Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. The gene coding for the S-layer protein (sap) was cloned on two contiguous fragments in Escherichia coli, and the complete sequence of the structural gene was determined. The protein, Sap, is composed of 814 residues, including a classical prokaryotic 29-amino-acid signal peptide. The mature form has a calculated molecular mass of 83.7 kDa and a molecular mass of 94 kDa on a sodium dodecyl sulfate-polyacrylamide gel. Sap possesses many charged residues, is weakly acidic, and contains only 0.9% methionine and no cysteine residues. The N-terminal region of Sap shares sequence similarities with the Acetogenium kivui S-layer protein, the Bacillus brevis middle wall protein, the Thermotoga maritima Omp␣ protein, and the Bacillus thuringiensis S-layer protein. Electron microscopy observations showed that this S-layer is not observed on B. anthracis cells in which sap has been deleted.Bacillus anthracis, the causative agent of anthrax, is a grampositive, spore-forming bacterium. Fully virulent bacilli are both capsulated and toxinogenic. The two toxins (lethal and edema toxins) are encoded by the virulence plasmid pXO1, and capsule synthesis is dependent on the presence of a second virulence plasmid, pXO2. When the capsule is absent, the cell wall of B. anthracis appears layered and is composed of small fragments displaying a highly patterned ultrastructure (8). Holt and Leadbetter (9) previously described a hexagonal lattice on the surface of vegetative cells of B. anthracis. This cell surface structure most likely represents what is called an S-layer, with p6 symmetry and a center-to-center spacing of the particles of 7 to 10 nm. S-layers, or surface arrays, have been found to be the outermost component of many archaebacteria and eubacteria. In most cases, single proteins compose these structures. When present, they represent 5 to 10% of the total cell protein, implying that their synthesis is energy-consuming for the bacterium. The fact that S-layers are found ubiquitously suggests that they play vital roles in the interaction between the cell and its environment. It has been suggested that the S-layer is an important virulence factor for bacteria such as Aeromonas salmonicida, Campylobacter fetus, and Rickettsia spp., protecting against complement killing, facilitating binding of the bacterium to host molecules, or enhancing its ability to associate with macrophages (see reference 21).Unlike most S-layers, that of B. anthracis is not the outermost component of the virulent bacilli since they are encapsulated; Azotobacter spp. are another of the rare examples of bacteria possessing both a capsule and an S-layer. The S-layer may have an important function in linking the capsule to the peptidoglycan wall or controlling the exchange of molecules with the environment. Bacillus thuringiensis, a closely related entomopathogen bacillus, possesses an S-layer (12). The B. thuringiensis S-layer is composed of linear arrays of sm...

Single-walled asolectin vesicles loaded with K+ at pH 7.00 released their K+ content upon incubation with tetanus toxin fragment B but only when the incubation was at pH below 5.00. Whole tetanus toxin exhibited only a weak releasing activity. Toxin light a chain and the carboxyl-terminal 48,000-dalton moiety of the heavy chain (fragment UC) were unable to provoke K+ release from vesicles at any pH. K+ release from lipid vesicles could also be detected with tetanus toxin heavy ,3 chain at low pH. Furthermore, using a detergent binding assay ([3H]Triton X-100), we have also shown that an hydrophobic domain, localized in the 50,000-dalton terminal polypeptide of tetanus toxin heavy chain, is detectable at pH 3.60 but not at pH 5.00. These results lead us to conclude that the ability of tetanus toxin fragment B to release K+ from asolectin vesicles at low pH is due to the 50,000-dalton amino-terminal polypeptide of the heavy chain present in toxin fragment B. We propose that this phenomenon is caused by channel formation across the vesicle membrane as has been observed for the 23,000-dalton amino-terminal moiety of diphtheria toxin fragment B.Tetanus toxin is a 150-kilodalton (kDal) protein produced by the anaerobic bacterium Clostridium tetani. This toxin causes spastic paralysis by acting on synapses at the level of spinal cord motoneurons (1). Tetanus toxin is a two-chain protein. A heavy chain (( chain, 100 kDal) is joined by a disulfide bridge to a light chain (a chain, 50 kDal) (2, 3). The heavy chain can be split by proteases to produce a nontoxic fragment (fragment C or IIC, =50 kDal) which retains ganglioside-binding properties (4, 5). The remainder of the heavy chain linked to the light chain has been called fragment B (3). It has been recently shown that fragment C is located at the COOH terminus of the 18 chain (6). A schematic representation of tetanus toxin is given in Fig. 1.Through its fragment IIC, tetanus toxin binds to ganglioside GDlb on presynaptic membranes (1, 4, 5), and there is good evidence now that toxin molecules block synaptic transmission by interfering with the release of neurotransmitter substances such as glycine, y-aminobutyric acid, norepinephrine, and acetylcholine (7, 8). However, nothing is known about the mode of action of tetanus toxin at the molecular level.We were struck by the analogy of architecture between tetanus and diphtheria toxins ( Fig. 1) and considered that this might be related to similarity in mode of action. The cytotoxic effect of diphtheria toxin requires the entry of its enzymatic fragment A into the cytosol of susceptible animal cells (9). Recent work strongly suggests that diphtheria toxin fragment A reaches the cell cytoplasm by crossing the lipid membrane through a pore formed at low pH by the hydrophobic region located in the NH2-terminal part of the toxin fragment B that is present in B45 (10-12), probably when endocytotic vesicles in which toxin is internalized fuse with primary lysosomes (13, 14).Here, we present evidence that the NH2-termi...

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The two protein exotoxins secreted by Bacillus anthracis are composed of three distinct components : protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitvo and in vivo.