A nutrient uptake role for bacterial cell envelope extensions

Jennifer K. Wagner, Sima Setayeshgar, Laura A. Sharon, James P. Reilly, and Yves V. Brun
PNAS August 1, 2006 103 (31) 11772-11777; https://doi.org/10.1073/pnas.0602047103

  1. Edited by A. Dale Kaiser, Stanford University School of Medicine, Stanford, CA, and approved June 8, 2006 (received for review March 13, 2006)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    Micrograph of a C. crescentus cell and models of nutrient uptake. (A) Transmission electron micrograph of C. crescentus. F, flagellum; S, stalk; H, adhesive holdfast; D, division site. (B) Models of nutrient uptake by the stalk and cell body. The model in the top part of the diagram (thick arrows) illustrates uptake of a nutrient molecule into the stalk periplasm, its binding to a periplasmic nutrient-binding protein, the diffusion of the nutrient-binding protein from the stalk periplasm to the cell body periplasm, and the uptake of the nutrient into the cell body cytoplasm by an ABC transporter. This periplasm-to-periplasm model is consistent with our results. The model on the bottom part of the diagram (thin arrows) illustrates nutrient uptake into the stalk core, followed by its diffusion into the cell body cytoplasm. Nutrient uptake in the cell body is also shown (intermediate-thickness arrows).

  • Fig. 2.
    Fig. 2.

    FDP uptake by cells and stalks. (A and C) Phase contrast. (B and D) Fluorescence. Cells (B) and stalks (D) after incubation with FDP are shown.

  • Fig. 3.
    Fig. 3.

    Periplasmic nutrient-binding protein localization in the stalk. Western blot analysis of periplasmic binding proteins in stalk (S) and cell body (B) fractions. PstS (CC1515) is the high-affinity phosphate-binding protein, PhnD (CC0362) is a putative phosphonate-binding protein, and PotF (CC3137) is a putative putrescine-binding protein. Lower blot shows the same membrane reprobed with an antibody raised against the abundant cytoplasmic protein FtsZ. Molecular weights are indicated to the left. NA1000 is the isogenic wild-type control.

  • Fig. 4.
    Fig. 4.

    ExbB and PstA localize to the cell body but not the stalk. (A and D) Phase contrast. (B and E) Fluorescence. (C and F) 2D deconvolved fluorescence. Localization of ExbB–M2 in YB4058 (B and C) and PstA–GFP in YB4062 (E and F) is shown.

  • Fig. 5.
    Fig. 5.

    Rate of diffusive uptake by discrete absorbers. (A) The number of particles per unit time absorbed by the cell body, I cell(N), is plotted for b cell = 0.25 μm and L cell = 1 μm, in units of the maximum rate of uptake by the cell body, I cell max. The radius of an individual absorber is s = 1 nm, a typical size for transport proteins (26). By increasing the number of absorbers on the cell body from N = 10,000 to N = 20,000, the rate of uptake is increased by only 5% of the maximum rate. (B) The number of particles per unit time absorbed by the stalk, I stalk(N), is plotted for b stalk = 50 nm and L stalk = 1 μm (red), 5 μm (green), and 10 μm (blue). Addition of N = 10,000 absorbers to stalks of length 1, 5, and 10 μm increases the rate of uptake by ≈50%, 140%, and 210% of I cell max. The absorbing surface is represented in red in the figures (Left).

Data supplements

  • Wagner et al. 10.1073/pnas.0602047103.

    Supporting Information

    Files in this Data Supplement:

    Supporting Figure 6
    Supporting Table 1
    Supporting Figure 7
    Supporting Text
    Supporting Figure 8
    Supporting Figure 9
    Supporting Table 2
    Supporting Table 3





    Supporting Figure 6

    Fig. 6. Abundance of PstA-GFP in stalks and cell bodies. Western blot analysis using anti-GFP antibody to detect PstA-GFP in purified stalks (lane 1), cell body lysates (lane 2), and cell body membrane preparations (lane 3) following growth in HIGG containing 30 μM phosphate. The same western blot following staining with Ponseau S protein stain (lanes 4, 5, and 6).





