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PHYSIOLOGY AND BIOTECHNOLOGY

Proteomic Analysis Reveals Differential Protein Expression by Bacillus cereus during Biofilm Formation

Marinda C. Oosthuizen, Bridgitta Steyn, Jacques Theron, Pascal Cosette, Denise Lindsay, Alexander von Holy, Volker S. Brözel
Marinda C. Oosthuizen
1School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050
2Laboratory for Biofilm Physiology, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa
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  • For correspondence: mcoosthuizen@excite.com
Bridgitta Steyn
2Laboratory for Biofilm Physiology, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa
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Jacques Theron
2Laboratory for Biofilm Physiology, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa
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Pascal Cosette
3UMR 6522 CNRS, Université de Rouen, Faculté des Sciences, 76821 Mont-Saint-Aignan Cedex, France
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Denise Lindsay
1School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050
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Alexander von Holy
1School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050
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Volker S. Brözel
2Laboratory for Biofilm Physiology, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa
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DOI: 10.1128/AEM.68.6.2770-2780.2002
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  • FIG. 1.
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    FIG. 1.

    Photomicrographs illustrating the development of B. cereus DL5 biofilms on glass wool in SONLB at 2 h (a), 18 h (b and c), and 42 h (d) after inoculation.

  • FIG. 2.
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    FIG. 2.

    Culturable counts (expressed as log10 CFU per milliliter of culture) (a) and cell volumes (expressed as cubic micrometers) (b) of B. cereus DL5 planktonic cells (P), PGW cells (SIP), and biofilm cells (A) grown for 2 and 18 h in SONLB at 37°C. Different letters indicate that differences are statistically significant (P < 0.05).

  • FIG. 3.
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    FIG. 3.

    Extracellular protein profiles of 18-h-old planktonic cells grown in the absence of glass wool (a) and combined PGW-biofilm cells (b) of B. cereus DL5 following separation by one-dimensional SDS-12% PAGE and silver staining. A BENCHMARK prestained protein ladder (GIBCO BRL) was used as the molecular weight (MW) marker (lane M). Unique bands are indicated by arrows.

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    FIG. 4.

    2DE proteome patterns for 2-h-old B. cereus DL5 planktonic cells (a), PGW cells (b), and biofilm cells (c) and 18-h-old B. cereus DL5 planktonic cells (d), PGW cells (e), and biofilm cells (f). Unique protein spots are indicated by arrows. The horizontal axes represent pIs of the isoelectric focusing gradients, and the vertical axes represent molecular masses (MW), based on comigration of Bio-Rad 2-D SDS-PAGE standards (Roche Diagnostics GmbH).

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  • TABLE 1.

    Protein expression in B. cereus DL5 planktonic, PGW, and biofilm cells following 2 and 18 h of culturing in the absence and presence of glass wool

    Protein expressionNo. of spots% of total spots (n = 345)
    Proteins unique to a specific growth phase
        Planktonic cells (2 h)10.3
        PGW cells (2 h)20.6
        Biofilm cells (2 h)154.4
        Planktonic cells (18 h)10.3
        PGW cells (18 h)72.3
        Biofilm cells (18 h)72.0
    Proteins up-regulated in a specific phase compared with the levels of the corresponding spot in other phases
        Planktonic cells (2 h)30.9
        PGW cells (2 h)102.9
        Biofilm cells (2 h)41.2
        Planktonic cells (18 h)72.0
        PGW cells (18 h)308.7
        Biofilm cells (18 h)195.5
    Proteins present in a specific phase regardless of time (2 h or 18 h)
        Planktonic cells00
        PGW cells10.3
        Biofilm cells51.5
    Proteins present in all phases and always at the same level (no up- or down-regulation)3510.1
    Proteins present in planktonic and PGW cells and absent in biofilm cells113.2
    Proteins present in PGW and biofilm cells and absent in planktonic cells00
    Proteins present in planktonic and biofilm cells and absent in PGW cells00
    After 2 h present only in biofilm cells; after 18 h present in PGW and biofilm cells20.6
    After 2 h present only in planktonic cells; after 18 h present in planktonic and PGW cells10.4
  • TABLE 2.

    Growth phase-related differences in the levels of expression of proteins selected for N-terminal protein sequencing

    Protein spotLevel of expression
    Planktonic cells (2 h)PGW cells (2 h)Biofilm cells (2 h)Planktonic cells (18 h)PGW cells (18 h)Biofilm cells (18 h)
    B5a0a00−2−1−1
    B11b012020
    D5h000−133
    E1d000011
    E7gAbA0A00
    E7h003033
    E9pAAAAAUc
    G1b012233
    • ↵ a A value of 0 indicates a level of synthesis similar to that of the corresponding spot in the 2-h planktonic growth phase (reference spot intensity). A positive value indicates up-regulation, and a negative value indicates repression or down-regulation.

    • ↵ b A, protein spot is absent.

    • ↵ c U, protein spot is unique to the growth phase.

  • TABLE 3.

    Summary of the proteins identified from 2DE gels of B. cereus DL5 whole-cell protein extracts

    Protein spotProtein identityFunctionN-terminal sequenceEstimated from gelCalculated from sequenceAccession no.
    Size (kDa)pISize (kDa)pIB. cereus databaseaSWISS-PROT protein database
    B5aPyruvate dehydrogenase E1 component, beta subunit (PdbB)Pyruvate + lipoamide → S-acetyl-dihydro-lipoamide + CO2QMTMIQAITD364.9035.234.75 RZC01921 P21882
    B11bUnknownMNVKRHM824.80
    D5h l-Lactate dehydrogenase (LctE)Pyruvate + NADH → l-lactate + NAD+MKKGINRVVL345.5034.794.99 RZC05932 P13714
    E1dHypothetical cytosolic proteinUnknownAKDFYSAIEDRRS245.6523.075.21 RZC03248 A83323
    E7gcOTCase l-Citrulline + Pi → carbamoyl phosphate + ornithineMLMTRPNLKGRS395.7037.465.35 RZC04697 O86132
    E7hcOTCase l-Citrulline + Pi → carbamoyl phosphate + ornithineMLMTRPNLKGRS395.8037.465.35 RZC04697 O86132
    E9pUnknownMPPVVI546.10
    G1bYhbH light-repressed protein AUnknownMKFNI236.2021.085.63 RZC04452 P28368
    • ↵ a Accession number obtained from the B. cereus database website (http://wit.Integratedgenomics.com/Public/IG_Release.html ).

    • b Accession number obtained from the SWISS-PROT protein database website (http://www.expasy.ch/sprot/ ).

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Proteomic Analysis Reveals Differential Protein Expression by Bacillus cereus during Biofilm Formation
Marinda C. Oosthuizen, Bridgitta Steyn, Jacques Theron, Pascal Cosette, Denise Lindsay, Alexander von Holy, Volker S. Brözel
Applied and Environmental Microbiology Jun 2002, 68 (6) 2770-2780; DOI: 10.1128/AEM.68.6.2770-2780.2002

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Proteomic Analysis Reveals Differential Protein Expression by Bacillus cereus during Biofilm Formation
Marinda C. Oosthuizen, Bridgitta Steyn, Jacques Theron, Pascal Cosette, Denise Lindsay, Alexander von Holy, Volker S. Brözel
Applied and Environmental Microbiology Jun 2002, 68 (6) 2770-2780; DOI: 10.1128/AEM.68.6.2770-2780.2002
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KEYWORDS

Bacillus cereus
Bacterial Proteins
biofilms
proteome

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