Citrate Transport And Regulation Of The Citrate Metabolism In Enterococcus faecalis

BLANCATO, VS; LOLKEMA, J; MAGNI, C

Tucumán

Simposio; II Simposio Internacional de Bacterias Lácticas y Primer encuentro Red BAL (Bacterias Lácticas) Argentina; 2006

Centro de Referencia para Lactobacilos

Citrate fermentation is an important industrial feature of Lactic Acid Bacteria. In Enterococcus this metabolism contribute to aroma development in traditional raw milk cheese manufacture. The characterization of the expression profile of the cit locus revealed two divergent operons, citHO and oadDBcitCDEFXoadAcitMG. The citH gene encodes a membrane protein that shows high homology to metal-citrate transporters, the citO gene product have high homology to transcriptional regulator proteins. The genes citD, citE and citF are located in a central position of the operon, they encode the three citrate lyase subunits and the accessories genes citC, citX and citG. Also, we identified four putative genes encoding for two alternative oxaloacetate decarboxylases,  oadABD and citM enzymes. In order 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 (EfCitH) catalyzed proton motive force driven uptake of the Ca2+-citrate complex with an affinity constant of 3.5 µM. Homologous exchange was catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to the specificity of the Bacillus subtilis Ca2+-citrate transporter belonging to the same family. By contrary, in spite of the high sequence identity with the Streptococcus mutans Fe3+-citrate transporter, no transport activity with Fe3+ (or Fe2+) could be detected. EfcitH catalyzes also the translocation of citrate in complex with Sr2+, Mn2+, Cd2+ and Pb2+ but not with Mg2+, Zn2+, Ni2+ and Co2+. The specificity appears to correlate with the size of the metal ion in the complex. EfCitO belongs to the FadR subfamily of the helix turn helix GntR family of bacterial regulators. Our experiments showed that CitO could bind to the divergent promoter region and activate the transcription of both cit operons. A CitO defective mutant constructed by insertional mutation, revealed a significant different growth pattern when citrate is present in the medium than the wild type strain, indicating that the defective CitO strain was unable to metabolize citrate. This data supports the idea that CitO is a new positive regulator involved in the regulation of the citrate fermentation pathway in E. faecalis.