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.
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.
Jorgelina
J
Morán-Barrio
Adriana S
AS
Limansky
Alejandro M
AM
Viale
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