The main objective of this project is to understand the regulation of the molecular events involved in the export of the heat stable enterotoxin (ST) of Escherichia coli. The information could be used to design a simpler diagnostic test for ST and could further lead to a new therapeutic approach for pathogenic gram negative bacteria that export virulence factors. The sequencing of the 1000 base pair DNA fragment that encodes for ST will unveil new restriction sites for further subcloning of the ST gene. The elimination of flanking non-ST sequences will facilitate the quantification of ST-mRNA by RNA-DNA hybridization techniques. The size of the ST-mRNA encoded by the cloned ST gene, the effect of the cAMP positive regulatory system on the ST enterotoxin activity, and the localization of the ST-mRNA (membrane bound or free polysomes), as well as the absence of ST-DNA dosage effect on extracellular ST will be analyzed by RNA-DNA hybridization. The subcloned ST gene will be placed under the strong promoter PL of bacteriophage Gamma; the expected increase in ST-mRNA, ST enterotoxin and peptide intermediates in export of ST should facilitate their characterization. The shared metabolic steps in the export of periplasmic and outer membrane proteins with exoproteins will be analyzed by introducing the ST plasmids, previously marked with DNA encoding for the intracellular enzyme dihydrofolate reductase, into export deficient mutants. The cellular localization of the ST peptides will be monitored after labeling the mutants with S35 cysteine or Na2-35S04 and analyzing in PAGE and immunologically, the cellular compartments (cytoplasm, periplasm, inner and outer membrane) of the bacteria. Whole cells and minicells treated with export inhibitors will also be used to characterize the peptide intermediates in export of ST. The role of the carboxy terminal end of the ST peptide in export and activity will be analyzed by the construction of mutants with synthetic oligonucleotides. Of particular interest is the participation of cysteines in these events.
