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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

Heat map of di/tripeptide utilization by PA14, the DppBCDF transporter mutant, the SBP penta mutant DppA1–A5, and by strains of the penta mutant complemented with individual SBPs.Each square represents the average respiratory activity of a strain in one well of the Biolog Phenotype MicroArray plates. The heat map is based on the values reflecting the extent of respiration after 24 hours at 37°C. Values exceeding 8000 reflect solid respiratory activity during the assay. Values below 2000 were considered as no respiratory activity.
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pone-0111311-g004: Heat map of di/tripeptide utilization by PA14, the DppBCDF transporter mutant, the SBP penta mutant DppA1–A5, and by strains of the penta mutant complemented with individual SBPs.Each square represents the average respiratory activity of a strain in one well of the Biolog Phenotype MicroArray plates. The heat map is based on the values reflecting the extent of respiration after 24 hours at 37°C. Values exceeding 8000 reflect solid respiratory activity during the assay. Values below 2000 were considered as no respiratory activity.

Mentions: In order to investigate the substrate profile of the ABC transporter DppBCDF, a deletion mutant of the dppBCDF operon was constructed (Figure 1A). We used Biolog phenotype MicroArrays to elucidate the effect of the dppBCDF deletion on the ability of the mutant to utilize dipeptides. Dipeptides that did not support respiratory activity of the mutant indicated deficiency of the mutant to utilize the respective dipeptide as nitrogen source. Biolog respiration curve comparison between the wild type and the dppBCDF-deficient mutant for all PM plates are provided in Dataset S2. A direct comparison of utilized dipeptides in terms of the extent of respiratory activity is demonstrated in the heat map in Figure 4.


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)

Heat map of di/tripeptide utilization by PA14, the DppBCDF transporter mutant, the SBP penta mutant DppA1–A5, and by strains of the penta mutant complemented with individual SBPs.Each square represents the average respiratory activity of a strain in one well of the Biolog Phenotype MicroArray plates. The heat map is based on the values reflecting the extent of respiration after 24 hours at 37°C. Values exceeding 8000 reflect solid respiratory activity during the assay. Values below 2000 were considered as no respiratory activity.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111311-g004: Heat map of di/tripeptide utilization by PA14, the DppBCDF transporter mutant, the SBP penta mutant DppA1–A5, and by strains of the penta mutant complemented with individual SBPs.Each square represents the average respiratory activity of a strain in one well of the Biolog Phenotype MicroArray plates. The heat map is based on the values reflecting the extent of respiration after 24 hours at 37°C. Values exceeding 8000 reflect solid respiratory activity during the assay. Values below 2000 were considered as no respiratory activity.
Mentions: In order to investigate the substrate profile of the ABC transporter DppBCDF, a deletion mutant of the dppBCDF operon was constructed (Figure 1A). We used Biolog phenotype MicroArrays to elucidate the effect of the dppBCDF deletion on the ability of the mutant to utilize dipeptides. Dipeptides that did not support respiratory activity of the mutant indicated deficiency of the mutant to utilize the respective dipeptide as nitrogen source. Biolog respiration curve comparison between the wild type and the dppBCDF-deficient mutant for all PM plates are provided in Dataset S2. A direct comparison of utilized dipeptides in terms of the extent of respiratory activity is demonstrated in the heat map in Figure 4.

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