Could transformation mechanisms of acetylase-harboring pMdT1 plasmid be evaluated through proteomic tools in Escherichia coli?

Luís Pinto, Alexandre Gonçalves, Hugo M. Santos, José Luis Capelo, Yolanda Saénz, María de Toro, Carmen Torres, Christophe Chambon, Michel Hébraud, Patrícia Poeta, Gilberto Igrejas

Research output: Contribution to journalArticlepeer-review

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. Biological significance 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.

Original languageEnglish
Pages (from-to)103-111
Number of pages9
JournalJournal of Proteomics
Volume145
DOIs
Publication statusPublished - 11 Aug 2016

Keywords

  • AAC-(6′)-lb-cr
  • Antibiotic resistance
  • Escherichia coli
  • MALDI-TOF MS and LC-MS/MS
  • Mechanisms of gene transfer
  • pMdT1 plasmid

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