad. Thermostability of Bacillus cereus penicillinase. J. Bacteriol. 91:257-261. 1966.-The extracellular penicillinase of Bacillus cereus, strain 13-10, exhibited an unusual thermostability. Whereas it was completely and irreversibly inactivated by heating at 70 C, it retained considerable activity when heated at 100 C for 30 min. The active enzyme remaining was completely stable to further heating at temperatures from 40 to 100 C for as long as 1 hr. Preparations of the enzyme heated to 100 C possessed pH (7.0) and temperature (37 C) optima identical with the unheated enzyme. Furthermore, both enzyme preparations exhibited identical combining capacity for the substrate (penicillin G), suggesting that the two preparations had similar hydrolytic properties. Our findings suggest that heating of penicillinase at 100 C results in the formation of a protein complex which is resistant to further denaturation by heat and other agents. Addition of certain metal ions to the enzyme solution before heat treatment increased the stability to heat at 100 C by virtue of their ability to induce complex formation. Pectin was shown to decrease thermostability, presumably by preventing aggregation of proteins present in the enzyme preparations. The well-known stabilizing effect of gelatin may be attributed to its role in enhancing complex formation.     

Microtitration of Bacillus cereus Hemolysin

A microtiter procedure for the quantitation of Bacillus cereus hemolysin is described.     

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.