Folding of periplasmic metallo B lactamase in Salmonella Typhimurium

BRAMBILLA, L.; MORÁN BARRIO, JORGELINA; VIALE, ALEJANDRO MIGUEL

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2007

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Protein biogenesis in all cells is assisted by different protein systems known as molecular chaperones, which prevent aggregation of non-native proteins favoring their productive folding or degradation. Although cytoplasmic chaperones are well characterized, few data exists on folding assistants present in the periplasm of Gram-negative bacteria. We studied periplasmic protein biogenesis using as a model the metallo-b-lactamse (MbL) GOB-18 form Elizabethkingia meningoseptica (Moran-Barrio et al, J.Biol.Chem., 282(25), 2007). We expressed the gob gene in Salmonella typhymurium, where the enzyme precursor is correctly processed to its periplasmic native form. Folding assistants were searched by MudJ-based mutagenesis and selected for clones with decreased cefotaxime resistance. From more than 10,000 mutants, five showed reduced antibiotic resistance and were further characterized by growth behavior under normal and stress conditions, SDS-PAGE, Southern-blot, and sequencing analyses. In four insertional mutants we found that the disrupted genes were not directly related to antibiotic resistance. On the contrary, the fifth mutant consistently showed much reduced growth rate under stress conditions, no significance differences in heat stress protein pattern, and an insertional disruption which could account for the reduced cefotaxime resistance phenotype and a MbL folding assistant.