Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml⁻¹, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.     

The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is a metalloprotease with gelatinolytic and collagenolytic activity

The integration site(s) of the IncJ element, R391, was localised to a specific region of the Escherichia coli chromosome, between the uxuA and serB loci (98.0-99.5 min), using classical Hfr mapping techniques. F-prime plasmid hosts, diploid for regions spanning the E. coli chromosome, were used as recipients in R391 and R997 conjugal transfer assays. Analysis of transconjugants revealed the integration of R391 and R997 into specific F-primes that contain the uxuA to serB region, but not F-primes that contain other regions of the chromosome. A comparison of the electrophoretic mobility of the original F-primes with those containing inserts demonstrated the integration of large elements, in excess of 85 kb. Linear integration of the IncJ elements into chromosomal DNA was demonstrated in recombination-deficient (recA) backgrounds in the absence of detectable autonomous stages. These observations account for the inability to isolate plasmid DNA from IncJ hosts, and suggests that the elements exhibit a conjugative transposon-like biology in E. coli.     

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.     

Inhibition of Bacillus cereus phospholipase C by univalent anions.

The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.     

Formation of model lipid bilayers at the silica-water interface by co-adsorption with non-ionic dodecyl maltoside surfactant

This present article describes a new and simple method for preparing model lipid bilayers. Stable and reproducible surface layers were produced at silica surfaces by co- adsorbing lipid with surfactant at the silica surface from mixed micellar solutions. The adsorption was followed in situ by use of ellipsometry. The mixed micellar solution consisted of a lipid (L-alpha-dioleoyllecithin) and a non-ionic sugar-based surfactant (n-dodecyl-beta-maltoside). The latter showed, by itself, no affinity for the surface and could, therefore, easily be rinsed off the surface after the adsorption step. By first adsorbing from solutions with high lipid and surfactant concentrations and then, in succession, rinsing and re-adsorbing from solutions with lower lipid-surfactant concentrations, a dense-packed lipid bilayer was produced at the silica surface. The same result can be achieved in a one-step process where the rinsing, after adsorption from the concentrated solution, is performed very slowly. The thickness of the adsorbed lecithin bilayer after this treatment found was to be about 44 +/- 3 A, having a mean refractive index of 1.480 +/- 0.004. The calculated surface excess of lipids on silica was about 4.2 mg m(-2), giving an average area per lipid molecule in the two layers of 62 +/- 3 A2. The physical characteristic of the adsorbed bilayer is in good agreement with previously reported data on bulk and surface supported lipid bilayers. However, in contrast to previous investigations, we found no support for the presence of a thicker multi-molecular water layer located between the lipid layer and the solid substrate.     

Bacillus cereus pore-forming toxins hemolysin II and cytotoxin K: polymorphism and distribution of genes among representatives of the cereus group

Abstract-Phylogenetic interrelation between 40 strains of the Bacillus cereus group has been established using BcREP fingerprinting. The PCR method has shown that the frequency of occurrence of the genes of cytotoxin K (cytK) and hemolysin II (hlyII) is 61% and 56%, respectively, and the gene of the hemolysin II regulator (hlyIIR) occurs together with hlyII. Comparison of the results of fingerprinting, PCR, and RFLP of the toxin genes showed that bacteria with the hlyII+ and cytK+ genotypes did not form separate clusters. However, microorganisms with the similar fingerprints were shown to have toxin genes of the same type. The proposed variant of RFLP analysis made it possible to clearly distinguish between the cytK1 and cytK2 genes. Twenty-three strains having the cytK genes carried no cytK1 dangerous for mammals. Additionally, the entire collection of microorganisms was tested for the ability to grow at 4 degrees C. This property was revealed for five strains, which should most likely be classified as B. weihenstephanensis. Two of the five psychrotolerant microorganisms carried the hemolysin II gene variant of the same type according to RFLP. None of the five strains had the cytK gene. These strains did not form close groups upon clustering by the applied method of Bc-REP fingerprints.     

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.     

Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure

Mutation E71A in the bacterial K⁺-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and ~2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the “flipped” conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open “flipped” state via electrostatic interaction in the proximity of the channel pore.     

Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol

Remsen, C. C. (Syracuse University, Syracuse, N.Y.), D. G. Lundgren, and R. A. Slepecky. Inhibition of the development of the spore septum and membranes in Bacillus cereus by beta-phenethyl alcohol. J. Bacteriol. 91:324-331. 1966.-The effect of phenethyl alcohol (PEA) upon the initial stages of sporulation in Bacillus cereus was studied with an electron microscope. PEA (0.35%) completely inhibited the development of the spore septum and forespore membranes. Some of the treated cells did form the axial filament of chromatin material regarded as the first stage in sporulation, but this was delayed by 4 to 5 hr compared with untreated cells. The definite effect upon these membrane systems lends support to the belief that the primary site of PEA inhibition may be upon the bacterial membrane. The fine-structure details observed during the initial stages of sporulation in untreated cells were in agreement with the structure published for other Bacillus species.     

The Bacillus cereus Hbl and Nhe Tripartite Enterotoxin Components Assemble Sequentially on the Surface of Target Cells and Are Not Interchangeable

Bacillus cereus is a spore-forming, Gram-positive bacterium commonly associated with outbreaks of food poisoning. It is also known as an opportunistic pathogen causing clinical infections such as bacteremia, meningitis, pneumonia, and gas gangrene-like cutaneous infections, mostly in immunocompromised patients. B. cereus secretes a plethora of toxins of which four are associated with the symptoms of food poisoning. Two of these, the non-hemolytic enterotoxin Nhe and the hemolysin BL (Hbl) toxin, are predicted to be structurally similar and are unique in that they require the combined action of three toxin proteins to induce cell lysis. Despite their dominant role in disease, the molecular mechanism of their toxic function is still poorly understood. We report here that B. cereus strain ATCC 10876 harbors not only genes encoding Nhe, but also two copies of the hbl genes. We identified Hbl as the major secreted toxin responsible for inducing rapid cell lysis both in cultured cells and in an intraperitoneal mouse toxicity model. Antibody neutralization and deletion of Hbl-encoding genes resulted in significant reductions of cytotoxic activity. Microscopy studies with Chinese Hamster Ovary cells furthermore showed that pore formation by both Hbl and Nhe occurs through a stepwise, sequential binding of toxin components to the cell surface and to each other. This begins with binding of Hbl-B or NheC to the eukaryotic membrane, and is followed by the recruitment of Hbl-L₁ or NheB, respectively, followed by the corresponding third protein. Lastly, toxin component complementation studies indicate that although Hbl and Nhe can be expressed simultaneously and are predicted to be structurally similar, they are incompatible and cannot complement each other.     

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.     

pH gradients through colonies of Bacillus cereus and the surrounding agar.

pH-sensitive microelectrodes, constructed with a tip diameter of about 4 microns, were deployed through 24 h and 48 h colonies of Bacillus cereus incubated on CYS medium (Casamino acids, yeast extract, salts), with and without glucose. Measurements of pH were used to construct pH profiles through the colony and the surrounding agar. pH gradients could be detected for at least 800 microns into the agar beneath a 24 h colony, and to approximately 10 mm horizontally away from the edge of the colony. In older colonies, the lateral gradient extended for over 20 mm. The pH of the underlying agar was increased by up to 1.45 pH units after 48 h growth without glucose. When colonies were grown with glucose, a significant area of acidification was observed within the colony in addition to a zone of alkalinization present at its periphery. Acidification was thought to be due to the anaerobic fermentation of glucose producing organic acids whilst alkalinization was due to the aerobic oxidation of amino acids releasing ammonia.     

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 Transformation of Quercetin by Bacillus cereus

Biotransformation of quercetin was examined with a number of bacterial cultures. In the presence of a bacterial culture (Bacillus cereus), quercetin was transformed into two crystalline products, identified as protocatechuic acid and quercetin-3-glucoside (isoquercitrin).     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。