High-throughput screening of dipeptide utilization mediated by the ABC transporter DppBCDF and its substrate-binding proteins DppA1-A5 in Pseudomonas aeruginosa

PLoS One. 2014 Oct 22;9(10):e111311. doi: 10.1371/journal.pone.0111311. eCollection 2014.

Abstract

In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1-A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Carrier Proteins / metabolism*
  • Dipeptides / metabolism*
  • Gene Order
  • Genetic Loci
  • High-Throughput Screening Assays*
  • Mutation
  • Nitrogen / metabolism
  • Operon
  • Pseudomonas aeruginosa / genetics
  • Pseudomonas aeruginosa / metabolism*
  • Substrate Specificity

Substances

  • ATP-Binding Cassette Transporters
  • Bacterial Proteins
  • Carrier Proteins
  • Dipeptides
  • Nitrogen

Grant support

The research leading to these results was conducted as part of the Translocation consortium (www.imi.europa.eu/content/translocation) and has received support from the Innovative Medicines Joint Undertaking under Grant Agreement No. 115525, resources which are composed of financial contribution from the European Union's seventh framework program (FP7/2007–2013) and EFPIA companies in kind contribution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.