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.     

Microtitration of Bacillus cereus Hemolysin

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

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.     

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。     

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.     

Cryoenzymology of Bacillus cereus beta-lactamase II

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads

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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.     

The Angiotensin II Type 1 Receptor C-Terminal Lys Residues Interact with Tubulin and Modulate Receptor Export Trafficking

The physiological and pathological functions of angiotensin II are largely mediated through activating the cell surface angiotensin II type 1 receptor (AT1R). However, the molecular mechanisms underlying the transport of newly synthesized AT1R from the endoplasmic reticulum (ER) to the cell surface remain poorly defined. Here we demonstrated that the C-terminus (CT) of AT1R directly and strongly bound to tubulin and the binding domains were mapped to two consecutive Lys residues at positions 310 and 311 in the CT membrane-proximal region of AT1R and the acidic CT of tubulin, suggestive of essentially ionic interactions between AT1R and tubulin. Furthermore, mutation to disrupt tubulin binding dramatically inhibited the cell surface expression of AT1R, arrested AT1R in the ER, and attenuated AT1R-mediated signaling measured as ERK1/2 activation. These data demonstrate for the first time that specific Lys residues in the CT juxtamembrane region regulate the processing of AT1R through interacting with tubulin. These data also suggest an important role of the microtubule network in the cell surface transport of AT1R.     

Lethal Toxin of Bacillus cereus I. Relationships and Nature of Toxin, Hemolysin, and Phospholipase

Bacillus cereus phospholipase was characterized as a phospholipase C by the analysis of lecithin degradation products by thin-layer and paper chromatography. Methanol in the growth menstruum inhibited completely the synthesis of phospholipase C, whereas the synthesis of lethal toxin and hemolysin were only partially inhibited. Dialysis of preformed B. cereus products against ethyl alcohol and methanol did not inactivate hemolytic, phospholipase C, or lethal activity. The hemolytic and lethal activities of culture filtrates were completely abolished by trypsin, but phospholipase C activity was resistant to inactivation. Lethal and phospholipase C properties of culture filtrates were resistant to inactivation at 45 C, whereas the hemolytic activity was completely destroyed. Lethal, hemolytic, and phospholipase C activities appeared simultaneously in a complex growth menstruum, but the kinetics of synthesis were different in all cases. Resolution of B. cereus filtrates on columns of Sephadex showed that the phospholipase C, hemolysin, and lethal toxin are distinct proteins. Evidence is also presented which suggests a correlation between the synthesis of B. cereus toxin and the period of transition from vegetative growth to sporulation. The activity of each B. cereus product was cation-independent, as opposed to cation-dependency of the phospholipase C and lethal activities of Clostridium perfringens alpha-toxin. Immunological cross-reactivity between the B. cereus products and C. perfringens alpha-toxin was not apparent; indeed, they were shown to be antigenically distinct.     

Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription

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几种芽胞杆菌溶血素BL基因及其溶血素的检测

用PCR方法对几种芽胞杆菌溶血素BL基因进行了检测,结果表明7株蜡样芽胞杆菌含有溶血素BL基因hblA、hblC、hblD,其他枯草芽胞杆菌、多粘类芽胞杆菌、地衣芽胞杆菌检测到部分溶血素基因;通过血平板培养的方法检测结果表明只有含有溶血素全部基因的菌株才会产生溶血环,从而为筛选不产生溶血素的有益芽胞杆菌奠定一定基础。     

Purification and cytotoxic properties of Bacillus cereus hemolysin II

The hemolysin II from Bacillus cereus, HlyII, is a member of the beta-barrel pore-forming toxin family of secreted microbial proteins that includes the Staphylococcus aureus alpha-toxin. Compared with other proteins of the family, hemolysin II has 90 extra amino acids at its C-terminus. To examine more closely the cytotoxic and pore-forming properties of the protein, we have cloned and expressed it in Escherichia coli. We developed a purification procedure for the matured HlyII protein from both culture media and cell extracts using a combination of cation exchange and affinity chromatography together with gel-filtration. In both cases, the fully processed HlyII protein was purified as confirmed by N-terminal sequence analysis. The HlyII protein exhibits cytolytic activity of different extent on erythrocytes from various kinds of mammals. The results presented here show for the first time that two types of human cells are sensitive to HlyII action. In view of its broad cytotoxic activity as well as the ability to interact with artificial membranes, we assume that HlyII needs no specific receptor to bind to cell membranes.