Transcript Elucidating the role of SgrT in the glucose

SgrT is a 43-amino acid protein encoded by the small regulatory RNA (sRNA) SgrS. SgrS is a
dual-function sRNA expressed in Escherichia coli under glucose-phosphate stress, a condition in
which the accumulation of phospho-sugars is bacteriostatic. Previous work from our lab has shown
that SgrT and the base pairing function of SgrS act independently to mitigate glucose-phosphate
stress. Expression of either SgrS base pairing or SgrT under stress conditions is sufficient to relieve
growth inhibition. Conversely, ectopic overexpression of either SgrS base pairing or SgrT inhibits
cell growth when glucose is the sole carbon source. Our lab has established that the base pairing
function of SgrS mediates these effects through preventing synthesis of the sugar transporters PtsG
and ManXYZ by repressing translation of their mRNAs. In contrast, SgrT has no effect on
transporter mRNA or protein levels and inhibits sugar uptake via an unknown mechanism. While
SgrS regulates at least two different sugar transporters, regulation by SgrT is specific to glucose
transport, suggesting that SgrT may target either PtsG (EIICBGlc) or Crr (EIIAGlc) - the glucose-specific
components of the glucose phoshotransferase system (PTS). Yeast two-hybrid analyses were used
to test the hypothesis that SgrT affects glucose transport through protein-protein interactions with
PtsG and Crr. Positive control interactions were demonstrated between PtsG and Mlc (a
transcription factor known to interact with PtsG) in this system. However, interactions between
SgrT and PtsG or Crr were not detected. As an unbiased approach to identification of the SgrT
target, we isolated seven mutants resistant to the effect of SgrT overexpression when grown on
glucose as a sole carbon source. None of the mutations isolated were linked to the ptsG or crr loci,
further suggesting that SgrT has a different target. These mutants were sequenced using Illumina
sequencing, and mutations were mapped with the program Breseq. Studies to discern which
mutations are responsible for the SgrT-resistant phenotype are in progress. Once a candidate SgrT
target is identified, future studies will investigate the mechanism by which SgrT acts on its target
and regulates glucose influx during glucose-phosphate stress.
PtsG EIIC
EIIC
EIIB
EIIB
Outer Membrane
Cytoplasmic Membrane
P
SgrT
P
P
EIIAGlc
P
P
P
P
HPr
P
EIIC
EIIC
EIIB
EIIB
Inhibit existing
transporters?
P
P
EI
P
SgrR*
sgrS
SgrS
sgrT
SgrS
PEP
ptsG mRNA
Hfq
RNaseE
Stop synthesis of
new transporters
During active transport through the glucose transporter PtsG (EIICBGlc), the PEP phosphotransferase (PTS)
system uses a signal cascade to phosphorylate incoming glucose or αMG resulting in accumulation of
glucose-6-phosphate or αMG-6-phosphate molecules. This accumulation induces the expression of SgrS,
which base pairs with ptsG mRNA in an Hfq dependent manner. This mRNA-sRNA complex is then targeted
for degradation by RnaseE. Translation of SgrT (encoded by sgrS) is also induced, which is thought to inhibit
preexisting transporters by an unknown mechamism.
Wadler C S , Vanderpool C K PNAS 2007
Previously, alleles were constructed to separate the function of SgrT from the base pairing function of SgrS.
Full-length sgrS is depicted by an arrow, the rectangle represents sgrT and “bp” stands for base pairing.
Truncations made by UAA stop codon insertions are illustrated as shortened rectangles and/or arrows.
Alleles were expressed in a ΔsgrS::kan, lacIq+ host strain, CV104, and induced with IPTG. Cultures were
grown in LB with amp and stress was induced with αMG. As shown, strains expressing SgrT alone, SgrS bp
alone (sgrSUAA) are able to recover growth in the presence of αMG as well as wild type, full length sgrS.
