title: Could transformation mechanisms of acetylase-harboring pMdT1 plasmid be evaluated through proteomic tools in Escherichia coli?
abstract: Escherichia coli is a commensal microorganism of the gastrointestinal tract of animals and humans and it is an excellent model organism for the study of antibiotic resistance mechanisms. The resistance transmission and other characteristics of bacteria are based on different types of gene transfer occurring throughout the bacterial evolution. One of which is horizontal gene transfer that allows us to understand the ability of bacteria to acquire new genes. One dimensional and two dimensional electrophoresis (2-DE) techniques were performed in order to identify and characterize the proteome of two E. coli strains: Electromax DH10B, a transformation-ready strain; and TF-Se20, the Electromax DH10B that contains the aac(6')-Ib-cr4-harboring pMdT1 plasmid. After 2-DE and subsequent analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), it was possible to identify 76 distinct proteins on the TF-Se20 strain, whereas 71 had a known function. From Electromax DH10B strain, 72 different proteins were identified of which 71 were associated with a biological process. The protein of interest, aminoglycoside N-(6')-acetyltransferase type 1, was identified by MALDI-TOF MS. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique was performed to determine its sequence. Seventy six percent of the acetylase sequence was reconstructed only in the TF-Se20 strain, representing the single protein associated to antibiotic resistance. MALDI-TOF MS and LC-MS/MS approaches allowed us to determine the total proteome of both strains, as well as the acetylase sequence. Both of them enhance the ability to obtain more accurate information about the mechanisms of antimicrobial resistance. The pMdT1 plasmid brings a new perspective in understanding the metabolic processes that lead to antibiotic resistance.
  
  
  
  This study highlights the importance of proteomics and bioinformatics in understanding mechanisms of gene transfer and antibiotic resistance. These two approaches allow to compare the protein expression in different samples, as well as different biological processes related to each protein.
authors.affiliation: Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Magalhães
authors.affiliation: Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Pinto
authors.affiliation: Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Gonçalves
authors.affiliation: UCIBIO-REQUIMTE, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal.
authors.forename: José Eduardo
authors.initials: JE
authors.lastname: Araújo
authors.affiliation: UCIBIO-REQUIMTE, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal; ProteoMass Scientific Society, Faculty of Sciences and Technology, Caparica, Portugal.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Santos
authors.affiliation: UCIBIO-REQUIMTE, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal; ProteoMass Scientific Society, Faculty of Sciences and Technology, Caparica, Portugal.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Capelo
authors.affiliation: Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Saénz
authors.affiliation: Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain; Instituto de Biomedicina y Biotecnologia de Cantabria IBBTEC, Universidad de Cantabria/CSIC, Santander, Spain.
authors.forename: María
authors.initials: M
authors.lastname: de Toro
authors.affiliation: Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain; Department of Food and Agriculture, Biochemistry and Molecular Biology, University of La Rioja, Logroño, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Torres
authors.affiliation: Institut National de la Recherche Agronomique, Centre Auvergne-Rhône-Alpes, Plate-Forme d'Exploration du Métabolisme composante protéomique (PFEMcp), France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chambon
authors.affiliation: Institut National de la Recherche Agronomique, Centre Auvergne-Rhône-Alpes, Plate-Forme d'Exploration du Métabolisme composante protéomique (PFEMcp), France; Institut National de la Recherche Agronomique, Centre Auvergne-Rhône-Alpes, UR454 Microbiologie, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Hébraud
authors.affiliation: UCIBIO-REQUIMTE, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal; Veterinary Science Department, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Poeta
authors.affiliation: Functional Genomics and Proteomics Unit, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; Department of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro, Vila Real, Portugal; UCIBIO-REQUIMTE, Faculty of Science and Technology, University NOVA of Lisbon, Caparica, Portugal. Electronic address: gigrejas@utad.pt.
authors.forename: Gilberto
authors.initials: G
authors.lastname: Igrejas
mesh_terms: D000115:Acetylesterase / Q000235:genetics*; D000595:Amino Acid Sequence; D019295:Computational Biology; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics*; D022761:Gene Transfer, Horizontal; D010957:Plasmids / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D014170:Transformation, Genetic
chemical_list: D000115:Acetylesterase
keywords: AAC-(6′)-lb-cr; Antibiotic resistance; Escherichia coli; MALDI-TOF MS and LC-MS/MS; Mechanisms of gene transfer; pMdT1 plasmid