IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Biosynthesis and secretion of a novel metallo-b-lactamase from an opportunistic pathogen in e. coli.
Autor/es:
MORAN-BARRIO, J.; GONZALEZ, J.; ADRIANA SARA LIMANSKY; VILA, A.; VIALE, A.
Lugar:
Buenos Aires
Reunión:
Congreso; . Congreso Sociedad Argentina de Microbiología General; 2006
Institución organizadora:
(SAMIGE)
Resumen:
In E. coli, secreted proteins with an ultimate destination to the periplasm are synthesized in the cytoplasm as precursors with a cleavable amino-terminal signal sequence. The translocation across the inner membrane is accomplished by the Sec machinery (SecYEG/SecA). In the cytoplasm, preproteins must be sorted to the membrane complex in an extended conformation, protected from aggregation and competent with the secretion process. s32-dependent cytoplasmic chaperones such as GroEL/GroES (Hsp60-type) and DnaK/DnaJ/GrpE (Hsp70-type), as well as s32-independent chaperones such as SecB and Trigger Factor (TF) had been proposed as candidates for this function. We use as a secretion model the expression in E. coli of the metallo-b-lactamase (MbL) GOB gene from the gram-negative opportunistic pathogen Elizabethkingia meningoseptica (family Flavobacteriaceae, class Flavobacteria). The GOB enzyme shares the common ab/ba sandwich fold characteristic of the MbLs. However, the primary structure shows some residue substitutions in highly conserved positions that build up the active site in most related MbLs, making the GOB lactamase a very interesting issue of study. Size exclusion chromatography and isoelectric focusing revealed that GOB is a monomeric enzyme with a pI of 8.3. Kinetic parameters additionally indicated a broad-substrate spectrum to b-lactam antibiotics. However, and contrasting other related di-zinc MbLs, GOB is a mono-zinc enzyme and uptake of an extra zinc ion is not essential for activity. Based on EXAFS data and 3D modeling, it is proposed that GOB has a novel mononuclear zinc active site different from other related MbLs. Genetic procedures indicated a fundamental role of the Sec machinery (SecA and the SecYEG translocon) for GOB secretion to the periplasm, and a minor role of SecB in this process. In addition, main cytoplasmic chaperones including DnaK and TF were required for GOB secretion, as judged by the low levels of MbL activity found in the corresponding mutants. Moreover, the use of double mutants deficient in SecA and TF provided evidence for a close cooperation between these two chaperones in GOB secretion. Conversely, GroEL/S deficient mutants showed minor effects on GOB secretion, indicating minor or no roles of these chaperonins in this process. Where and how the uptake of the zinc ion takes place and whether specific periplasmic components are involved in productive GOB folding are under study