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Antimicrobial Agents and Chemotherapy, July 2009, p. 2908-2917, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.01637-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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 {triangledown}

Jorgelina Morán-Barrio, Adriana S. Limansky, and Alejandro M. Viale*

Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina

Received 12 December 2008/ Returned for modification 1 April 2009/ Accepted 6 May 2009

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 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 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.

* Corresponding author. Mailing address: Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina. Phone: 54-341-4351235. Fax: 54-341-4390465. E-mail: viale{at}ibr.gov.ar

{triangledown} Published ahead of print on 11 May 2009.

Antimicrobial Agents and Chemotherapy, July 2009, p. 2908-2917, Vol. 53, No. 7
0066-4804/09/$08.00+0     doi:10.1128/AAC.01637-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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