Substrate
ΔsgrS::kan
vector
ΔsgrS::kan sgrT
Preferred
Transporter
LB
++++
++++
glucose
++++
+
PtsG
N-acetyl glucosamine
+++
+++
ManXYZ
mannose
+++
+++
ManXYZ
fructose
++++
++++
FruBKA
trehalose
++++
++++
FruBKA
Because SgrS negatively regulates the major glucose transporter mRNA, its overexpression inhibits cell
growth on glucose. This has also been shown to occur when SgrT is overexpressed. However, SgrS has also
been shown to negatively regulate the mannose transporter mRNA. To qualitatively test if SgrT may inhibit
other transporters, E. coli expressing either a vector control or SgrT were grown on a variety of carbon
sources that are preferentially transported by the glucose, mannose or fructose transporter (as shown).
When SgrT was overexpressed, cells only experienced significant growth inhibition on minimal glucose
medium suggesting that SgrT is glucose specific and therefore most likely targets the glucose transporter
PtsG. Also, the trehalose permease relies on the glucose-specific EIIAGlc protein for translocation suggesting
the glucose specificity of SgrT is due to the EIICBGlc (PtsG) protein and not EIIAGlc.
800
ΔsgrS PsgrS-lacZ
0' Vector pBRCS12
120' Vector pBRCS12
700
Miller Units
600
0' SgrT pBRCS1
500
120' SgrT pBRCS1
400
0' Vector pBRCS12
300
200
120' Vector pBRCS12
100
0' SgrT pBRCS1
0
120' SgrT pBRCS1
CL104 +2DG
Above: Cells expressing SgrT are
unable to inhibit 2DG-mediated
induction of PsgrS-lacZ through
ManXYZ.
Right: Cells lacking ManXYZ do
not experience 2DG-mediated
induction of PsgrS-lacZ despite the
presence of SgrT showing that
2DG is specific to this transporter.
ΔsgrS, ΔmanXYZ PsgrS-lacZ
800
0' Vector pBRCS12
120' Vector pBRCS12
700
0' SgrT pBRCS1
600
Miller Units
CL104 -2DG
To ask whether SgrT can inhibit
ManXYZ transport, we measured 2deoxyglucose (2DG - which is solely
transported through ManXYZ)
induction of PsgrS-lacZ activity in the
presence or absence of PlacO-sgrT in a
ΔsgrS +/- ΔmanXYZ background.
500
120' SgrT pBRCS1
400
0' Vector pBRCS12
300
120' Vector pBRCS12
200
100
0' SgrT pBRCS1
0
CL105 -2DG
CL105 +2DG
120' SgrT pBRCS1
Active Glucose Transport
EIICGlc
Inactive Glucose Transport
LacY
EIIBGlc
LacY
EIIBGlc
P
EIIAGlc
EIICGlc
EIIAGlc
P
P
EIIAGlc
P
lacZ
PtsG activity
LacZ activity
P
PtsG activity
lacZ
LacZ activity
Wadler C S , Vanderpool C K PNAS 2007
ΔsgrS::kan, lacIq+ cells expressing either a vector control or SgrT were grown in media containing glucose,
lactose and IPTG and were assayed for β-galactosidase activity. When SgrT is expressed, LacZ activity is
high indicating that SgrT is somehow interfering with inducer exclusion and therefore acting on PtsG at the
level of transport activity.
SgrT most likely targets glucose-specific PTS components:
EIICBGlc or EIIAGlc
Test using yeast two-hybrid system:
SgrT + Mlc
SgrT + EIIA
SgrT + EIIB391-477 (wt)
SgrT + EIIBC421S
SgrT + EIIBR424A
InvitrogenTM
Mlc is a transcriptional repressor of ptsG. During active
glucose transport, Mlc is inactivated via sequestration to
the membrane through interactions with EIIBGlc
Seitz, S., et al. ( J Biol Chem, 2003) found EIIBC421D
constitutively binds Mlc whereas this interaction is
abrogated with the mutation EIIBR424A these will serve as
positive and negative controls, respectively.
Plumbridge, J. Microbiology, 2000.
