Cholera is a major cause of illness and death in many developing countries. Currently available vaccines provide only short lived modest protection. Vibrio cholerae produces and secretes a potent toxin that is effector of the diarrhea produced by this organism. In addition to cholera toxin, the organism produces and secrets a variety of other proteins. Understanding the secretory apparatus involved in secretion of proteins from V. cholerae will enable the construction of V. cholerae as a bacterial vaccine vector. Using transposon mutagenesis secretory deficient mutants will be generated in V. cholerae. colonies will be tested for defect in secretion of cholera toxin, protease, and DNase. A chromosomal DNA library will be prepared from the parent strain to complement the excretory deficient phenotype. The gene(s) coding for the secretory apparatus will be characterized. In addition, a gene coding for a DNase gene from V. cholerae was cloned. A chromosomal library from V. cholerae will be transformed into E. coli containing the cloned DNase gene. Gene allowing for the DNAse to be secreted from E. coli will be analyzed.
