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

Pletzer D, Lafon C, Braun Y, Köhler T, Page MG, Mourez M, Weingart H - PLoS ONE (2014)

Bottom Line: We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides.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.

View Article: PubMed Central - PubMed

Affiliation: Jacobs University Bremen, School of Engineering and Science, Bremen, Germany.

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.

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Related in: MedlinePlus

Global utilization pattern of dipeptides by PA14, the dipeptide transporter DppBCDF and the SBPs DppA1–A5.The rings show the total number of dipeptides utilized by the wild-type strain PA14 (outer purple ring), transported by the dipeptide transporter system DppBCDF (middle blue ring), and recognized by the substrate-binding proteins (inner dark green ring). The oval rings (green) present different pools of dipeptides (nonpolar, acidic, uncharged) containing a specific amino acid side-chain either at the N- or C-terminal end of the dipeptide (see Table 2). Dipeptides containing positively charged amino acid residues were excluded from the analysis (see Materials and Methods).
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pone-0111311-g003: Global utilization pattern of dipeptides by PA14, the dipeptide transporter DppBCDF and the SBPs DppA1–A5.The rings show the total number of dipeptides utilized by the wild-type strain PA14 (outer purple ring), transported by the dipeptide transporter system DppBCDF (middle blue ring), and recognized by the substrate-binding proteins (inner dark green ring). The oval rings (green) present different pools of dipeptides (nonpolar, acidic, uncharged) containing a specific amino acid side-chain either at the N- or C-terminal end of the dipeptide (see Table 2). Dipeptides containing positively charged amino acid residues were excluded from the analysis (see Materials and Methods).

Mentions: The remaining set of dipeptides was classified based on the properties of the side-chain group of the single amino acid residues (acidic, basic, uncharged polar or nonpolar) and the ability of PA14 to utilize them as nitrogen source (Table 1, Figure 3). Since dipeptides contain two amino acids, we clustered each dipeptide into two groups. For example, if a dipeptide contains one nonpolar and one uncharged amino acid (N- or C-terminally attached), this dipeptide was clustered into the pool of ‘nonpolar side-chains’ as well as into the pool of ‘uncharged side-chains’. This classification allowed us to identify whether specific side-chain groups are preferred by the dipeptide permease. Based on this grouping, we clustered 173 dipeptides as nonpolar, 43 as acidic, and 75 as uncharged. These clusters were then further divided based on the position of the amino acid residue in the dipeptide; either N- or C-terminal (Table 2). Basic amino acids were excluded from our analysis (see Materials and Methods).


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

Pletzer D, Lafon C, Braun Y, Köhler T, Page MG, Mourez M, Weingart H - PLoS ONE (2014)

Global utilization pattern of dipeptides by PA14, the dipeptide transporter DppBCDF and the SBPs DppA1–A5.The rings show the total number of dipeptides utilized by the wild-type strain PA14 (outer purple ring), transported by the dipeptide transporter system DppBCDF (middle blue ring), and recognized by the substrate-binding proteins (inner dark green ring). The oval rings (green) present different pools of dipeptides (nonpolar, acidic, uncharged) containing a specific amino acid side-chain either at the N- or C-terminal end of the dipeptide (see Table 2). Dipeptides containing positively charged amino acid residues were excluded from the analysis (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206461&req=5

pone-0111311-g003: Global utilization pattern of dipeptides by PA14, the dipeptide transporter DppBCDF and the SBPs DppA1–A5.The rings show the total number of dipeptides utilized by the wild-type strain PA14 (outer purple ring), transported by the dipeptide transporter system DppBCDF (middle blue ring), and recognized by the substrate-binding proteins (inner dark green ring). The oval rings (green) present different pools of dipeptides (nonpolar, acidic, uncharged) containing a specific amino acid side-chain either at the N- or C-terminal end of the dipeptide (see Table 2). Dipeptides containing positively charged amino acid residues were excluded from the analysis (see Materials and Methods).
Mentions: The remaining set of dipeptides was classified based on the properties of the side-chain group of the single amino acid residues (acidic, basic, uncharged polar or nonpolar) and the ability of PA14 to utilize them as nitrogen source (Table 1, Figure 3). Since dipeptides contain two amino acids, we clustered each dipeptide into two groups. For example, if a dipeptide contains one nonpolar and one uncharged amino acid (N- or C-terminally attached), this dipeptide was clustered into the pool of ‘nonpolar side-chains’ as well as into the pool of ‘uncharged side-chains’. This classification allowed us to identify whether specific side-chain groups are preferred by the dipeptide permease. Based on this grouping, we clustered 173 dipeptides as nonpolar, 43 as acidic, and 75 as uncharged. These clusters were then further divided based on the position of the amino acid residue in the dipeptide; either N- or C-terminal (Table 2). Basic amino acids were excluded from our analysis (see Materials and Methods).

Bottom Line: We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides.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.

View Article: PubMed Central - PubMed

Affiliation: Jacobs University Bremen, School of Engineering and Science, Bremen, Germany.

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.

Show MeSH
Related in: MedlinePlus