V. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is... Show moreV. cholerae is a gastrointestinal pathogen which causes extreme watery diarrhea and results in over 120,000 deaths per year worldwide. It is especially prevalent in developing countries that lack proper water treatment and in areas struck by natural disasters such as hurricanes. P. aeruginosa is an opportunistic pathogen that is ubiquitous in nature, and increasingly found in hospitals burn wards, sinks, catheters and other surgical equipment. Both bacteria are developing increased antibiotic resistance through several mechanisms, with one of the most common ones being the formation of a complex exopolysaccharide matrix known as a biofilm. In this study, using metabolic inhibition, we determined that Na+-NQR is essential for the growth of V. cholerae and P. aeruginosa in both nutrient rich and physiological conditions. We were also able to confirm that inhibition of this enzyme, in both growth conditions, resulted in decreased biofilm production, subsequently eliminating one of the main mechanisms for antibiotic resistance of these bacteria. M.S. in Biology, May 2017 Show less
ApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As... Show moreApbE is a novel enzyme that transfers flavin cofactors into subunits NqrB and NqrC of the sodium-dependent NADH dehydrogenase (Na+-NQR). As the first enzyme of the bacterial respiratory chain, the function of Na+-NQR affects the survival and development of pathogenicity in many disease-causing bacteria, including Vibrio cholerae. Our preliminary studies indicate that His257 plays a key role in the catalytic activity of ApbE, and that it is an essential component in the transfer of FMN to NqrC. In order to further study how His257 is specifically involved in the catalytic reaction of ApbE, we produced and characterized four mutants: H257G, H257E, H257K, and H257T; in the presence of the activator, K+. Our data showed that mutants H257E and H257K present minimal flavin transfer activity. Interestingly, the mutants H257G and H257T showed activity several times higher compared to the other mutants, however, their activities were still smaller when compared to wild-type. The data suggests that His257 has a very important role for ApbE activity, but that it is not essential. Furthermore, steady-state kinetics showed that the mutants have similar substrate KM values with the wild-type. In addition, double reciprocal plots from bi-substrate titrations showed that ApbE follows a sequential kinetic mechanism where a ternary complex is formed during the reaction. Show less