    Supporting Figure 7

    Fig. 7. Sketch of random walk trajectories of two substrate particles (green), demonstrating the tendency for diffusing particles to explore a given region of space thoroughly before moving on. The steady state rate of absorption by discrete absorbers (red) is doubled when two absorbers (e.g., absorbers 1 and 2) are placed sufficiently far apart that they are unlikely to be encountered by the same diffusing particle. On the other hand, a third absorber (e.g., absorber 3) placed close to the first two will not triple the rate of absorption, as it will be correlated with the existing absorbers.





    Supporting Figure 8

    Fig. 8. Absorption current to elongated and stalked cells. (A) The number of particles per unit time absorbed by the cell body of length Lcell = 3 mm (red), 5 mm (green) and 10 mm (blue) and bcell = 0.25 mm is plotted as function of the number of discrete absorbers, N, in units of the maximum current to the cell body of typical dimensions (given by Lcell = 1 mm and bcell = 0.25 mm). (B) The number of particles absorbed per unit time by a stalked cell is plotted as function of the number of discrete absorbers, N, in units of the maximum current to the cell body of typical dimensions. For the stalked cell, the cell body has typical dimensions (given by Lcell = 1 mm and bcell = 0.25 mm), and the stalk length is given by Lstalk = 2 mm (red), 4 mm (green) and 9 mm (blue) with bstalk = 50 nm. The absorbers are distributed uniformly on the surface of the stalked cell. We note that corresponding curves in (A) and (B) indicate that the rates of uptake with N absorbers for a stalked cell and an elongated cell of the same total length are comparable.





    Supporting Figure 9

    Fig. 9. The maximum rate of diffusive uptake per unit area (A) and per unit volume (B) (in units of 4pDc0 = 1) is plotted as function of total length of the receptor for a stalked cell (with cell length Lcell = 1 mm, cell radius bcell = 0.25 mm and stalk radius bstalk = 50 nm) and an elongated cell (with bcell = 0.25 mm). We note that stalked cells are capable of a larger rate of uptake per unit volume and area than elongated cells of the same overall length.





    Table 1. Proteins identified in the stalk fraction by 2D-LC MS

    Gene

    Protein

    Localization (a)

    # of Peptides

    CC0116

    hypothetical protein

    U(-)

    3

    CC0125

    hypothetical protein

    U(+)

    3

    CC0163

    hypothetical protein

    OM

    22

    CC0170

    hypothetical protein

    U(+)

    15

    CC0171

    TonB-Dependent Receptor

    OM

    40

    CC0201

    OmpA Family Protein

    OM

    8

    CC0210

    TonB-Dependent Receptor

    OM

    38

    CC0214

    TonB-dependent receptor

    OM

    2

    CC0252

    penicillin-binding protein, 1A family

    IM(1)

    2

    CC0288

    hypothetical protein

    OM

    14

    CC0292

    phosphate ABC transporter, ATP-binding protein (pstB)

    CY

    10

    CC0322

    biopolymer transport protein ExbD

     

    4

    CC0351

    conserved hypothetical protein

    OM

    5

    CC0361

    phosphonates ABC transporter, ATP-binding protein (phnC)

    IM(1)

    5

    CC0362

    phosphonates ABC transporter, periplasmic phosphonates-binding protein (phnD)

    PP

    9

    CC0375

    conserved hypothetical protein

    U(+)

    2

    CC0394

    hypothetical protein

    U(+)

    8

    CC0454

    TonB-Dependent Receptor

    OM

    42

    CC0455

    alkaline phosphatase, putative

    U(+)

    8

    CC0472

    ubiquinol-cytochrome c reductase, iron-sulfur subunit (petA)

    IM(2)

    2

    CC0486

    hypothetical protein

    U(+)

    2

    CC0510

    acetyl-CoA acetyltransferase (phbA)

