Characterization Of The Citrate Transporter And Regulation Of Citrate Metabolism In Enterococcus faecalis

BLANCATO, VS; LOLKEMA, J; MAGNI, C

Rosario

Congreso; XLII Reunión Anual de SAIB; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In Enterococcus citrate fermentation contribute to aroma development in traditional raw milk cheese manufacture. The expression pattern of the cit locus showed two divergent operons, citHO and oadDBcitCDEFXoadAcitMG. The genes citD citE and citF encode the three citrate lyase subunits, with the genes oadABD and citM encoding two alternative oxaloacetate decarboxylases were identified. The citH gene encodes a membrane protein homologous to Me2+-citrate transporters, to characterize the citrate uptake in E. faecalis, the citH gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter CitH catalyzed proton motive force driven uptake of the Ca2+-citrate complex (KM 3.5 µM). Citrate in complex with Sr2+, Mn2+, Cd2+ and Pb2+ is substrate of CitH while complexes with Mg2+, Zn2+, Ni2+ and Co2+ are not. On another hand, the citO gene product has high homology to GntR transcriptional regulator proteins. We constructed a CitO defective strain and it was unable to metabolize citrate. CitO was expressed in E. coli and purified; band shift experiments showed that CitO could bind to the divergent promoter region. This supports the idea that CitO is a new positive regulator involved in the regulation of the citrate fermentation pathway in E. faecalis.