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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

Effect of the localization of an amino acid residue on utilization of a dipeptide.The middle square boxes represent the specific amino acid. The background color of the box gives information about the chemical nature of the specific side-chain group. The left and right arms of a box indicate the localization of the amino acid either at the N- or C-terminal end of the dipeptide. The numbers within the arms indicate how many dipeptides of this group were utilized by the different strains. The colors within the arms indicate the percentage of dipeptides used by a strain (see legend) related to the total amount of dipeptides used by the wild type.
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pone-0111311-g005: Effect of the localization of an amino acid residue on utilization of a dipeptide.The middle square boxes represent the specific amino acid. The background color of the box gives information about the chemical nature of the specific side-chain group. The left and right arms of a box indicate the localization of the amino acid either at the N- or C-terminal end of the dipeptide. The numbers within the arms indicate how many dipeptides of this group were utilized by the different strains. The colors within the arms indicate the percentage of dipeptides used by a strain (see legend) related to the total amount of dipeptides used by the wild type.

Mentions: A closer look at single amino acid residues of the dipeptides revealed that only 1 out of 7 dipeptides that contained an Asn residue at the C-terminal end was utilized by the transporter (Figure 5). Moreover, only 2 out of 10 dipeptides containing Tyr at the N-terminal end could be used. Dipeptides containing Leu residues were poor substrates of the DppBCDF transporter. However, a localization of the Leu residue at the C-terminal end leads to a slightly increased utilization of these dipeptides by the ABC transporter. Moreover, less than 50% of dipeptides containing Ala or Gln at the N-terminal end were utilized by the transporter system (Figure 4, Figure 5). In contrast, almost all Thr-containing dipeptides were utilized regardless of their localization at the N- or C-terminal end. A similar observation was made with dipeptides containing Asp and Glu, which have negatively charged side chains. Several other dipeptides containing nonpolar amino acid residues such as Gly, Ile, Met, Pro, Thr, and Trp were good transporter substrates. While most of those residues were attached at the N-terminal end, Met and Pro showed higher preference of being utilized when residues were at the C-terminal end (Figure 5).


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)

Effect of the localization of an amino acid residue on utilization of a dipeptide.The middle square boxes represent the specific amino acid. The background color of the box gives information about the chemical nature of the specific side-chain group. The left and right arms of a box indicate the localization of the amino acid either at the N- or C-terminal end of the dipeptide. The numbers within the arms indicate how many dipeptides of this group were utilized by the different strains. The colors within the arms indicate the percentage of dipeptides used by a strain (see legend) related to the total amount of dipeptides used by the wild type.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111311-g005: Effect of the localization of an amino acid residue on utilization of a dipeptide.The middle square boxes represent the specific amino acid. The background color of the box gives information about the chemical nature of the specific side-chain group. The left and right arms of a box indicate the localization of the amino acid either at the N- or C-terminal end of the dipeptide. The numbers within the arms indicate how many dipeptides of this group were utilized by the different strains. The colors within the arms indicate the percentage of dipeptides used by a strain (see legend) related to the total amount of dipeptides used by the wild type.
Mentions: A closer look at single amino acid residues of the dipeptides revealed that only 1 out of 7 dipeptides that contained an Asn residue at the C-terminal end was utilized by the transporter (Figure 5). Moreover, only 2 out of 10 dipeptides containing Tyr at the N-terminal end could be used. Dipeptides containing Leu residues were poor substrates of the DppBCDF transporter. However, a localization of the Leu residue at the C-terminal end leads to a slightly increased utilization of these dipeptides by the ABC transporter. Moreover, less than 50% of dipeptides containing Ala or Gln at the N-terminal end were utilized by the transporter system (Figure 4, Figure 5). In contrast, almost all Thr-containing dipeptides were utilized regardless of their localization at the N- or C-terminal end. A similar observation was made with dipeptides containing Asp and Glu, which have negatively charged side chains. Several other dipeptides containing nonpolar amino acid residues such as Gly, Ile, Met, Pro, Thr, and Trp were good transporter substrates. While most of those residues were attached at the N-terminal end, Met and Pro showed higher preference of being utilized when residues were at the C-terminal end (Figure 5).

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