The Membrane Proteome of Shewanella oneidensis MR-1

C.S. Giometti, T. Khare, N. VerBerkmoes, E. O’Loughlin, K. Nealson

ERSP PI Meeting

April, 2006

Oak Ridge National Laboratory

University of Southern California

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Shewanella MR-1 is in Touch with its Environment

In natural habitats, MR-1 interacts with insoluble metals as terminal electron acceptors – the cell membrane is the point of contact.

From: J. Fredrickson, PNNLUranium

From: E. O’Loughlin, ANLLepidocrocite

From: K. Kemner, ANL

Lepidocrocite + U,S,orP

From: K. Nealson, USC

MagnetiteFrom: PNNLHematite

From: S. LowerGoethite

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Gram Negative Microbe Membrane

•Membrane has 3 layers•Proteins in each layer serve unique functions•Proteins are essential in cell interaction with the environment

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What Are the Proteins in These Membrane Compartments?

CymA

Cct

?

NADH Dehydrogenase

MQ

MtrA

OM

Periplasm

CM

MtrC MtrB

Fe(III)

(Belieav, Myers and Myers)

Mutation work has revealed some of the players, but there is more to do.

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Identification of Expressed Membrane Proteins

Step #1: Business as usual – identify proteins expressed in response to soluble electron acceptors

MR-1 grown aerobically or anaerobically with fumarate

Step #2: Move closer to natural environment – identify proteins expressed in response to insoluble electron acceptors

MR-1 grown anaerobically with insoluble iron oxide as electron acceptor•Goethite•Lepidocrocite•Ferrihydrite

In collaboration with E. O’Loughlin, ANL

GOETHITEGOETHITE

INOCULATED WITH

S. oneidensis

Goethite GoethiteS. oneidensis

Ferrihydrite FerrihydriteS. oneidensis

Lepidocrocite LepidocrociteS. oneidensis

GOETHITEGOETHITE

INOCULATED WITH

S. oneidensis

Goethite GOETHITEGOETHITE

INOCULATED WITH

S. oneidensis

Goethite Goethite S. oneidensis

Ferrihydrite FerrihydriteS. oneidensis

Lepidocrocite LepidocrociteS. oneidensis

Ferrihydrite FerrihydriteS. oneidensis

Lepidocrocite LepidocrociteS. oneidensis

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Analysis: Two Complementary Proteomics Approaches

2DE with LC/MS-MS of particular proteins for the assessment of relative abundance and the identification of specific proteins showing differential expression (ANL)

0 20 40 60 80 100 1200.0

2.0x108

4.0x108

6.0x108

8.0x108

1.0x109

1.2x109

Inte

nsit

y (

arb

itra

ry u

nit

s)

Time (min)

“Shotgun” Proteomics with LC-LC/MS-MS for the identification of ALL proteins present in membrane preparations

(ORNL – N. VerBerkmoes/ R. Hettich)

0 20 40 60 80 100 1200.0

2.0x108

4.0x108

6.0x108

8.0x108

1.0x109

1.2x109

Inte

nsi

ty (

arb

itra

ry u

nit

s)

Time (min)

Denatured proteins

Tryptic peptides

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Enrichment of outer and inner membrane proteins

The challenge is to separate the two membrane

components and then released the proteins from those components while

minimizing cross contamination.

EDTA-lysozyme-Brij lysis protocol (Myers and Myers 1992)

Sucrose Gradient Centrifugation

Denature/solubilize membrane proteins

200 400 600 800 10000.0

2.0x105

4.0x105

6.0x105

8.0x105

Inte

nsity

m/z

Trypsin digest membrane proteins

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Outer and Inner Membrane Proteins: Aerobic vs. Anaerobic With Fumarate

OMP/Anaerobic

IMP/Anaerobic

Formate dehydrogenase subunit

Decaheme cytochrome c

Outer membrane protein precursor mtrB

Methyl-accepting chemotaxis protein

Flagellin

Major outer membrane lipoprotein

ATP synthase F1, alpha subunit

Fumarate reductase

Bacterioferritin subunit 1

16 kDa Heat shock protein A

TonB dependent receptor

Outer membrane protein tolC

OMP/Aerobic

IMP/Aerobic

Outer membrane porin

Differential protein

expression observe in

both membrane

compartments.

Association of specific proteins with outer or inner membranes observed.

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2DLC/MS-MS (LTQ) analysis of outer and inner membrane preparations from cells grown aerobically provided additional identifications.

Outer membrane prep: 76 (2 or more peptides); 1307 (1 peptide)

80% overlap with inner membrane identificationsInner membrane prep: 877 (2 or more peptides);

1333 (1 peptide)58% overlap with outer membrane identifications

Outer and Inner Membrane Proteins: LC/LC-MS/MS Analysis

Mixtures are too complex to elucidate the proteins actually in contact with the extracellular

environment.

Cell surface protein enrichment is needed.

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Approaches to Determining Surface Location of Proteins

Radiolabeling with I-125 –radioisotope; intracellular labeling Proteinase K digestion – antibodies to identify specific proteins lost Biotinylation – nonisotopic method of tagging all proteins; less intracellular labeling

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Intact Cells are Biotinylated

Syto9-Green fluorescence/Live cells

Propidium iodide-Red fluorescence/Dead cells

Live/Dead kit, Molecular probes

•Cells are labeled in culture

•Zwittergent 3-14 is used to release membrane proteins

•Biotinylated proteins are captured by avidin affinity chromatography

•Biotinylated proteins are analyzed by 2DE and 2DLC/MS-MS

Outer membrane porin

Biotinylation increased sensitivity of detection.

