Elucidating the Role of the Novel Peptide SgrT in the Glucose-Phosphate Stress

Response of Escherichia coli

Department of Microbiology University of Illinois Urbana-Champaign

Chelsea R. Lloyd* and Carin K. Vanderpool

Abstract 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.

The glucose-phosphate stress responseEIIC

EIIBP

sgrS sgrT

PtsG

SgrR*HfqSgrS

RNaseE

EIIC

EIIBP

SgrS ptsG mRNA

Outer MembraneCytoplasmic Membrane

P

P

P

EI

HPr

EIIAGlc

P

PEP

P

P

P

EIIC

EIIB

EIIC

EIIBSgrT

P

P

Stop synthesis of

new transporters

Inhibit existing

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.

SgrS and SgrT independently mitigate glucose-phosphate stress

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.

Overexpression of SgrT inhibits cell growth on minimal glucose medium

Substrate ΔsgrS::kan vector

ΔsgrS::kan sgrT

Preferred Transporter

LB ++++ ++++

glucose ++++ + PtsG

N-acetyl glucosamine

+++ +++ ManXYZ

mannose +++ +++ ManXYZ

fructose ++++ ++++ FruBKA

trehalose ++++ ++++ FruBKABecause 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.

SgrT does not inhibit ManXYZ transport activity

CL104 -2DG CL104 +2DG0

100

200

300

400

500

600

700

800ΔsgrS PsgrS-lacZ

0' Vector pBRCS12

120' Vector pBRCS12

0' SgrT pBRCS1

120' SgrT pBRCS1

0' Vector pBRCS12

120' Vector pBRCS12

0' SgrT pBRCS1

120' SgrT pBRCS1

Mill

er U

nits

CL105 -2DG CL105 +2DG0

100

200

300

400

500

600

700

800

ΔsgrS, ΔmanXYZ PsgrS-lacZ 0' Vector pBRCS12

120' Vector pBRCS12

0' SgrT pBRCS1

120' SgrT pBRCS1

0' Vector pBRCS12

120' Vector pBRCS12

0' SgrT pBRCS1

120' SgrT pBRCS1

Mill

er U

nits

To ask whether SgrT can inhibit ManXYZ transport, we measured 2-deoxyglucose (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.

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.

Measuring PtsG activity via inducer exclusion

EIIBGlc

EIICGlc

LacY

EIIAGlc

P

P

EIIBGlc

EIICGlc

LacY

P

EIIAGlc

PlacZ

Active Glucose Transport Inactive Glucose Transport

lacZ

EIIAGlc

P

PtsG activity LacZ activity PtsG activity LacZ activity

SgrT inhibits PtsG transport activityMeasuring PtsG activity via inducer exclusion

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.

What is the target of SgrT regulation?SgrT most likely targets glucose-specific PTS components:

EIICBGlc or EIIAGlc

Test using yeast two-hybrid system:

Plumbridge, J. Microbiology, 2000.

InvitrogenTM

SgrT + MlcSgrT + EIIA SgrT + EIIB391-477 (wt)SgrT + EIIBC421S

SgrT + EIIBR424A

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.

SgrT does not interact with EIIAGlc or EIIBGlc in vivo

0

50

100

150

200

250 SgrT Yeast Two-Hybrid InteractionsStrong +

Weak +

-

SgrT + EIIA

SgrT + EIIB

EIIB + Mlc

EIIB R424A + Mlc

EIIB C421S + Mlc

Mill

er U

nits

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.

SgrT suppressor mutants do not map to ptsG or crr lociTsup3

rsxC

yehB

glcB

yhcD

yhgA

ugpB

dtpB

mdtF

ubiB

yjjP

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.

Example of one suppressor strain: TSup3

Linkage experiments reveal no single mutation is responsible for SgrT suppression

SM

WT Δgene::kan

Lose Suppression PhenotypeGrowth Inhibition on Glc

WT Δgene::kan Δgene::kan SM

Suppression Phenotype RemainsRobust Growth on Glc

Mutation is in candidate gene Mutation is in another gene/multiple genes

SgrT

ampR

pBRCV7

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.

SgrT may target the PtsG linker region

Gabor, E., et al., The phosphoenolpyruvate-dependent glucose-phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell. Eur J Cell Biol. 90(9): p. 711-20.

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 375-477 are currently being tested.

Screening for SgrT suppressors in PtsG

sgrT

ampR

pBRCV7

mutptsG

camR

pZACL1

Ptet PlacO

CL108mal::lacIq+ PsgrS-lacZ

sgrS::tet ptsG::kan

screen

PCR mutagenize ptsG

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.

Conclusions• 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

Future directions• Confirm the target of SgrT regulation• Elucidate the mechanism of SgrT action• Investigate the structure of SgrT

Acknowledgments

Vanderpool LabCarin Vanderpool

Present Members:Richard HorlerGreg RichardsJen Han RiceYan Sun Divya BalasubramanianMax Bobrovskyy

Past Member:Caryn Wadler

Committee MembersCharles Miller – ChairJohn CronanJeffrey GardnerWilliam Metcalf

Special ThanksJeffrey BarrickSlauch Lab – Koh Eun Narm

Funding