| The human pathogen, Vibrio cholerae, is a Gram-negative motile bacterium that causes the disease cholera. New variants of the pathogen continually evolve, able to circumvent established immunity and resist prophylactics. With a fatality ratio above 20%, cholera infections result in more than 100,000 deaths per year. This dissertation studies the two general diffusion porins of V. cholerae, OmpU and OmpT. Porins are trans-membrane, beta-barrels that span the outer membrane. The monomers associate to form trimers, allowing for the permeation of small hydrophilic solutes.;The second part of this dissertation looks at the electrophysiological modulation of OmpT by bile acids. Chenodeoxycholate and deoxycholate are both strong blockers of OmpT. Taurodeoxycholate has a mild modulatory effect on OmpT. This modulation is concentration and voltage dependent. These results show that these bile acids are able to block OmpT, presumably as they transit through the pore. Other bile acids, glycodeoxycholate, lithocholate, and cholate were ineffective. The presented results highlight the important differences in OmpU and OmpT and explain the imperative need for differential genetic regulation of these porins between the aquatic and the human intestinal environments for bacterial survival.;Using a homology model of OmpU based on the E. coli porins, multiple OmpU mutant phenotypes were investigated; including sensitivity to beta-lactam antibiotics, growth sensitivity to deoxycholate, and biofilm induction by deoxycholate. The results indicate that specific residues play a variety of roles in controlling the passage of various compounds. Mutations of the arginine residues on the barrel wall affect pore size and growth in deoxycholate. Sensitivity to large cephalosporins is affected by the mutation of D116 to alanine. This residue is located on the L3 loop in the constriction zone of the porin. Using electrophysiological studies, this mutant was also shown to affect the propensity of the channel to close, and strongly diminished the cation selectivity of the porin. The L3 residue mutations also effected biofilm induction in the presence of deoxycholate. Based on the homology model, these results lead to the conclusion that the mutated residues influence the pore characteristics of OmpU in various ways, with dependence on the permeating molecule. |
