Elaboration of Bacillus anthracis Antigens in a New, Defined Culture Medium

Abstract

Improved culture conditions and a new, completely synthetic medium (R medium) were developed to facilitate the production of Bacillus anthracis holotoxin antigens. Levels of these antigens up to fivefold greater than the highest previously reported values were recovered with the described system. Cultures of Sterne, V770-NP1-R, and Vollum 1B strains of B. anthracis were monitored for growth, pH change, glucose utilization, supernatant protein concentration, lethal toxin activity, and protease activity.

Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion

“…anthracis Sterne (pXO1 +; strain 7702) and its cured pXO1 derivative (pXO1-; strain 7700) [15] were used in this study. Cells were grown in a 5% CO 2 incubator in R-medium [10] supplemented with 0:8% sodium bicarbonate, except when mentioned otherwise, and with 5 tzg/ml kanamycin when the strains were harboring a recombinant plasmid. 2 mM Alanine was added to the medium when spores were germinated.…”

“…Medium composition is known to influence toxin production [10] and gene expression. In Bacillus subtilis, catabolite repression of several genes (e.g.…”

“…Toxin gene expression and capsule formation are both influenced in vitro by the medium composition and the presence of CO 2. PA, EF and LF are poorly produced in rich media, but are readily synthesized in minimal medium if bicarbonate is added or if the culture is grown under CO 2 [10].…”

Monoclonal antibodies against spore antigens of Bacillus anthracis

“…For use in immunoblotting, spores were extracted in 5 mM Tris HCI buffer, pH 9.8, containing 1% (w/v) SDS, 50 mM 2-mercaptoethanol, and 10 mM disodium ethylene diamine tetraacetate. Vegetative cell preparations were grown in nutrient broth in shake flasks sealed with cotton plugs or in R medium [16] in screw capped flasks, overnight at 37 ° C. Cells were then washed three times in phosphate buffered saline (PBS) pH 7.5, and inactivated by addition of formaldehyde to 1% (v/v) and incubation for at least two days at room temperature.…”

Evaluation of Bacillus subtilis strain IS53 for the production of Bacillus anthracis protective antigen

“…Cultures were incubated with shaking (150 rev min-') at 37°C for 14 h. Growth was monitored by optical density at 540 nm. The following media, with and without the addition of yeast extract (Difco Laboratories Ltd, Surrey), were examined for their ability to support growth and the production of PA: Ristroph medium (Ristroph and Ivins 1983), modified Ristroph (Leppla 1988), further modified Ristroph (Leppla 1991), synthetic N medium (Belton and Strange 1954) and minimal medium (Anagnostopoulos and Spizizen 1961). All media were supplemented with kanamycin to 10 mg I-' and were filter sterilized.…”