Secretion of GOB Metallo-β-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-β-lactamases (MβLs) are zinc-dependent enzymes produced by many clinically relevant gram-negative pathogens that can hydrolyze most β-lactam antibiotics. MβLs 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 MβL GOB-18 demands cooperation between a main chaperone system of the bacterial cytoplasm, DnaK, and the Sec secretion machinery. Using the expression of the completegob-18 gene from the gram-negative opportunistic pathogenElizabethkingia meningoseptica inEscherichia 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 β-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.