The primary goal of the proposed investigation is the detailed elucidation of the molecular aspects of toxin regulation in V. cholerae. Biochemical and genetic methods will be used to locate the DNA sites involved in formation of the cholera toxin operon (ctx) promoter and control sites. We will attempt to define the precise molecular mechanism by which the positive regulatory gene toxR, activates ctx transcription. The regulatory role played by the repetitive sequence TTTTGAT will be studied by localized and mismatch primer mutagenesis. The nutritional and physical factors which regulate toxin production will be correlated with specific transcriptional effects on ctx or toxR. We will continue to study ctx amplification as a genetic mechanism controlling toxin production in E1 Tor strains of V. cholerae. In this regard, we will test the repetitive sequence RS1 for its ability to mediate various recombinational events including transposition, duplication and site-specific recombination. The knowledge gained from all the above studies will be used to devise methods with which to study toxin regulation and ctx amplification in vivo. Specifically, we will study how the intestine provides a selective environment for increased toxin expression by defining which genes (ctxA, ctxB or toxR) are necessary for in vivo selection of genetic reversion and amplification events. We will apply what we have learned about ctx regulation and expression to the development of improved live and dead, oral vaccines against cholera. Mutations will be constructed in the cloned recA gene of V. cholerae and then recombined back in place of the rec+ gene of the prototype live oral vaccine strain 0395-N1. In addition to recA, streptomycin-dependent, DAP- and ga1E mutations will also be incorporated into 0395-N1 derivatives as a means of limiting the persistence of this strain in humans and the environment. Finally, derivatives of 0395-N1 which are suitable for use as a dead, oral cholera vaccines will be constructed which hyperproduce the B subunit of the toxin in both a secreted and nonsecreted form.
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