Enterotoxinogenic E.coli (BTEC) which produce heat-labile enterotoxin (LT are among the leading causes of diarrheal disease in man and agricultural animals. Investigation of the genetic regulation of LT will lead hopefully lead to a clearer understanding of how ETEC strains interacts with the host and causes disease as well as to the development of improved vaccines. Information gained with LT may provide information that is usefiil for other enteric diseases caused by other E. coli strains and Shigella whose virulence factors are also thermoregulated by the same regulatory protein, H-NS. Electrophoretic mobility shift assays (EMSA) and in vitro transcription experiments will be used to determine if H-NS mediates its temperature control on LT expression by directly binding to LT operon DNA, specifically the downnstream regulatory element (DRE). Further characterization of the LToperon DNA will be achieved by isolating mutants with altered LT expression that have been obtained by carrying out chemical mutagenesis and then oligonucleotide-directed mutagenesis. H- NS interactions with the mutated DNA will be evaluated by EMSA and in vitro transcription if appropriate. Several H-NS-sensitive virulence factors have been shown to have their expression alterated by changes in DNA topolgy. Supercoiling effects on LT expression will be investigated by evaluating the effects of gyrase inhibitors , by using strains that have mutations in the genes encoding top oisomerase l and the two subunits of gyrase, and by evaluating environmental conditions beside temperature which are known to alter supercoiling Since many H-NS binding sites have been shown to display intrinsic curvature, this characteristic of LT operon DNA will also be investigated. The LT DRE will be studied to see if this DNA fragment moves aberrantly through agarose or acrylamide gels at low temperatures. If the LT DRE is found to be curved, the antibiotic distamycin can be used to determine if this curvature is essential for binding of H-NS or other regulatory proteins (yet to be determined).
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