INVESTIGADORES
BLANCATO Victor Sebastian
congresos y reuniones científicas
Título:
Citrate Transport And Regulation Of The Citrate Metabolism In Enterococcus faecalis
Autor/es:
BLANCATO, VS; LOLKEMA, J; MAGNI, C
Lugar:
Tucumán
Reunión:
Simposio; II Simposio Internacional de Bacterias Lácticas y Primer encuentro Red BAL (Bacterias Lácticas) Argentina; 2006
Institución organizadora:
Centro de Referencia para Lactobacilos
Resumen:
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.