PERIPLASMATIC PROTEINS OF GRAM-NEGATIVE BACTERIA INVOLVED IN THE BIOGENESIS OF METALLO-b-LACTAMASES

BRAMBILLA L.; MORAN BARRIO, J; VIALE AM.

Rosario

Congreso; Quinto Congreso Argentino de Microbiología General (SAMIGE).; 2008

Sociedad Argentina de Microbiología General

All proteins fold to a characteristic native functional conformation, a situation that requires in vivo participation of molecular chaperones which help promoting proper protein folding and prevent aggregation of misfolded states. Although a plethora of studies exist on the role of cytoplasmic chaperones, much less is known about periplasmic folding assistants. We analyzed here whether the biogenesis of metallo-b-lactamases (MbLs) requires assistance in the periplasmic compartment of Gram-negative bacteria. We first studied the biogenesis of the MbL GOB in Escherichia coli strains deficient in otherwise suggested periplasmic chaperones such as DsbA, DsbC, DsbG, PpiA, PpiD, SurA, FkpA and DegP. Productive folding of this enzyme was evaluated by expressing the cognate gene (including its leader sequence) in the above mutants and analyzing its effects on b-lactam antibiotic resistance. Expression of the gob gene in any of these mutants did not result in a significant decrease of cefotaxime resistance when compared to the wild-type strain, suggesting that none of the above periplasmic proteins participate individually in GOB folding, or the presence of redundant functions among them. In order to identify novel periplasmatic folding assistants, we generated MudJ random insertion mutants in GOB-producing Salmonella enterica var. Typhimurium 13544 and selecting for clones with reduced cefotaxime resistance. One insertion was localized in the phsB gen, which codes for a FeS protein and forms part of the phsABC operon that encodes the subunits of thiosulfate reductase responsible for H2S production during anaerobic respiration. Our results suggest that this FeS protein may aldo function in the productive folding of metalloproteins such as MbLs in the periplasm of Gram-negative bacteria.