Limits...
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.

Show MeSH
Utilization of dipeptides by strains of the SBP penta mutant complemented with individual SBPs.The numbers in parentheses indicate the total number of dipeptides analyzed. There are no significant differences among the complemented SBPs DppA1 to DppA4 as determined by single factor ANOVA. DppA1 to DppA4 show significant difference to DppA5 (p<0.05) as determined by a two-sided t test with equal variances.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206461&req=5

pone-0111311-g006: Utilization of dipeptides by strains of the SBP penta mutant complemented with individual SBPs.The numbers in parentheses indicate the total number of dipeptides analyzed. There are no significant differences among the complemented SBPs DppA1 to DppA4 as determined by single factor ANOVA. DppA1 to DppA4 show significant difference to DppA5 (p<0.05) as determined by a two-sided t test with equal variances.

Mentions: We observed that all SBPs are able to bind and deliver dipeptides with different side chains to the ABC transporter. DppA2 was able to recognize more dipeptides than any other SBP, about 15% of these dipeptides contain acidic residues (Figure 6). We observed the tendency that all SBPs, except DppA5, have a higher affinity for dipeptides containing acidic residues at the C-terminus than for dipeptides containing acidic residues at the N-terminus.


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)

Utilization of dipeptides by strains of the SBP penta mutant complemented with individual SBPs.The numbers in parentheses indicate the total number of dipeptides analyzed. There are no significant differences among the complemented SBPs DppA1 to DppA4 as determined by single factor ANOVA. DppA1 to DppA4 show significant difference to DppA5 (p<0.05) as determined by a two-sided t test with equal variances.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111311-g006: Utilization of dipeptides by strains of the SBP penta mutant complemented with individual SBPs.The numbers in parentheses indicate the total number of dipeptides analyzed. There are no significant differences among the complemented SBPs DppA1 to DppA4 as determined by single factor ANOVA. DppA1 to DppA4 show significant difference to DppA5 (p<0.05) as determined by a two-sided t test with equal variances.
Mentions: We observed that all SBPs are able to bind and deliver dipeptides with different side chains to the ABC transporter. DppA2 was able to recognize more dipeptides than any other SBP, about 15% of these dipeptides contain acidic residues (Figure 6). We observed the tendency that all SBPs, except DppA5, have a higher affinity for dipeptides containing acidic residues at the C-terminus than for dipeptides containing acidic residues at the N-terminus.

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