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

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

La Plata, Buenos Aires

Congreso; Segundo Congreso Argentino de Microbiología General (SAMIGE); 2005

Sociedad Argentina de Microbiología General

Although the necessary information for a protein to reach its subcellular localization and native structure is contained in the amino acid sequence, in vivo this process requires the participation of folding assistants. In E. coli, secreted proteins to the periplasm are synthesized as precursors with a cleavable amino terminal signal sequence. Early steps in secretion involve the translocation of precursors across the inner membrane, a process requiring the Sec translocation machinery. Precursors must remain in an unfolded, secretion-competent state in the cytoplasm, which is accomplished by SecB and yet unidentified chaperones. We studied here by genetic procedures whether the main cytoplasmic chaperones cooperate in vivo with the Sec machinery, using as a model the expression in E. coli of the gene of the metallo-b-lactamase (MBL) GOB from the gram-negative pathogen Elizabethkingia meningoseptica. The use of E. coli chaperone Sec mutants demonstrated a fundamental role of this machinery (SecA and the SecYEG translocon) in GOB secretion. In addition, main cytoplasmic chaperones including the DnaK system and Trigger Factor (TF) were required for GOB secretion, as judged by the low levels of MbL activity found in the corresponding mutants. Moreover, the use of secA-tig double mutants provided evidence for a close cooperation between these two chaperones in GOB secretion. Conversely, mutants in GroEL/S had minor effects on GOB secretion, suggesting minor or no roles of these chaperonins in this process. In summary, the overall data revealed that the DnaK system and other functional homologs of the bacterial cytoplasm are required components in the secretion of MbLs in Gram-negative bacteria.