Construction of Bacillus anthracis mutant strains producing a single toxin component

Abstract

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.

The vacuolar ATPase proton pump is required for the cytotoxicity of Bacillus anthracis lethal toxin

“…PA and LF were obtained from the culture supernatant fluids of the Bacillus anthracis strains RP42 and RP4, respectively [21], and purified as described in [22]. Briefly, culture supernatants equilibrated in 20 mM Tris-HCl, pH 6.5 buffer were loaded onto the Mono-Q HR column (Pharmacia, Uppsala, Sweden).…”

In-vitro characterisation of the phagocytosis and fate of anthrax spores in macrophages and the effects of anti-PA antibody

“…Two double-mutant derivatives of Sterne strain 7702 were also provided by M. Mock (Pasteur Institute, Paris, France). Strain RP4 has deletions in the loci encoding PA and oedema factor (EF) and produces only the lethal factor (LF) component of anthrax lethal toxin; RP42 has deletions of both the EF and LF genes and produces only PA [8,9]. All growth media for strains RP4 and RP42 contained erythromycin 10 mg=L and kanamycin 20 mg=L.…”

Use of a photoactivatable lipid to probe the topology of PA63 of Bacillus anthracis in lipid membranes

“…The anthrax protective antigen (PA) was produced by a double mutant strain of B. anthracis, in which the genes coding for LF (lef) and EF (cya) were inactivated by intragenic deletion. This strain was capable of producing only a single toxin component (PA) and was used to avoid any contamination from the two other proteins EF and LF [12]. The production and the purification of PA were carried out as previously described [12,13].…”

“…This strain was capable of producing only a single toxin component (PA) and was used to avoid any contamination from the two other proteins EF and LF [12]. The production and the purification of PA were carried out as previously described [12,13]. PA ∆FFD was obtained from a recombinant strain carrying a deletion of the F313FD residues [14] and purified using the same protocol as PA.…”