Characterization of the Genes Involved in Citrate Fermentation in Enterococcus faecalis

BLANCATO, VS; REPIZO, GD; MAGNI, C

Iguazú (Misiones)

Congreso; XL Reunión Anual de SAIB; 2004

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Enterococci compose the microbial association of a variety of fermented foods such as cheese, fermented sausages and fermented vegetables. However, members of the genus Enterococcus have distinguished themselves from other lactic acid bacteria by their role in human infection, harboring a number of identified virulence factors, and for their acquired resistance to antibiotics. The purpose of the present work was to study the Citrate metabolism in E. faecalis. We characterized a cluster of 12 genes involved in the citrate fermentation. On the basis of their homology with previously characterized proteins we identified the citrate transporter (citH), the subunits of citrate lyase (citD, citE, citF) and the accessory genes (citC, citX and citG). Furthermore, we identified two oxalacetate decarboxylases (OAD): a soluble protein named citM and the membrane-bound biotin-dependent OAD complex (oadA, oadB, oadD). Finally, we found a transcriptional regulator (citR). Uptake experiments in E. coli expressing citH, shown specific requirement of Ca2+ for citrate uptake. Citrate lyase activity was determined in total extracts of E. faecalis, showing higher levels in those extracts obtained from cultures grown in presence of citrate. CitM was expressed in E. coli, purified as a his-tagged protein, and the kinetics parameters were determined (Km value for oxaloacetate was 0.453 mM and Vmax 17.7 μmol/min). Transcriptional analysis shown divergent operons citHR and oadABcitCDEFXoadDcitMG which are inducible by citrate.