300
SgrT Yeast Two-Hybrid Interactions
Strong +
250
Miller Units
Weak +
200
150
SgrT + EIIA
SgrT + EIIB
100
50
EIIB + Mlc
EIIB R424A + Mlc
EIIB C421S + Mlc
0
Yeast strain PJ69-4a was transformed with Invitrogen™ controls Krev1 + RalGDS-wt (strong +),
Krev1 + RalGDS-m1 (Weak +), Krev1 + RalGDS-m2 (-) or the aforementioned constructs. Interaction
strength was quantified by measuring the specific activity of a lacZ reporter gene. When compared with
both our negative controls, it appears that SgrT does not interact with either EIIAGlc or EIIBGlc in vivo.
Tsup3
rsxC
yehB
glcB
yhcD
yhgA
ugpB
dtpB
mdtF
ubiB
yjjP
Example of
one suppressor
strain: TSup3
Ten independent cultures of CS216 (E. coli sgrS::tet, mal::lacIq+) were
subjected to NTD mutagenesis, then transformed with a plasmid
expressing SgrT. Cells were then grown on minimal glucose medium
and colonies with robust growth were selected as SgrT suppressors.
Seven suppressor strains (Tsup1,2,3,4,5,7,8) were isolated and sent
for whole-genome sequencing. Sequences were analyzed with the
program Breseq (created by Jeffrey Barrick), which mapped point
mutations in the genome.
Suppressor strains had as few as ten point mutations to as many as
200. The only common mutation between all strains was in yjjP. All
silent mutations and consensus mutations in DJ480 (the parent
strain) and MG155 (the reference genome) were omitted. The ptsG
and crr genes were wild type in all suppressor strains.
SgrT
SM
ampR
pBRCV7
WT
Δgene::kan
Lose Suppression Phenotype
Growth Inhibition on Glc
WT
Δgene::kan
Mutation is in candidate gene
Suppression Phenotype Remains
Robust Growth on Glc
SM
Δgene::kan
Mutation is in another gene/multiple genes
Each single mutation in TSup3 was tested via linkage experiments in which P1 lysates of
downstream deletion genes were transduced into the Tsup3 strain, then transformed with
pBRCV7 and tested on glucose plates for loss of suppression. No single mutation lost
suppression and was not therefore not responsible for the suppression phenotype.
Multiple mutations may be cooperatively responsible for suppression.
A recent review by Gabor et al.
described an SgrT suppressor
mutation they had isolated in the
linker region of PtsG: P384R
My yeast two-hybrid construct
contains residues 391-477 and new
constructs containing residues 375477 are currently being tested.
Gabor, E., et al., The phosphoenolpyruvate-dependent glucose-phosphotransferase system from Escherichia coli K12 as the center of a network regulating carbohydrate flux in the cell. Eur J Cell Biol. 90(9): p. 711-20.
Ptet
PlacO
mutptsG
sgrT
camR
pZACL1
ampR
pBRCV7
CL108
mal::lacIq+
PsgrS-lacZ
sgrS::tet ptsG::kan
PCR mutagenize ptsG
screen
We will mutagenize ptsG by PCR, then
screen for activation of our PsgrS-lacZ fusion
on MacConkey-lactose-αMG plates. Red
colonies will signify ptsG mutants that are
resistant to SgrT inhibition.
• SgrT serves an independent function in the glucose-phosphate stress
response
• SgrT is glucose-specific
• SgrT acts on the level of PtsG transport activity
• SgrT most likely targets the PtsG linker region or EIIC domain
• Confirm the target of SgrT regulation
• Elucidate the mechanism of SgrT action
• Investigate the structure of SgrT
Vanderpool Lab
Carin Vanderpool
Present Members:
Richard Horler
Greg Richards
Jen Han Rice
Yan Sun
Divya Balasubramanian
Max Bobrovskyy
Committee Members
Charles Miller – Chair
John Cronan
Jeffrey Gardner
William Metcalf
Special Thanks
Jeffrey Barrick
Slauch Lab – Koh Eun Narm
Funding
Past Member:
Caryn Wadler