Secretion of GOB metallo-beta-lactamase in Escherichia coli depends strictly on the cooperation between the cytoplasmic DnaK chaperone system and the Sec machinery: completion of folding and Zn(II) ion acquisition occur in the bacterial periplasm.
Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes produced by many clinically relevant gram-negative pathogens that can hydrolyze most beta-lactam antibiotics. MbetaLs are synthesized in the bacterial cytoplasm as precursors and are secreted into the periplasm. Here, we report that the biogenesis process of the recently characterized MbetaL GOB-18 demands cooperation between a main chaperone system of the bacterial cytoplasm, DnaK, and the Sec secretion machinery. Using the expression of the complete gob-18 gene from the gram-negative opportunistic pathogen Elizabethkingia meningoseptica in Escherichia coli as a model system, we found that the precursor of this metalloenzyme is secreted by the Sec pathway and reduces cell susceptibility to different beta-lactam antibiotics. Moreover, acting with different J proteins such as cytoplasmic DnaJ and membrane-associated DjlA as cochaperones, DnaK plays an essential role in the cytoplasmic transit of the GOB-18 precursor to the Sec translocon. Our studies also revealed a less relevant role, that of assisting in GOB-18 secretion, for trigger factor, while no significant functions were found for other main cytoplasmic chaperones such as SecB or GroEL/ES. The overall findings indicate that the biogenesis of GOB-18 involves cytoplasmic interaction of the precursor protein mainly with DnaK, secretion by the Sec system, and final folding and incorporation of Zn(II) ions into the bacterial periplasm.
Morán-Barrio
Jorgelina
J
Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Limansky
Adriana S
AS
Viale
Alejandro M
AM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002439:Cefotaxime / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D041965:Flavobacteriaceae / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D001618:beta-Lactamases; C063842:dnaK protein, E coli; D002439:Cefotaxime