Gene structure and functional analysis of the cluster encoding the enzymes of the citrate fermentation pathway from Enterococcus faecalis

BLANCATO, VS; MAGNI, C

Egmond aan Zee, Holanda

Simposio; 8th symposium on Lactic Acid Bacteria; 2005

Enterococci compose the microbial association of a variety of fermented foods such as cheese, fermented sausages and vegetables. The purpose of this 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 we identified the transporter (citH) and the regulatory protein (citO) genes located upstream and divergent to the citrate-lyase subunits (citD, citE, and citF), the accessory genes involved in the synthesis of an active citrate-lyase complex (citC, citX and citG), the soluble oxaloacetate decarboxylase (citM) and the genes encoding for the membrane oxaloacetate decarboxylase complex (oadB, oadA, oadD). Transcriptional analysis revealed two divergent operons, citHO and oadDBcitCDEFXoadAcitMG. The transcriptional initiation start sites of these two operons were mapped by primer extension. Both polycistronic mRNA were detected on RNA isolated from cells grown in presence of citrate and strongly repressed by glucose. According with that, citrate-lyase activity was induced by citrate and repressed by glucose. Uptake experiments in E. coli expressing citH, showed that CitH catalyzed the transport of Ca+2-citrate complex. Biochemical characterization of CitM showed that it is an enzyme with specific oxaloacetate decarboxylase activity. Finally, genetics and biochemical studies suggest that CitO is a regulator involved in regulation of the citrate fermentation in E. faecalis. Results obtained in the present study not only elucidate the pathway of citrate metabolism in Enterococcus, but also the mechanism of activation of the gene expression by citrate and the potential mechanism of Catabolic Repression.