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Supplementary Figure 2 from Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

Supplementary Figure 3 from Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

Supplementary Figure 1 from Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

Supplementary Methods and Materials from Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

The recent completion of the Pseudomonas Genome Project, in conjunction with the Pseudomonas Community Annotation Project (PseudoCAP) has fast-tracked our ability to apply the tools encompassed under the term 'proteomics' to this pathogen. Such global approaches will allow the research community to answer long-standing questions regarding the ability of Pseudomonas aeruginosa to survive diverse habitats, its high intrinsic resistance to antibiotics and its pathogenic nature towards humans. Proteomics provides an array of tools capable of confirming the expression of Open Reading Frames (ORF), the relative levels of their expression, the environmental conditions required for this expression and the sub-cellular location of the encoded gene-products. Since proteins are important cellular effectors, the biological questions we pose can be defined in terms of changes in protein expression detectable by separation to purity using two-dimensional gel electrophoresis (2-DGE) and relation to gene sequences via mass spectrometry. As such, we can compare strains with well-characterized phenotypic differences, growth under a variety of stresses, protein interactions and complexes and aid in defining proteins of unknown function. While the complete genome has only recently been finished, a number of studies have already utilized this information and examined various protein gene-products using proteomics. This review summarizes the application of proteomics to P. aeruginosa and highlights potential areas of future research, including overcoming the traditional technical limitations associated with 2-DGE. More focused approaches that target sub-cellular fractions ('sub-proteomes') prior to 2-DGE can provide further functional information. A review of current and previous proteomic projects on P. aeruginosa is presented, as well as theoretical considerations of the importance of sub-proteomic approaches to enhance these investigations.

Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors. These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells. We report here comparisons of outer membrane and extracellular protein-enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P. aeruginosa separated by two-dimensional (2-D) gel electrophoresis. Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated. Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2-D gels. Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles. Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin-binding protein, as well as several previously designated 'hypothetical' proteins. LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study. The significant number of active extracellular proteases (including LasB itself) may account for this processing. Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits. These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells. These studies will aid in elucidating the differences between invasive and cytotoxic P. aeruginosa at the proteome level.