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D. De Biase - The glutamate-based acid resistance system of Escherichia coli
intergenic region. In fact, the data from gadX::lacZ
transcriptional fusions indicate that gadX transcription
from its own promoter is not significantly affected
by either the pH of the growth medium or any of
the regulators of the GDAR system. This reinforces
our previous findings that the intracellular levels of
gadX mRNA, and consequently of the GadX protein,
are mainly controlled at the transcriptional level by
H-NS at the PgadX and PgadAX promoters. We
showed that the GadX/GadW-dependent circuit is
primarily controlled at the level of the gadX-gadW
intergenic region, where the gadY gene is located and
transcribed divergently from gadW. The gadY promoter
responds positively to the acidic pH in the stationary
phase and requires GadX for maximal activity.
Indeed, we identified a GadX/GadW binding site
in the gadY-gadW intergenic region. This binding
site, spanning position -61 to -21 with respect to the
+1 of gadY, positively affects gadY transcription and
negatively affects gadW transcription from one of its
promoters. Our analysis of gadW transcription
revealed that although this regulator has a minor role
in the GDAR system, its expression indirectly affects
the gadX mRNA levels by controlling transcription
from the gadY promoter. In fact, binding of the RNA
polymerase to the gadY promoter favours the transcription
of the divergent gadW gene, whereas gadW
transcription has the opposite effect on the activity of
the gadY promoter.
Another important finding was the identification of
a GadX/GadW consensus sequence. By aligning the
GadX/GadW-binding site on the gadY promoter
with the GadX/GadW-binding sites previously identified
in the gadA and gadBC 5’ regulatory regions,
we generated a 42-bp GadX/GadW consensus
sequence. DNAse I footprinting analyses confirmed
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that a 42-bp GadX/GadW-binding site is also present
in the AFI regulatory regions of the slp-yhiF,
hdeAB and gadE-mtdEF operons. The 42-bp long
GadX/GadW-binding site clearly arises from the
tandem arrangement of two 21-bp sequences, in
which nine positions are highly-to-strictly conserved.
The distance of the GadX/GadW-binding
site from the transcriptional start site of the target
genes varies significantly.
In a previous study we showed that at least in vitro
GadX is able to upregulate the gadA gene by itself
and can also displace H-NS from the gadA promoter,
thereby allowing its transcription even when H-NS
is already bound. It was therefore suggested that in
vivo, the most important role played by GadX
(GadW) on the gadA promoter is as a H-NS countersilencer.
Remarkably, we could demonstrate that the
5’ regulatory regions of the transcriptional units slpyhiF,
hdeAB-yhiD, hdeD, gadE-mtdEF, gadY and
gadAX in the AFI were direct targets of the activators
GadX and GadW. Molecular analysis in the
presence of GadX (GadW) and H-NS is necessary to
establish whether there is functional interplay
between these proteins in the AFI. Overall, our findings
suggest a possible role for GadX and GadW in
the AFI as antirepressors of H-NS.
Selected publications
Tramonti A, De Canio M, De Biase D.
GadX/GadW-dependent regulation of the
Escherichia coli acid fitness island: transcriptional
control at the gadY-gadW divergent promoters and
identification of four novel 42-bp GadX/GadW-specific
binding sites. Mol Microbiol. 2008, 70:965-82.