    CY

    2

    CC0600

    hypothetical protein

    U(+)

    6

    CC0685

    chaperonin, 60 kDa (groEL)

    CY

    4

    CC0702

    serine protease

    PP

    2

    CC0722

    TonB-Dependent Receptor

    OM

    26

    CC0792

    flagellin FljM (fljM)

    PP

    3

    CC0797

    1,4-beta-D-glucan glucohydrolase D (celD)

    PP

    5

    CC0806

    efflux system protein

    OM

    2

    CC0925

    OmpA Related Protein

    OM

    42

    CC0927

    signal peptide peptidase SppA (sppA)

    IM(3)

    9

    CC0983

    TonB-Dependent Receptor

    OM

    7

    CC0991

    TonB-Dependent Receptor

    OM

    8

    CC0995

    TonB-Dependent Receptor

    OM

    35

    CC1007

    S-layer protein RsaA (rsaA)

    EX

    4

    CC1038

    hypothetical protein

    U(+)

    3

    CC1056

    hypothetical protein

    U(+)

    3

    CC1099

    TonB-Dependent Receptor

    OM

    8

    CC1113

    TonB-Dependent Receptor

    OM

    20

    CC1131

    TonB-Dependent Receptor

    OM

    2

    CC1132

    conserved hypothetical protein

    U(+)

    5

    CC1240

    translation elongation factor EF-Tu (tufB)

    CY

    4

    CC1295

    3-phytase, fusion, putative

    U(+)

    5

    CC1318

    outer membrane protein TolC, putative

    OM

    8

    CC1335

    TPR domain protein

    OM

    2

    CC1353

    hypothetical protein

    U(-)

    9

    CC1375

    conserved hypothetical protein

    U(+)

    4

    CC1460

    flagellin FljL (fljL)

    PP

    2

    CC1507

    conserved hypothetical protein

    U(+)

    4

    CC1543

    rod shape-determining protein MreB (mreB)

    CY

    2

    CC1559

    signal peptidase I

    IM(1)

    3

    CC1565

    alkaline phosphatase D (phoD)

    U(+)

    9

    CC1579

    superoxide dismutase, Cu-Zn (sodC)

    PP

    2

    CC1653

    PQQ enzyme repeat family protein

    U(+)

    6

    CC1666

    TonB-Dependent Receptor

    OM

    6

    CC1695

    conserved hypothetical protein

    U(+)

    3

    CC1750

    TonB-Dependent Receptor

    OM

    32

    CC1879

    conserved hypothetical protein

    U(-)

    2

    CC1894

    rotamase family protein

    PP

    2

    CC1914

    conserved hypothetical protein

     

    4

    CC1915

    outer membrane protein

    OM

    14

    CC1970

    TonB-Dependent Receptor

    OM

    8

    CC2139

    metallo-beta-lactamase superfamily protein

    U(+)

    2

    CC2149

    TonB-Dependent Receptor

    OM

    32

    CC2160

    hypothetical protein

    U(-)

    10

    CC2190

    hypothetical protein

    CY

    6

    CC2212

    hypothetical protein

    U(+)

    3

    CC2257

    hypothetical protein

    U(+)

    8

    CC2287

    TonB-Dependent Receptor

    OM

    7

    CC2294

    outer membrane protein

    PP

    3

    CC2296

    hypothetical protein

    U(+)

    2

    CC2336

    MotA/TolQ/ExbB proton channel family protein

    IM(4)

    4

    CC2456

    carboxypeptidase (cpsA)

    U(-)

    2

    CC2476

    hypothetical protein

    U(+)

    12

    CC2477

    hypothetical protein

    U(-)

    11

    CC2539

    conserved hypothetical protein

    U(+)

    5

    CC2544

    aminopeptidase, putative

    EX

    2

    CC2578

    peptidase, M16 family

    U(+)

    23

    CC2672

    Xaa-Pro dipeptidase, putative

    U(+)

    3

    CC2758

    serine protease HtrA (htrA)

