HSP70 cytosolic chaperones involved in the secretion of metallo-b-lactamases in Gram-negative bacteria

MORAN BARRIO, J; LIMANSKY, A. S; VIALE, A. M

Pinamar

Congreso; XLI Reunión Anual SAIB; 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

In gram-negative bacteria, periplasmic proteins are synthesized in the cytoplasm as precursors and directed to the general secretory pathway (Sec) for their secretion. Whether and how the main cytoplasmic chaperones participate in this process is still obscure. We studied here the putative cooperations of the DnaK, GroE, and TF systems with the Sec pathway in E. coli by genetic procedures, using as a model the metallo-b-lactamase (MbL) GOB from the gram-negative pathogen Elizabethkingia meningoseptica. The coding GOB sequence was cloned and expressed in E. coli mutants in the different chaperone systems, and their capability to secrete an active MbL enzyme was analyzed by a plate dilution assay. Mutants in SecA or SecYEG showed very poor MbL activity in comparison to wt strains, indicating fundamental roles of the Sec pathway in proper GOB secretion. In turn, mutants in cytoplasmic chaperones including DnaK, DnaJ and Trigger Factor (TF) were also deficient in MbL secretion, indicating their importance in this process. The use SecA-TF double mutants provided further evidence for synergistic effects between these chaperones in GOB secretion. The overall data further highlights the existence of complex interactions between the Sec machinery with Hsp70/DnaK cytoplasmic chaperones and functional analogs in the secretion of MbLs in gram-negative bacteria.