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
Effect of the chemical nature of amino acid residues located either at the N- or C-terminal end of dipeptides on the utilization by strains of the SBP penta mutant complemented with individual SBPs.The numbers indicate dipeptides that enabled a more than 2-fold increase in respiratory activity of SBP mutant complemented with an individual SBPs compared to the respiratory activity of the mutant carrying an empty plasmid. The SBP penta mutant was able to utilize these dipeptides.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111311-g007: Effect of the chemical nature of amino acid residues located either at the N- or C-terminal end of dipeptides on the utilization by strains of the SBP penta mutant complemented with individual SBPs.The numbers indicate dipeptides that enabled a more than 2-fold increase in respiratory activity of SBP mutant complemented with an individual SBPs compared to the respiratory activity of the mutant carrying an empty plasmid. The SBP penta mutant was able to utilize these dipeptides.

Mentions: The utilization of dipeptides was not strongly influenced by the type of amino acid residues located at the N- or C-terminal end. Although, it appears that DppA1, DppA3, and DppA4 prefer dipeptides with acidic residues at the C-terminal end (Table 3). Moreover, the SBP penta mutant was still able to utilize 118 clustered dipeptides as N source. In order to investigate whether the SBPs are able to increase the utilization of those peptides, we searched for dipeptides that increased the respiratory activity of the complemented strains at least 2-fold compared to the penta mutant. We found that DppA2 is able to increase the utilization of about 30% of all dipeptides, while the other SBPs were able to increase the utilization of less than 20% of dipeptides (Figure 7).


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 chemical nature of amino acid residues located either at the N- or C-terminal end of dipeptides on the utilization by strains of the SBP penta mutant complemented with individual SBPs.The numbers indicate dipeptides that enabled a more than 2-fold increase in respiratory activity of SBP mutant complemented with an individual SBPs compared to the respiratory activity of the mutant carrying an empty plasmid. The SBP penta mutant was able to utilize these dipeptides.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111311-g007: Effect of the chemical nature of amino acid residues located either at the N- or C-terminal end of dipeptides on the utilization by strains of the SBP penta mutant complemented with individual SBPs.The numbers indicate dipeptides that enabled a more than 2-fold increase in respiratory activity of SBP mutant complemented with an individual SBPs compared to the respiratory activity of the mutant carrying an empty plasmid. The SBP penta mutant was able to utilize these dipeptides.
Mentions: The utilization of dipeptides was not strongly influenced by the type of amino acid residues located at the N- or C-terminal end. Although, it appears that DppA1, DppA3, and DppA4 prefer dipeptides with acidic residues at the C-terminal end (Table 3). Moreover, the SBP penta mutant was still able to utilize 118 clustered dipeptides as N source. In order to investigate whether the SBPs are able to increase the utilization of those peptides, we searched for dipeptides that increased the respiratory activity of the complemented strains at least 2-fold compared to the penta mutant. We found that DppA2 is able to increase the utilization of about 30% of all dipeptides, while the other SBPs were able to increase the utilization of less than 20% of dipeptides (Figure 7).

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