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									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
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, PNNL
                                                                             Uranium




                                                   From: K. Kemner, ANL
                                                   Lepidocrocite + U,S,orP




                                                        From: S. Lower
                           From: K. Nealson, USC        Goethite
                                                                                         From: PNNL
                           Magnetite                                                     Hematite
From: E. O’Loughlin, ANL
Lepidocrocite




                                                                                                          2
Gram Negative Microbe Membrane

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




                                                          3
What Are the Proteins in These Membrane Compartments?

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


                                            MtrC     ?                       OM
                                                               MtrB



                                                   MtrA
                                                                           Periplasm
                                      Cct

                                            CymA


                     NADH                                 MQ                   CM
                     Dehydrogenase




  (Belieav, Myers and Myers)


                                                                                       4
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
                                                  GOETHITE
                                    GOETHITE
                                     Goethite         Goethite     Ferrihydrite   Ferrihydrite    Lepidocrocite    Lepidocrocite
                                      Goethite   INOCULATED
                                                      Goethite     Ferrihydrite   Ferrihydrite    Lepidocrocite   Lepidocrocite
                                                    S.oneidensis
                                                   S. WITH
                                                      oneidensis                  S.oneidensis
                                                                                  S. oneidensis                    S. oneidensis
                                                                                                                  S. oneidensis
                                                  S. oneidensis




                                                      In collaboration with E. O’Loughlin, ANL




                                                                                                                                   5
Analysis: Two Complementary Proteomics Approaches




                                                                                             Intensity (arbitrary units)
                                                                                                                                    9
                                                                                                                           1.2x10

                                                                                                                                    9
                                                                                                                           1.0x10

                                                                                                                                    8
                                                                                                                           8.0x10

                                                                                                                                    8
                                                                                                                           6.0x10

                                                                                                                                    8
                                                                                                                           4.0x10

                                                                                                                                    8
                                                                                                                           2.0x10

                                                                                                                              0.0
                                                                                                                                        0       20   40     60   80    100   120

                                                                                                                                                          Time (min)




                                                                                                                                                                                         Tryptic peptides




                                                               Intensity (arbitrary units)
                                                                                                                                            9
                                                                                                                           1.2x10

                                                                                                                                            9
                                                                                                                           1.0x10

                                                                                                                                            8
                                                                                                                           8.0x10

                                                                                                                                            8
                                                                                                                           6.0x10
 Denatured proteins                                                                                                                         8
                                                                                                                           4.0x10

                                                                                                                                            8
                                                                                                                           2.0x10

                                                                                                                                        0.0
                                                                                                                                                0           20         40          60   80   100   120

                                                                                                                                                                             Time (min)
 2DE with LC/MS-MS of particular proteins for the
   assessment of relative abundance and the         “Shotgun” Proteomics with LC-LC/MS-MS for the
   identification of specific proteins showing      identification of ALL proteins present in membrane
   differential expression (ANL)                    preparations
                                                    (ORNL – N. VerBerkmoes/ R. Hettich)




                                                                                                                                                                                                            6
Enrichment of outer and inner membrane proteins

                               EDTA-lysozyme-Brij lysis protocol (Myers and Myers 1992)
The challenge is to separate
     the two membrane
    components and then
                                            Sucrose Gradient Centrifugation
 released the proteins from
  those components while
      minimizing cross
       contamination.




                                       Denature/solubilize        Trypsin digest
                                       membrane proteins        membrane proteins




                                                                                               5
                                                                                      8.0x10

                                                                                               5
                                                                                      6.0x10




                                                                          Intensity
                                                                                               5
                                                                                      4.0x10

                                                                                               5
                                                                                      2.0x10


                                                                                         0.0
                                                                                                   200   400   600    800   1000

                                                                                                                m/z




                                                                                                                                   7
Outer and Inner Membrane Proteins:
Aerobic vs. Anaerobic With Fumarate
                                                             Outer
Association of     TonB dependent receptor                   membrane    Decaheme
                                                                         cytochrome c
                                                             protein
specific                                                     precursor
                                                                                           Formate
                                                             mtrB
proteins with     Outer
                                                                                           dehydrogenase
                                                                                           a subunit

outer or inner    membrane
                  protein
membranes         tolC
                                          Major outer
                                          membrane
observed.        Outer
                                          lipoprotein

                 membrane
                 porin          Flagellin



                                             OMP/Aerobic                      OMP/Anaerobic

                                                                                                             Differential
                                     Methyl-accepting                                                          protein
                                     chemotaxis protein                                 Fumarate
                                                                                        reductase            expression
                            ATP synthase                                                                     observe in
                            F1, alpha subunit
                                                                                                                 both
                                                                                                             membrane
                                      16 kDa Heat
                                      shock protein A                                                      compartments.
                  Bacterioferritin
                  subunit 1
                                               IMP/Aerobic                      IMP/Anaerobic


                                                                                                                            8
Outer and Inner Membrane Proteins:
LC/LC-MS/MS Analysis

     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 identifications
         Inner membrane prep: 877 (2 or more peptides);
                                  1333 (1 peptide)
               58% overlap with outer membrane identifications


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

      Cell surface protein enrichment is needed.
                                                                     9
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




                                                                                        10
Intact Cells are Biotinylated

 •Cells are labeled in culture
 •Zwittergent 3-14 is used to release membrane
 proteins
 •Biotinylated proteins are captured by avidin affinity
                                                                                  Live/Dead kit, Molecular probes
 chromatography
                                                                  Syto9-Green fluorescence/Live cells
 •Biotinylated proteins are analyzed by 2DE and
                                                                  Propidium iodide-Red fluorescence/Dead cells
 2DLC/MS-MS


  Ton B dependent receptor


                                                                                                        Biotinylation
                                                                                                        increased sensitivity
                                                                                                        of detection.

