Proteomic analysis of the Escherichia coli outer membrane

Mark P. Molloy*, Ben R. Herbert, Martin B. Slade, Thierry Rabilloud, Amanda S. Nouwens, Keith L. Williams, Andrew A. Gooley

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    412 Citations (Scopus)

    Abstract

    Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two- dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.

    Original languageEnglish
    Pages (from-to)2871-2881
    Number of pages11
    JournalEuropean Journal of Biochemistry
    Volume267
    Issue number10
    DOIs
    Publication statusPublished - 2000

    Keywords

    • Escherichia coli
    • proteome
    • outer membrane proteins
    • two-dimensional electrophoresis
    • integral membrane proteins

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