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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.
Escherichia coli has been adopted as a model organism for structural and functional studies aimed at understanding the biophysical and biochemical organization of proteins in Gram-negative cell walls. The cell walls of these bacteria comprise of three morphologically defined layers . The innermost layer is the cytoplasmic membrane that regulates passage of metabolites into and out of the cytoplasm. External to this membrane, a thin peptidoglycan layer encases the cytoplasmic membrane providing mechanical rigidity. The most exterior layer, the outer membrane is an additional barrier consisting of lipids, polysaccharides and proteins that easily distinguishes Gram-negative from Gram-positive bacteria that lack this additional membrane.
Studies of the outer membrane has revealed the presence of both integral and lipid-linked membrane proteins. In an early account using two-dimensional electrophoresis with carrier ampholyte generated pH gradients, Ames and Nikaido  provided an indication of the complexity of the E. coli cell envelope, reporting 150 proteins from this preparation. Some bacterial membrane proteins are expressed in high copy number (e.g. OmpA is present at 105 copies per cell ), which have aided their detection and biochemical characterization. These include the porins (e.g. OmpC, OmpF), a family of integral outer membrane proteins (OMPs) that form a hydrophilic channel, allowing nonspecific diffusion of small molecules across the outer membrane barrier (reviewed in [4–6]).Proteins responsible for transport of specific nutrients have also been identified. These include channel-forming transmembrane proteins for the passage of phosphate (PhoE), maltose and maltodextrins (LamB), and nucleosides (Tsx). Other transmembrane proteins have been implicated as high affinity receptors for the transport of ferric iron (FepA, FhuA), vitamin B12 (BtuB), and fatty acids (FadL). Furthermore, some of these OMPs are target receptors for bacteriophages and colicins. Finally, due to their location as interfaces between the cell and the environment, OMPs are important candidate antigens for developing strategies aimed at providing protection against bacterial pathogens [7–10].
The elucidation in 1997 of the nucleotide sequence encoding the E. coli K-12 genome is an invaluable resource for microbiologists . One of the opportunities this achievement presents is the chance to fully define the protein composition of a bacterial outer membrane. As of July 1999, all but approximately 10% of the open-reading frames (ORFs) had been annotated in the SWISS-PROT protein database (A. Bairoch, personal communication). At the present time, examination of the predicted ORFs incorporated into SWISS-PROT Release 37, indicates that E. coli K-12 has the potential to express 62 OMPs (discounting OMPs derived from plasmids). Proteins are annotated as OMPs based upon literature reports, computer prediction programs or by sequence similarity with known related OMPs (A. Bairoch, personal communication). When the known lipoproteins, which are attached by a N-acyl diglyceride linkage are omitted, 39 integral or transmembrane proteins are predicted in the E. coli outer membrane. However, for many of these ORFs it is not known whether they are transcriptionally active, with the potential gene-products known only as ‘hypothetical’ proteins. Furthermore, it is of interest to microbiologists to understand which gene-products are expressed under various states of growth and environmental stresses. One approach to examine these issues is to isolate and identify the entire complement of membrane proteins in parallel using a combination of 2D electrophoresis and mass spectrometry.
Until recently such an approach has been impractical due to technical difficulties in both extracting and solubilizing membrane proteins. This issue was highlighted by investigators recording proteins identified on 2D gels, and comparing these to proteins predicted by genome sequencing . Interestingly, for each of the three model organisms examined (E. coli, Bacillus subtilis, Saccharomyces cerevisiae) the majority of identified proteins displayed hydrophilic hydropathies, while hydrophobic proteins were vastly under-represented on these same gels. In addition, examination of current E. coli 2D gel reference maps (e.g. SWISS-2DPAGE) indicates the absence of many predicted OMPs. The confounding element in this case is that unlike the hydrophobic protein cluster missing from 2D gels , E. coli OMPs possess hydrophilic hydropathy values comparable to many cytoplasmic proteins that are easily separable by 2D electrophoresis. This suggests that extraction and solubilization of OMPs prior to 2D electrophoresis is an important area that requires exploration.
Solving this aspect of the puzzle is important, as excellent progress in downstream steps has been achieved. These include improved solubilization of some intractable proteins, combined with reproducible, high loading 2D gels [13,14] and improved protein microanalysis techniques such as mass spectrometry [15,16], intertwined with powerful database identification packages. This science has been formalized as ‘proteomics’. Put simply, proteomics is a formalized approach for obtaining a rapid ‘snap-shot’ of the protein complement of a tissue, cell or cell component [17,18]. Such an approach is powerful in that it allows a parallel assessment of temporal protein fluxes. This is an important concept in view of the dynamic nature of protein expression. Undoubtedly, changes in protein expression are essential in any study aimed at investigating cellular networks.
In this report we describe a proteomic approach to study the protein composition of the E. coli outer membrane. We propose a simple method for the rapid isolation, separation by 2D electrophoresis and identification of predominantly integral OMPs. We compare predicted and experimentally identified E. coli OMPs and evaluate the potential of a protein-based approach for large-scale analyses of membrane proteins. In addition we present two cases demonstrating the value of a proteomic approach for generating insight into protein function. In the first instance we propose a role for a previously hypothetical protein (YbiL) as an iron-binding protein related to FoxA, while in the second experiment we identify two temperature inducible OMPs. Finally, the significance of a proteomic approach for the study of bacterial OMPs is discussed.