             Outer membrane porin




                    Outer membrane prep – no biotin   Western Blot with Neutravidin-HRP – biotin tag


                                                                                                                          11
LC/LC-MS/MS Confirms Biotinylation Captures Outer
Membrane Proteins
outer membrane porin, putative            decaheme cytochrome c (omcA)                     conserved hypothetical protein
outer membrane protein TolC               hypothetical protein                             conserved hypothetical protein
outer membrane protein OmpH               cytochrome c                                     conserved hypothetical protein
TonB-dependent receptor, putative         cytochrome c oxidase, cbb3-type, subunit II      periplasmic glucan biosynthesis protein, putative
TonB-dependent receptor domain protein    MSHA pilin protein MshA (mshA)                   survival protein surA (surA)
tolB protein (tolB)                       agglutination protein (aggA)                     decaheme cytochrome c (omcB)
TPR domain protein                        conserved hypothetical protein                   polyamine ABC transporter, periplasmic
                                                                                           polyamine-binding protein
peptidase, M16 family                     peptidoglycan-associated lipoprotein (pal)       flagellar hook-associated protein FliD
peptidase, M13 family                     cytochrome c (cytcB)                             conserved hypothetical protein
heme transport protein (hugA)             outer membrane protein precursor MtrB            ferric alcaligin siderophore receptor
MotA/TolQ/ExbB proton channel family      multidrug resistance protein, AcrA/AcrE family   periplasmic nitrate reductase (napA)
protein
sulfate ABC transporter, periplasmic      lipoprotein, putative                            OmpA family protein
sulfate-binding protein
major outer membrane lipoprotein,         ATP synthase F1, beta subunit                    thiol:disulfide interchange protein DsbE
putative
ubiquinol-cytochrome c reductase, iron-   formate dehydrogenase, alpha subunit
sulfur subunit (petA)




                                                                                                                                               12
Biotinylated Proteins From Aerobic and Anaerobic
with Fumarate Growth




                    Aerobic                   Fumarate




                                                         13
Differentially Expressed Surface Proteins (2DLC/MS-MS
   Detected in Anaerobic but not Aerobic Cells                                     Significantly More Abundant in Anaerobic Cells
                                                                                                              hemolysin protein putative
                     phage shock protein A
                                                                                                           Hypothetical protein (gi7587156)
                    decaheme cytochrome c
                                                                                                              adhesion protein putative
              general secretion pathway protein D
                                                                                                           hypothetical protein (gi7587828)
                      TPR domain protein                                                                       outer membrane protein

              transcriptional regulator RpiR family                                              anaerobic dimethyl sulfoxide reductase B subunit

           conserved hypothetical protein (gi7597239)                                            anaerobic dimethyl sulfoxide reductase A subunit

                                                                                                   conserved hypothetical protein (gi7589906)
                      PqiB family protein
                                                                                                            cytochrome c551 peroxidase
                            flagellin
                                                                                                             16 kDa heat shock protein A
           conserved hypothetical protein (gi7589906)                                              RNA pseudouridylate synthase family protein

                 outer membrane protein TolC                                                                universal stress protein family

           conserved hypothetical protein (gi7595997)                                              conserved hypothetical protein (gi7599217)

                                                                                                    formate dehydrogenase iron-sulfur subunit
                      agglutination protein
                                                                                                           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



                                                                                                                                                    14
       Proteomics of MR-1 Grown with Insoluble Iron Oxides



                              GOETHITE                      Ferrihydrite                                Lepidocrocite
                    GOETHITE Goethite
                    Goethite INOCULATED     Ferrihydrite    S. oneidensis               Lepidocrocite   S. oneidensis
                                WITH
                                   S.
                              S. oneidensis
                               oneidensis




            8                                                                  10                                                           25


                                                                               8                                                            20
            6
Fe(II) mM




                                                                   Fe(II) mM




                                                                                                                                Fe(II) mM
                                                                               6                                                            15
            4
                                                                               4                                                            10

            2
                                      Goethite                                 2                            Lepidocrocite                   5            Ferrihydrite
                                                                               0
            0                                                                                                                               0
                0     10         20         30         40                           0                   2               4   6                    0   1   2        3     4   5
                               Time (d)                                                                        Time (d)                                      Time (d)




                                                                                                                                                                                15
Differential Protein Expression Obvious from Total
Lysate 2DE Patterns




              Goethite         Lepidocrocite         Ferrihydrite




                                                                    16
Surface Proteome Also Distinctive




          Goethite       Lepidocrocite   Ferrihydrite




                                                        17
A Model of the Membrane Proteome Required for Fe(III) reduction



                                                        Fe(III)


                                   MtrC     ??                      OM
                                                      MtrB



                                          MtrA
                                                                  Periplasm
                             Cct

                                   CymA


             NADH                                MQ                   CM
             Dehydrogenase




                                                                              18
Summary

 •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.



                                                                                                                    19
Acknowledgement



    ERSP (NABIR) Funding
                            Collaborators:   E.J. O’ Loughlin (ANL)
                                             Robert Hettich (ORNL)
                                             Nathan VerBerkmoes (ORNL)
ANL Protein Mapping Group                    K. Nealson (USC)




                                             K.M. Kemner (ANL)




                                                                         20

								
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