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A completely synthetic gene encoding fragment C, a ϳ50-kDa fragment, of botulinum neurotoxin serotype A was constructed from oligonucleotides. The gene was expressed in Escherichia coli, and full-sized product was produced as judged by Western blot (immunoblot) analysis. Crude extracts of E. coli expressing the gene were used to vaccinate mice and evaluate their survival against challenge with active toxin. Mice given three subcutaneous vaccinations were protected against an intraperitoneal administration of 10 6 50% lethal doses (LD 50) of serotype A toxin. The same mice survived when challenged with 3 LD 50 of botulinum toxin serotype E but died when challenged with 10 LD 50 of serotype E or 3 LD 50 of serotype B. Purified fragment C was compared with the botulinum toxoid vaccine in a vaccination and challenge study. Fragment C was as efficacious in protecting against challenge with active botulinum neurotoxin serotype A as the toxoid vaccine. This recombinant protein product has many properties that make it a good candidate for human use to protect against botulinum toxin.

Escherichia coli B dependence on CO2 for growth was demonstrated. At suboptimal CO2 concentrations the rate of growth was controlled by CO2 concentration. Many techniques used to minimize the lag period preceding culture growth were described in the early microbiological literature, whereas others are a part of the legacy of unpublished observations from many laboratories. The appearance of a lag period after successive transfers of cultures in the same medium always was an enigma that has been subject to a number of theories, but each hypothesis was unable to account for some experimental observations. While determining conditions to obtain reproducible growth of the obligate autotrophic aerobe, Alcaligenes eutrophus, we found that a specific concentration of one component, bicarbonate, produced immediate growth without the customary lag period (6). This prompted a comparative study with an organism at the opposite end ofthe nutritional scale. Anaerobically grown Streptococcus sanguis responded in the same way, but CO2 was the active species (7). In this report we show that growth of a washed stationary-phase inoculum of the facultative aerobe, Escherichia coli, also will grow without a lag period if a suitable concentration of CO2 is provided. With all three organisms the initial and subsequent exponential growth rate was controlled solely by the concentration of CO2 or bicarbonate. These findings indicate that the effect of CO2 or bicarbonate on the conventional lag period and on the growth rate is not an unusual phenomenon and suggest that CO2 or bicarbonate may have the same regulatory function in all microorganisms.Carbon dioxide or its hydrated form, bicarbonate, has been a recognized requirement for cell growth since Valley and Rettger (8) perfonned an extensive survey and demonstrated that none of the more than 100 microorganisms tested would grow if CO2 were excluded. More recently the basis for the CO2 requirement by heterotrophic microorganisms was established; C02-fixing enzymes were isolated and their functions in various metabolic pathways are now known. The qualitative CO2 requirement for growth is not the subject of this report. We wish to emphasize the fundamental physiological effect of C02 concentration on regulation of the growth rate. The lag period appears to be the initial manifestation of the same effect.Walker (9) first showed that added CO2 shortened the lag period of E. coli, and Neidhardt et al. (5) recently reported that the lag period was virtually eliminated when bicarbonate was incorporated in the medium. Quantitative demonstration ofthe effect ofC02 on growth requires a minimal medium and a system for accurately maintining low concentrations of C02. Small incremental increases in C02 concentration must be tested to assure detection of the optimum, for the optimum may span a narrow range of concentrations (6,7).A vessel with a 1-liter working capacity was designed to receive three separate gases (N2, 02, and CO2) which were mixed in the vessel to fonn an atmosphere of any desir...

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