Ton B dependent receptor

Western Blot with Neutravidin-HRP – biotin tagOuter membrane prep – no biotin

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formate dehydrogenase, alpha subunitubiquinol-cytochrome c reductase, iron-sulfur subunit (petA)

ATP synthase F1, beta subunitmajor outer membrane lipoprotein, putative

lipoprotein, putativesulfate ABC transporter, periplasmic sulfate-binding protein

multidrug resistance protein, AcrA/AcrE familyMotA/TolQ/ExbB proton channel family protein

outer membrane protein precursor MtrBheme transport protein (hugA)

cytochrome c (cytcB)peptidase, M13 family

peptidoglycan-associated lipoprotein (pal)peptidase, M16 family

conserved hypothetical proteinTPR domain protein

agglutination protein (aggA)tolB protein (tolB)

MSHA pilin protein MshA (mshA)TonB-dependent receptor domain protein

cytochrome c oxidase, cbb3-type, subunit IITonB-dependent receptor, putative

cytochrome couter membrane protein OmpH

hypothetical proteinouter membrane protein TolC

decaheme cytochrome c (omcA)outer membrane porin, putative

LC/LC-MS/MS Confirms Biotinylation Captures Outer Membrane Proteins

polyamine ABC transporter, periplasmic polyamine-binding protein

flagellar hook-associated protein FliD

decaheme cytochrome c (omcB)

survival protein surA (surA)

periplasmic glucan biosynthesis protein, putative

conserved hypothetical protein

conserved hypothetical protein

conserved hypothetical protein

ferric alcaligin siderophore receptor

periplasmic nitrate reductase (napA)

OmpA family protein

thiol:disulfide interchange protein DsbE

conserved hypothetical protein

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Biotinylated Proteins From Aerobic and Anaerobic with Fumarate Growth

FumarateAerobic

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Differentially Expressed Surface Proteins (2DLC/MS-MS

Detected in Anaerobic but not Aerobic Cells

phage shock protein A

decaheme cytochrome cdecaheme cytochrome c

general secretion pathway protein D

TPR domain protein

transcriptional regulator RpiR family

conserved hypothetical protein (gi7597239)

PqiB family protein

flagellin

conserved hypothetical protein (gi7589906)

outer membrane protein TolC

conserved hypothetical protein (gi7595997)

agglutination protein

Significantly More Abundant in Anaerobic Cells

hemolysin protein putative

Hypothetical protein (gi7587156)

adhesion protein putative

hypothetical protein (gi7587828)

outer membrane protein

anaerobic dimethyl sulfoxide reductase B subunit

anaerobic dimethyl sulfoxide reductase A subunit

conserved hypothetical protein (gi7589906)

cytochrome c551 peroxidase

16 kDa heat shock protein A

RNA pseudouridylate synthase family protein

universal stress protein family

conserved hypothetical protein (gi7599217)

formate dehydrogenase iron-sulfur subunit

hypothetical protein (gi7597502)

putative lipoprotein, putative

Similar Abundance in Aerobic and Anaerobic Cells

General diffusion Gram-negative porins, putative

ATP synthase F1 epsilon subunit

ATP synthase F1 beta subunit

ATP synthase F1 gamma subunit

ATP synthase F1 alpha subunit

ATP synthase F0 B subunit

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Proteomics of MR-1 Grown with Insoluble Iron Oxides

GOETHITEGOETHITE INOCULATED

WITH S. oneidensis

Goethite GoethiteS.

oneidensis

Ferrihydrite

FerrihydriteS. oneidensis Lepidocrocite

LepidocrociteS. oneidensis

Time (d)

0 10 20 30 40

Fe(

II) m

M

0

2

4

6

8

Goethite

Time (d)

0 1 2 3 4 5

Fe(

II) m

M

0

5

10

15

20

25

Ferrihydrite

Time (d)

0 2 4 6

Fe(

II) m

M

0

2

4

6

8

10

Lepidocrocite

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Differential Protein Expression Obvious from Total Lysate 2DE Patterns

Lepidocrocite FerrihydriteGoethite

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Surface Proteome Also Distinctive

Goethite FerrihydriteLepidocrocite

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A Model of the Membrane Proteome Required for Fe(III) reduction

?

CymA

Cct

?

NADH Dehydrogenase

MQ

MtrA

OM

Periplasm

CM

MtrC MtrB

Fe(III)

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•Biotinylation of intact cells provides a valuable tool for identifying the proteins exposed at the surface of the cell.

•The membrane proteome of S. oneidensis MR-1 is dynamic, varying in content in response to terminal electron acceptors.

•Surface membrane proteome of cells grown with insoluble iron oxides is significantly different from that of cells grown with soluble terminal electron acceptors.

•A number of proteins in addition to c-type cytochromes comprise the variable component of the membrane surface.

“The effects of Omp35 on anaerobic electron acceptor use are therefore likely indirect. The results demonstrate the ability of non-electron transport proteins to influence anaerobic respiratory phenotypes.” From

Maier and Myers BMC Microbiology 2004

•Understanding the membrane protein configuration of microbes in the environment will be an important component of determining the fate and transport of contaminants.

Summary

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Acknowledgement

ERSP (NABIR) Funding

Collaborators: E.J. O’ Loughlin (ANL)

Robert Hettich (ORNL)

Nathan VerBerkmoes (ORNL)

K. Nealson (USC)

K.M. Kemner (ANL)

ANL Protein Mapping Group