    U(+)

    10

    CC2819

    TonB-Dependent Receptor

    OM

    9

    CC2820

    TonB-Dependent Receptor

    OM

    5

    CC2924

    TonB-Dependent Receptor

    OM

    32

    CC2944

    pilus assembly protein CpaD (cpaD)

    U(+)

    4

    CC2945

    pilus assembly protein CpaC (cpaC)

    OM

    4

    CC3007

    hypothetical protein

    U(+)

    6

    CC3013

    TonB-Dependent Receptor

    OM

    16

    CC3107

    peptidase, M20/M25/M40 family

    EX

    5

    CC3137

    spermidine/putrescine ABC transporter, periplasmic spermidine/putrescine-binding protein (potF)

    PP

    2

    CC3146

    TonB-Dependent Receptor

    OM

    20

    CC3147

    TonB-Dependent Receptor

    OM

    4

    CC3200

    translation elongation factor G (fusA)

    CY

    2

    CC3229

    OmpA Family Protein

    OM

    7

    CC3230

    tolB protein (tolB)

    PP

    5

    CC3232

    ExbD/TolR family protein

    IM(1)

    4

    CC3233

    MotA/TolQ/ExbB proton channel family protein

    IM(3)

    4

    CC3272

    glycerophosphoryl diester phosphodiesterase (glpQ)

    PP

    13

    CC3277

    penicillin binding protein, 1A family

    U(-)

    2

    CC3322

    peptidoglycan-binding protein, putative

    IM(0)

    3

    CC3376

    hypothetical protein

    U(+)

    2

    CC3392

    conserved hypothetical protein

    U(+)

    2

    CC3407

    cytochrome c oxidase, subunit II (coxB

    IM(3)

    2

    CC3444

    hypothetical protein

    U(-)

    8

    CC3447

    ATP synthase F1, beta subunit (atpD)

    U(+)

    3

    CC3455

    conserved hypothetical protein

    U(-)

    2

    CC3489

    penicillin-binding protein AmpH, putative

    PP

    2

    CC3494

    OmpA Family Protein

    OM

    6

    CC3504

    peptidase M13 family protein

    U(+)

    2

    CC3552

    conserved hypothetical protein

    U(+)

    2

    CC3559

    hypothetical protein

    U(-)

    2

    CC3584

    peptidase, M16 family

    U(+)

    6

    CC3688

    prolyl oligopeptidase family protein

    PP

    2

    (a) The localization of proteins was predicted with PSORT and is indicated with the following symbols: IM, inner membrane with predicted number of transmembrane domains in parentheses; OM, outer membrane; U(-), unknown with no predicted signal peptide; U(+), unknown with a predicted signal peptide; EX, extracellular; PP, periplasm; and CY, cytoplasmic.





    Table 2. Strains and plasmids

     

    Strain

    Description or construction

    Source or reference

    E. coli

    		  

    YB4055

    S17-1 pNTPSexbBM2-2

    This study

    YB4068

    S17-1 pMRpstCAGFP

    This study

    YB4625

    S17-1 (pJM21CC3137M2) (pLVC9)

    This study

    YB4627

    S17-1 (pJM21CC0362M2) (pLVC9)

    This study

    YB4631

    S17-1 (pJM21CC1515M2) (pLVC9)

    This study

    C. crescentus

    		  

    NA1000

    syn-1000, previously called CB15N, a synchronizable derivative of CB15

    Ref. 1

    YB776

    NA1000 (pstS::miniTn5lacZ)

    Ref. 2

    CB15NY111d1

    Stalk shedding mutant

    Ref. 3

    YB2811

    CB15NY111d1 pstS::miniTn5lacZ

    Ref. 4

    YB4058

    NA1000 exbBM2-2

    This study

    YB4634

    NA1000 CC3137::pJM21CC3137M2

    This study

    YB4635

    NA1000 CC0362::pJM21CC0362M2

    This study

    YB4637

    NA1000 CC1515::pJM21CC1515M2

    This study

    YB4062

    NA1000 pMRpstCAGFP

    This study

    YB4042

    NA1000 ∆bla ∆rsaA

    This study

       

