We will study the genetic basis for the coordinate regulation of bacterial virulence determinants utilizing three experimental organisms-Vibrio cholerae, Bordetella pertussia and Escherchia coli. Our goal is to define the molecular mechanism by which groups of virulence genes in each organism are concomitantly expressed or repressed. The experimental approach to this goal will be similar for each of the genetic systems examined and will include the following steps: 1. Analysis of nutritional and physical parameters that regulate virulence; 2. Isolation of TnphoA gene fusions that are regulated by these parameters; 3. Confirmation that the expected virulence regulatory gene controls the expression of these TnphoA gene fusions; 4. Identification of the promoter and control sites for selected TnphoA fusions by standard methods (i.e., DNA sequencing, S1 and primer extention mRNA mapping, deletion, chemical, and oligonucleotide-directed mutagenesis, and protein-DNA binding studies). For V. cholerae we will expand our analysis of the toxR coordinate regulatory system to include other possible ToxR-regulated virulence properties (i.e., expression of pili, OMPs, neuraminadase, protease, hemolysis, and motility). In B. pertussis we will continue our analysis of vir positive and negative coordinate regulation to include promoter structure and genetic analysis of the modulation response. For V. cholerae and E. coli we will study iron-regulatory responses focusing on the genes for Shiga- like toxin type 1 (sltAB) as well as other virulence factors (e.g., hemolysin, invasive properties, and adherence factors) potentially controlled by the iron-responsive fur gene products of each organism. Where possible, we will apply data we collect to the development and improvement of cholera and pertussis vaccines.
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