A PERIPLASMIC PROTEIN INFLUENCING GOB METALLO-BETA-LACTAMASE BIOGENESIS IN GRAM NEGATIVE BACTERIA

BRAMBILLA, L.; VIALE A.M.

Tucumán

Congreso; XLV. Reunión anual de Sociedad Argentina de Investigacion en Bioqumica y Biologia Molecular; 2009

SAIB

<!-- /* Font Definitions */ @font-face {font-family:"TimesNewRomanPSMT,Italic"; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-charset:0; mso-generic-font-family:roman; mso-font-format:other; mso-font-pitch:auto; mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Metallo-b-lactamases (MbL) can hydrolyze most clinically employed b-lactam antibiotics, representing the main mechanism of resistance in Gram-negative pathogens. We have recently reported that GOB MbL is secreted in E. coli by the Sec machinery in an extended conformation, adopting the proper folding and Zn (II) ion in the bacterial periplasm. The aim of our work is the identification and cooperation of proteins influencing GOB biogenesis in the periplasm of Gram-negative bacteria. For this purpose we expressed the gob-18 gene in Salmonella enterica and used MudJ random mutagenesis, searching for insertion mutants displaying decreased cefotaxime (CTX) resistance. One of these mutants was affected in the expression of dacD, which codes for a periplasmic protein proposed to act as a penicillin-binding protein with poor DDcarboxypeptidase activity. The mutation produced no effect on the intrinsic CTX resistance of the bacteria. The Escherichia coli DdacD mutant expressing gob-18 also showed decreased CTX resistance. Analysis of periplasmic fractions from wild type and DdacD cells by SDS-PAGE indicated several changes in the periplasmatic protein profile of the mutant, and immunoblots with anti-GOB revealed lower quantities of this protein in DdacD cells. The overall results suggest a new function for DacD in either the secretion or stability of MbLs in the bacterial periplasm.