    Plasmids

    		  

    pNTPS138

    Kanr, sacB

    M.R.K. Alley, unpublished

    pJM21+

    Ampr, M2 epitope fusion vector

    Ref. 5

    pLVC9

    Cmr Kans derivative of pGJ28 carrying ColE1 mob

    G. Warren, unpublished

    pNTPSexbBM2-2

    Cloned PstI/BamHI fragment from FexbBM2 and RExbBM2 amplification into pJM21+. Subcloned PstI/SpeI fragment into pNTPS138 (PstI/EcoRI) with the product of F2ExbDM2-2 and R2ExbDM2-2 (SpeI/EcoRI).

    This study

    pMRpstCAGFP

    Amplified pstCA from the 1B1 cosmid using FpstCpromH and RpstApa1 and GFP from EGFP-N2 using eGFPN2up and EGFPendH. Cut pstCA product with HindIII/ApaI and GFP product with ApaI/EcoRI. Cloned into HindIII/EcoRI site of pMR10.

    This study

    pJM21CC0362M2

    Cloned HindIII/ BamHI fragment from FCC0362M2 and RCC0362 amplification into pJM21+.

    This study

    pJM21CC1515M2

    Cloned HindIII/ BamHI fragment from FCC1515M2 and RCC1515 amplification into pJM21+.

    This study

    pJM21CC3137M2

    Cloned HindIII/ PstI fragment from FCC3137M2 and RCC3137 amplification into pJM21+.

    This study

    References

    1. Evinger, M. & Agabian, N. (1977) J. Bacteriol. 132, 294-301.

    2. Gonin, M., Quardokus, E. M., O'Donnol, D., Maddock, J. & Brun, Y. V. (2000) J. Bacteriol. 182, 337-347.

    3. Poindexter, J. S. (1978) J. Bacteriol. 135, 1141-1145.

    4. Ireland, M. M., Karty, J. A., Quardokus, E. M., Reilly, J. P. & Brun, Y. V. (2002) Mol. Microbiol. 45, 1029-1041.

    5. Alley, M. R. K., Maddock, J. R. & Shapiro, L. (1993) Science 259, 1754-1757.

     





    Table 3. Oligonucleotides used in this study

    Oligonucleotide

    Sequence 5’à3’

    EGFPN2up

    CGCTACCGGACTCAGATCTCGA

    EGFPendH

    TGTGGTATGGAAGCTTATGATCTAGA

    FExbBM2

    GCCTCGCAGGCTGCAGAGCTGTCGCG

    RExbBM2

    GCGGCCATGGGGATCCCGCCCCCTTG

    F2ExbDM2-2

    AGGGGGCGTAACTAGTATGGCCGCCA

    R2ExbDM2-2

    TCAGCCATGCGAATTCCCCTACTGGC

    FpstCpromH

    CATTGTCAAAAGCTTCATGAACCG

    RpstApa1

    ATGGACAAGCGGGCCCGCGGCGCTCG

    FCC0362M2

    CGGTGATCAGAAGCTTCCATCAGGC

    RCC0362M2

    CTCATCAAGGGGATCCGTTGGCCGG

    FCC1515M2

    TGCCGAACGAAGCTTTCCTGGCCT

    RCC1515M2

    GCTTTAGCCAGGATCCGTTCTTCGGC

    F3137M2

    CGCGGACCTGAAGCTTCTGTCGGCCA

    R3137M2

    TCGCGCCGCCTGCAGAGCCGCCCG

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A nutrient uptake role for bacterial cell envelope extensions
Jennifer K. Wagner, Sima Setayeshgar, Laura A. Sharon, James P. Reilly, Yves V. Brun
Proceedings of the National Academy of Sciences Aug 2006, 103 (31) 11772-11777; DOI: 10.1073/pnas.0602047103
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