IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The Enterococcus faecalis cit operons are regulated by multiple cre sites
Autor/es:
SUÁREZ, CA; BLANCATO, VS; MAGNI, C
Lugar:
Tucumán - Argentina
Reunión:
Simposio; III Simposio Internacional de Bacterias Lácticas y Segundo encuentro Red BAL (Bacterias Lácticas) Argentina; 2009
Institución organizadora:
CERELA
Resumen:
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In E. faecalis the genes encoding the enzymes involved in
citrate metabolism are organized in two divergent operons: citHO and
oadHDB-citCDEFX-oadA-citMG. Both
operons are specifically activated by the addition of citrate to the medium.
This activation is mediated by binding of CitO, a transcriptional regulator of
the GntR family, to the cis-acting sequences located in the cit
intergenic region. Early studies indicated that citrate and glucose could not
be co-metabolized suggesting some form of catabolite repression. Carbon
catabolite repression (CCR) is the general phenomenon to adjust expression of
catabolic genes in response to the availability of rapidly metabolizable carbon
sources. In Gram-positive bacteria CCR is exerted through binding of the CcpA
protein to cis-acting catabolite responsive element (cre). Our previous results indicated that citrate
metabolism is repressed by PTS sugars. Detailed sequence analysis of the
promoter regions revealed the presence of multiple cre sites, two located downstream of PcitHO (precisely 96 and 208
bp) and one downstream of PcitCL
(at 98 bp), strongly suggesting that the
pleiotropic transcriptional factor CcpA, could be involved in the repression of the citrate
metabolism.
In this work, we
shown by western blot analysis of cell extracts of E. faecalis grown
with different initial concentrations of glucose, that the level of the
activator CitO is
tightly regulated by the availability of preferential carbon sources. In
accordance, cell extracts showed a reduction in the citrate lyase activity.
Also, we analyzed the role of the cre
sites in the mechanism of transcription regulation. Gel mobility shift assays
using DNA fragments corresponding to the individual cre sites, confirmed
that the E. faecalis CcpA protein is capable of binding to each of them.
To determine if the binding of CcpA to the cre sites downregulates
the expression of the operons, a set
of DNA fragments covering alternative versions of the cit promoters (cre
sites deleted or mutated) were fused to the promoterless lacZ reporter
gene of pTCV-lac vector. The level of accumulated β-galactosidase
activity of the resulting strains, grown in LB supplemented with glucose and
citrate, suggested that the cre sites are indeed involved in the
regulation of the cit promoters. Moreover, we found that either of the cre
sites found in the PcitHO promoter were able to
repress this operon, and all the cre sites worked independently in the
conditions assayed. These results confirm the presence of multiple cre
sites involved in the CCR, which suggest a more complex mechanism implicated in
the fine tuning of the regulation mediated by CcpA.