Effect of the method of agitation on the accumulation of protective antigen in cultures of Bacillus anthracis.
Accumulation of protective antigen was essentially independent of culture volume, provided the organisms were kept in suspension by stirring. Agitation by orbited shaking was unsatisfactory.
Cloning and characterization of a gene whose product is a trans-activator of anthrax toxin synthesis
The 184-kb Bacillus anthracis plasmid pXO1, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to measure pag promoter activity, we cloned in Bacillus subtilis a gene (atxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted M(r) of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis. The cloned atxA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. beta-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate.
Studies on Immunity in Anthrax VIII. Relationship of Complement-Fixing Activity to Protective Activity of Culture Filtrates
Summary Complement fixation reactions were obtained with rabbit antisera and culture filtrates or partially purified preparations of protective antigen of Bacillus anthracis . By selection of appropriate antisera and adjustment of conditions, fixation by heat-stable antigens of no protective activity could be minimized or eliminated. Fixation titers were compared with the activity of the antigen preparations in active immunization of guinea pigs. A method was developed for deriving a dose-response curve from the mean reciprocal survival time after challenge of immunized guinea pigs. By use of this curve the results of the immunization assays were converted to relative potency units of protective antigen. There was significant correlation between the complement fixation titers and the immunizing activity of the preparations. The deviations were no greater than would be expected in view of the limited precision of the in vivo assay when practical numbers of guinea pigs are used. The significance of the results is discussed.