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Pseudomonas: Biology and Diversity |
Department of Microbiology1 and the W. M. Keck Biomedical Mass Spectrometry Laboratory, Biomolecular Research Facility2, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA3
Author for correspondence: Joanna B. Goldberg. Tel: +1 804 243 2774. Fax: +1 804 982 1071. e-mail: jbg2b{at}virginia.edu
Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates. To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography–tandem mass spectrometry (µLC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections. Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis. Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels. Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain. The proteins were identified by µLC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.
Keywords: genome analysis, proteomics, peptide sequencing, outer-membrane proteins, genotypic and phenotypic comparison
Abbreviations: 2-D, two-dimensional; CF, cystic fibrosis; IPG, immobilized pH gradient; µLC/MS/MS, micro-capillary column liquid chromatography–tandem mass spectrometry; REA, restriction endonuclease analysis; RAPD, random amplified polymorphic DNA
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