Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 µg.

Identification of anthranilate and benzoate metabolic operons of Pseudomonas fluorescens and functional characterization of their promoter regions.

BACKGROUND: In an effort to identify alternate recombinant gene expression systems in Pseudomonas fluorescens, we identified genes encoding two native metabolic pathways that were inducible with inexpensive compounds: the anthranilate operon (antABC) and the benzoate operon (benABCD). RESULTS: The antABC and benABCD operons were identified by homology to the Acinetobacter sp. anthranilate operon and Pseudomonas putida benzoate operon, and were confirmed to be regulated by anthranilate or benzoate, respectively. Fusions of the putative promoter regions to the E. coli lacZ gene were constructed to confirm inducible gene expression. Each operon was found to be controlled by an AraC family transcriptional activator, located immediately upstream of the first structural gene in each respective operon (antR or benR). CONCLUSION: We have found the anthranilate and benzoate promoters to be useful for tightly controlling recombinant gene expression at both small (< 1 L) and large (20 L) fermentation scales.

Biodesulfurization of fossil fuels.

Biotechnological techniques enabling the specific removal of sulfur from fossil fuels have been developed. In the past three years there have been important advances in the elucidation of the mechanisms of biodesulfurization; some of the most significant relate to the role of a flavin reductase, DszD, in the enzymology of desulfurization, and to the use of new tools that enable enzyme enhancement via DNA manipulation to influence both the rate and the substrate range of Dsz. Also, a clearer understanding of the unique desulfinase step in the pathway has begun to emerge.