- Top of page
- Materials and methods
We have developed a simple and rapid method for the isolation and separation of OMPs from E. coli. The most abundant proteins isolated with this technique were identified as transmembrane cell surface proteins, many previously missing from E. coli 2D gel reference maps. Significantly, the isolation and separation procedure appeared equally applicable when trialed with the Gram-negative microbe P. aeruginosa. Furthermore, this protocol is amenable to micropreparative separations. This is important in the context of high-throughput analysis, where each protein spot can then be subjected to a microanalytical identification/characterization technique, removing any need to pool identical protein spots from multiple gels. We view this study as an important first step to encourage proteomic-based investigations of bacterial membranes. As we have demonstrated such an approach can provide profound results (i.e. identification of a new iron receptor) even with widely studied model species such as E. coli.
One of the key procedures for the isolation of integral OMPs is incubating the cells in an alkaline solution of sodium carbonate. This approach avoids the need to isolate membranes by traditional methods such as density centrifugation, which is often time-consuming. In the initial report describing carbonate treatment, Fujiki et al.  reported that this procedure converted closed microsomal vesicles into open sheets, concomitantly stripping loosely attached peripheral proteins. In contrast to eukaryotic glycosylphosphatidylinositol linked proteins that are removed with carbonate treatment, a number of E. coli N-acyl diglyceride attached lipoproteins were recovered with the integral OMPs, albeit in much lower abundance than the integral OMPs (Table 2). It is anticipated that both the OM and CM are insoluble with this treatment. Nonetheless, our 2D gel indicates that OMPs are preferentially solubilized by the IEF solution, with proteins of the CM (predicted with considerably higher hydrophobic characteristics) most likely remaining insoluble. This is despite using a solubilizing solution amenable to 2D electrophoresis that we consider contains some of the most powerful reagents, outside of SDS. SDS/PAGE of the insoluble pellet showed the presence of some additional bands not detected in a sample that provided Fig. 1 (data not shown).
We recovered 21 out of 37 of all the predicted E. coli integral OMPs between pH 4–7 and independent of molecular mass. This takes into account 10 hypothetical proteins that were not observed. If one considers that these ORFs are not transcriptionally active under the conditions described or are not authethic transcripts, then we identified 78% of the integral OMPs with known transcriptionally activity. This data supports the view that during exponential growth in a defined minimal media, E. coli K-12 transcribes and translates greater than three-quarters of its genes encoding authentic integral OMPs. In addition, it is important for protein-based analyses of membrane proteins to note this level of protein coverage. We view this result as encouraging, and anticipate that with correct experimental design the large-scale analysis offered by proteomics will support investigation of complex biological networks.
At this point it is prudent to offer explanations for the missing 22% of predicted proteins. Large molecular mass proteins (> 80 kDa) are commonly missing from micropreparative 2D maps. The three large predicted proteins not detected here (NfrA, 108 kDa; FimD, 92 kDa; SfmD, 92 kDa) may have been lost in the separation procedure, either at the point of IPG reswelling or during IEF by precipitation at the isoelectric point. Of the remaining three unidentified proteins less than 80 kDa, OmpN is known as a quiescent porin, whose expression is diminished by the presence of the classical porins, OmpC and OmpF . It is likely the levels of expression of OmpN are too low for detection in the W3110 strain used in this experiment. Finally, GspD and HofQ, both annotated as general secretion pathway proteins are known only from their DNA sequence. These proteins may not be expressed in minimal media where secretion may be low, or alternatively are only lowly expressed thereby escaping detection. Of the hypothetical proteins, clearly further work is required to understand their absence. Monitoring of mRNA expression may confirm if these proteins are transcriptionally active, and therefore could be expected on 2D gels. We were surprised that we did not identify YiaT (pI 4.48, 25 kDa) as it is expected in a region of the gel where its presence should be obvious. This may indicate YiaT is not expressed, or at best lowly expressed in W3110 under the growth conditions described here.
In an interesting aspect of this study we monitored OMP expression under conditions of iron limitation. This led to the observation of increased expression of a protein (YbiL) with no previous known function. This response was in accord with expression increases from other known outer membrane iron binding proteins and implicates YbiL as an iron receptor. This approach of monitoring comparative expression levels has been valuable, enabling us to postulate a role for a previously hypothetical protein. Importantly, this opinion is supported by amino acid sequence similarity with other proteins of known function (Table 4). This provides an example of how proteomics can be used to enhance and strengthen information generated by genomics. In a second experiment we were able to readily document changes in protein composition of the outer membrane as cells were cultured at different temperatures. This approach identified an OMP (Ag43) that had previously shown phase-variable expression , although it had not been reported that Ag43 is repressed at low growth temperatures. This provides one example of how such an approach is a useful first step to identify interesting proteins prior to launching more direct functional studies. The protease OmpT was also missing from cells cultured at 21 °C. In this case, previous reports have shown OmpT to display temperature dependant expression [33,34]. Thus, our approach has demonstrated both discovery and confirmation of a previous finding.
The 2D gel (Fig. 1) exhibits a unique library of OMPs. We are of the opinion that there is great potential for uses of such a library in biochemistry and biotechnology. For example, the 2D gel pattern represents a bacterial ‘finger-print’ that may be useful in discerning bacterial species. Additionally, these proteins could be tested for their ability to interact with various ligands. To extrapolate from the current report, it should be possible to create a similar library of OMPs from many other Gram-negative bacteria. Even for species with undefined genomes, the near exclusive isolation of integral OMPs using this method means an abundant protein spot on a gel is highly likely to be an integral OMP and is worth the effort to identify and characterize it, albeit using techniques that are slower than PMF (i.e. Edman sequencing) or more complex (i.e. MS/MS). Continuing with this argument, for the immunologist there is enormous potential to use this approach to ‘fast-track’ identification of likely candidate antigens that may elicit immune responses in invaded host organisms. Integral and lipid-linked OMPs may be key molecules in designing strategies to guard against bacterial infection.