Large-molecular-weight plasmids were isolated from virulent and avirulent strains of Bacillus anthracis. Each strain contained a single plasmid species unique from the others with respect to molecular weight. Bacterial strains were cured of their resident extrachromosomal gene pools by sequential passage of cultures at 42.5 degrees C. Coincidental to the curing of plasmids was a loss of detectable lethal toxin and edema-producing activities and a dramatic decrease in lethal factor and protective antigen serological activities. The involvement of these plasmids in the production of toxin was firmly established by transformation of heat-passaged cells with plasmid DNA purified from the parent strain. The ability to produce parent strain levels of toxin was restored, and the plasmid DNA similar in molecular weight to that isolated from the parent was reisolated in all transformants examined. The exact role these plasmids play in the production of toxin remains to be elucidated. Two additional strains of B. anthracis, designated Pasteur vaccine strains, were examined for the ability to produce toxin and for the presence of plasmid DNA. Both strains were found to be nontoxigenic and contained no detectable plasmid elements. It is therefore likely that we, like Pasteur, cured B. anthracis strains of temperature-sensitive plasmids which code for toxin structural or regulatory proteins.

A chemically defined medium containing 18 amino acids, inorganic salts, rhamnose, choline, and ferric pyrophosphate has been developed. The final concentrations of salts and amino acids were modeled after yeast extract. This medium supported the growth of four serogroups of Legionella pneumophila. Growth in shake cultures at 37 degrees C produced a lag time of approximately 5 h and a generation time of 4 h with a maximum growth yield of 10 9 colony-forming units per ml. A soluble brown pigment was observed in the stationary phase of growth. The optimal pH was 6.3. Rhamnose and choline were stimulatory; arginine, serine, threonine, cysteine, valine, and methionine were essential. Supplemental iron was not required to attain maximum growth, but iron deprivation caused an extended lag phase.

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.

The medium described is a simple yeast extract broth capable of growing large number of Legionella neumophila, the causative organism of Legionnaires disease. Filtration was chosen as a means of sterilization, since medium that was autoclaved did not support growth without the presence of Norite A. The filtered medium gave rapid cell growth and maintained the initial antigen production. The observed generation time was 99 min with a maximum cell population of 2 X 10(2) COLONY-FORMING UNITS PER ML IN APPROXIMATELY 40 H.

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