Microtitration of Bacillus cereus Hemolysin

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

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.     

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.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Phospholipase C from Bacillus cereus. Evidence for essential lysine residues.

1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.     

蜡样芽胞杆菌B905 hblA基因的初步分析

采用血平板培养的方法对蜡样芽胞杆菌905菌株溶血素BL检测,并通过PCR方法克隆其基因,结果表明该菌株产生溶血环且含有hblA、hblC、hblD溶血素BL全部基因;采用同源重组法构建了该菌株hblA基因缺失突变体,结果该菌株的溶血活性并未发生改变,可能是由于该菌株溶血素基因的结构与Handelsman构建所用的菌株Bacillus cereus UW85有一定的差异,或者是由于突变位点在阅读框内后端,未能真正破坏其表达。还需要进一步对其进行研究。     

Semiautomated Metabolic Staining Assay for Bacillus cereus Emetic Toxin

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.     

X-ray crystal structure of the B component of Hemolysin BL from Bacillus cereus

Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.     

C-Terminal Domain of Bacillus cereus Hemolysin II Is Able to Interact with Erythrocytes

Russian Journal of Bioorganic Chemistry - Hemolysin II (HlyII) is one of the pathogenic factors of Bacillus cereus. With respect to the prototype of β-barrel toxins, the α-toxin of S....     

Toxin production by Bacillus cereus dairy isolates in milk at low temperatures.

A total of 136 strains of Bacillus cereus isolated from milk and cream were evaluated for toxin production based on HeLa S3, Vero, and human embryonic lung (HEL) cell cytotoxicity in vitro. HEL cell monolayers were more susceptible than the other two cell lines. The percentage of isolates exhibiting HEL cytotoxicity was similar (43.0 and 48.4%) when the strains were grown in brain heart infusion broth containing 0.1% glucose (BHIG) at 7 and 24 h, respectively, at 30 degrees C. In milk, only 21.8% of isolates showed HEL cytotoxicity at 7 h, and the number increased significantly to 73.2% at 24 h at 30 degrees C. Further, 102 toxin-positive isolates were acclimatized to grow at 8 degrees C in milk. Ninety-four (92.2%) of the strains produced HEL cytotoxicity of various degrees with no strict correlation to bacterial cell numbers and also elicited vascular permeability reaction in rabbit skin. Under aerated growth conditions (agitation, 200 rpm) B. cereus elicited cytotoxicity in BHIG and in milk at temperatures of 30, 15, and 8 degrees C. However, in nonaerated (stagnant) cultures toxin production was diminished (BHIG) or completely lost (milk) at all temperatures. Toxin production at 8 degrees C was evaluated in two different types of commercial cardboard milk packages by inoculation with a potent toxigenic dairy isolate. No detectable HEL cytotoxicity was observed in milk in any of the packages either at stagnant conditions or during mechanical shaking. However, the same strain produced cytotoxin in whipped cream at 8 degrees C.     

beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis.

The kinetic model of Ragan & Cottingham [(1985) Biochim. Biophys. Acta 811, 13-31] for electron transport through a mobile pool of quinone predicts that, under certain conditions, the normal linear dependence of electron flow on the degree of reduction (or oxidation) of the quinone should no longer be found. These conditions can be met by reconstituted NADH: cytochrome c reductase (Complex I-III from bovine heart) when electron flow is rate-limited by a low concentration of cytochrome c. We show that, in such a system, the dependence of activity (varied by inhibition with rotenone) on the steady-state level of quinone reduction is indeed non-linear and very closely accounted for by the theory.     

Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties.

A sphingomyelinase was purified 980-fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)2SO4 precipitation and chromatography on CM-Sephadex, DEAE-cellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholipases. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (Mr = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.     

Production and Characterization of Monoclonal Antibodies against the Hemolysin BL Enterotoxin Complex Produced by Bacillus cereus

A total of five hybridoma cell lines that produced monoclonal antibodies against the components of the hemolysin BL (HBL) enterotoxin complex and sphingomyelinase produced by Bacillus cereus were established and characterized. Monoclonal antibody 2A3 was specific for the B component, antibodies 1A12 and 8B12 were specific for the L₂ component, and antibody 1C2 was specific for the L₁ protein of the HBL enterotoxin complex. No cross-reactivity with other proteins produced by different strains of B. cereus was observed for monoclonal antibodies 2A3, 1A12, and 8B12, whereas antibody 1C2 cross-reacted with an uncharacterized protein of approximately 93 kDa and with a 39-kDa protein, which possibly represents one component of the nonhemolytic enterotoxin complex. Antibody 2A12 finally showed a distinct reactivity with B. cereus sphingomyelinase. The monoclonal antibodies developed in this study were also successfully applied in indirect enzyme immunoassays for the characterization of the enterotoxic activity of B. cereus strains. About 50% of the strains tested were capable of producing the HBL enterotoxin complex, and it could be demonstrated that all strains producing HBL were also highly cytotoxic.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Multiple-Locus Sequence Typing and Analysis of Toxin Genes in Bacillus cereus Food-Borne Isolates

In the present study we characterized 47 food-borne isolates of Bacillus cereus using multilocus sequence typing (MLST). Newly determined sequences were combined with sequences available in public data banks in order to produce the largest data set possible. Phylogenetic analysis was performed on a total of 296 strains for which MLST sequence information is available, and three main lineages—I, II, and III—within the B. cereus complex were identified. With few exceptions, all food-borne isolates were in group I. The occurrence of horizontal gene transfer (HGT) among various strains was analyzed by several statistical methods, providing evidence of widespread lateral gene transfer within B. cereus. We also investigated the occurrence of toxin-encoding genes, focusing on their evolutionary history within B. cereus. Several patterns were identified, indicating a pivotal role of HGT in the evolution of toxin-encoding genes. Our results indicate that HGT is an important element in shaping the population structure of the B. cereus complex. The results presented here also provide strong evidence of reticulate evolution within the B. cereus complex.