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Volume 154, Issue 3

Characterization of a ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system Free

Abstract

We describe an ATP-binding cassette (ABC) transporter in KT2440 that mediates the uptake of glutamate and aspartate. The system (AatJMQP, for cidic mino acid ransport) is encoded by an operon involving genes PP1071–PP1068. A deletion mutant with inactivated solute-binding protein (KT) failed to grow on Glu and Gln as sole sources of carbon and nitrogen, while a mutant lacking a functional nucleotide-binding domain (KT) was able to adapt to growth on Glu after an extended lag phase. Uptake of Glu and Asp by either mutant was greatly impaired at both low and high amino acid concentrations. The purified solute-binding protein AatJ exhibited high affinity towards Glu and Asp ( =0.4 and 1.3 μM, respectively), while Gln and Asn as well as dicarboxylates (succinate and fumarate) were bound with much lower affinity. We further show that the expression of AatJMQP is controlled by the -dependent two-component system AauRS. Binding of the response regulator AauR to the promoter was examined by gel mobility shift assays and DNase I footprinting. By screening, the AauR-binding motif (the inverted repeat NNNN) was detected in further KT2440 genes with established or putative functions in acidic amino acid utilization, and also occurred in other pseudomonads. The products of these AauR-responsive genes include the H/Glu symporter GltP, a periplasmic glutaminase/asparaginase, AnsB, and phosphoenolpyruvate synthase (PpsA), a key enzyme of gluconeogenesis in Gram-negative bacteria. Based on these findings, we propose that AauR is a central regulator of acidic amino acid uptake and metabolism in pseudomonads.

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Keyword(s): ABC, ATP-binding cassette and PAAT, polar amino acid uptake transporter
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2008-03-01
2024-04-26
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References

  1. Bagdasarian M., Timmis K. N. 1982; Host-vector systems for gene cloning in Pseudomonas . Curr Top Microbiol Immunol 96:47–67
     [Google Scholar]
  2. Banerjee S., Kundu T. K. 2003; The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription. Nucleic Acids Res 31:3236–3247
     [Google Scholar]
  3. Barrios H., Valderrama B., Morett E. 1999; Compilation and analysis of sigma54-dependent promoter sequences. Nucleic Acids Res 27:4305–4313
     [Google Scholar]
  4. Booth I. R., Kleppang K. E., Kempsell K. E. 1989; A genetic locus for the GltII-glutamate transport system in Escherichia coli . J Gen Microbiol 135:2767–2774
     [Google Scholar]
  5. Chao Y. P., Patnaik R., Roof W. D., Young R. F., Liao J. C. 1993; Control of gluconeogenic growth by pps and pck in Escherichia coli . J Bacteriol 175:6939–6944
     [Google Scholar]
  6. Copeland R. A. 2000 Enzymes: a Practical Introduction to Structure, Mechanism and Data Analysis New York: Wiley-VCH;
     [Google Scholar]
  7. Davidson A. L., Chen J. 2004; ATP-binding cassette transporters in bacteria. Annu Rev Biochem 73:241–268
     [Google Scholar]
  8. Fath M. J., Kolter R. 1993; ABC transporters: bacterial exporters. Microbiol Rev 57:995–1017
     [Google Scholar]
  9. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. 1986; Molecular cloning of the RP4 DNA primase region in a multirange tacP expression vector. Gene 48:119–131
     [Google Scholar]
  10. Harms E., Wehner A., Jennings M. P., Pugh K. J., Beacham I. R., Röhm K. H. 1991; Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts. Protein Expr Purif 2:144–150
     [Google Scholar]
  11. Hediger M. A. 1994; Structure, function and evolution of solute transporters in prokaryotes and eukaryotes. J Exp Biol 196:15–49
     [Google Scholar]
  12. Higgins C. F. 1992; ABC transporters: from microorganisms to man. Annu Rev Cell Biol 8:67–113
     [Google Scholar]
  13. Hosie A. H., Allaway D., Jones M. A., Walshaw D. L., Johnston A. W., Poole P. S. 2001; Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake. Mol Microbiol 40:1449–1459
     [Google Scholar]
  14. Janausch I. G., Zientz E., Tran Q. H., Kröger A., Unden G. 2002; C4-dicarboxylate carriers and sensors in bacteria. Biochim Biophys Acta 1553:39–56
     [Google Scholar]
  15. Kalman M., Gentry D. R., Cashel M. 1991; Characterization of the Escherichia coli K12 gltS glutamate permease gene. Mol Gen Genet 225:379–386
     [Google Scholar]
  16. Licht A., Brantl S. 2006; Transcriptional repressor CcpN from Bacillus subtilis compensates asymmetric contact distribution by cooperative binding. J Mol Biol 364:434–448
     [Google Scholar]
  17. Linton K. J., Higgins C. F. 1998; The Escherichia coli ATP-binding cassette (ABC) proteins. Mol Microbiol 28:5–13
     [Google Scholar]
  18. Morett E., Bork P. 1998; Evolution of new protein function: recombinational enhancer Fis originated by horizontal gene transfer from the transcriptional regulator NtrC. FEBS Lett 433:108–112
     [Google Scholar]
  19. Pridmore R. D. 1987; New and versatile cloning vectors with kanamycin-resistance marker. Gene 56:309–312
     [Google Scholar]
  20. Quentin Y., Fichant G., Denizot F. 1999; Inventory, assembly and analysis of Bacillus subtilis ABC transport systems. J Mol Biol 287:467–484
     [Google Scholar]
  21. Raunser S., Appel M., Ganea C., Geldmacher-Kaufer U., Fendler K., Kühlbrandt W. 2006; Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii . Biochemistry 45:12796–12805
     [Google Scholar]
  22. Reizer J., Reizer A., Saier M. H. Jr 1994; A functional superfamily of sodium/solute symporters. Biochim Biophys Acta 1197133–166
     [Google Scholar]
  23. Saier M. H. 2000; Families of transmembrane transporters selective for amino acids and their derivatives. Microbiology 146:1775–1795
     [Google Scholar]
  24. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  25. Saurin W., Dassa E. 1994; Sequence relationships between integral inner membrane proteins of binding protein-dependent transport systems: evolution by recurrent gene duplications. Protein Sci 3:325–344
     [Google Scholar]
  26. Saurin W., Hofnung M., Dassa E. 1999; Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters. J Mol Evol 48:22–41
     [Google Scholar]
  27. Schellenberg G. D., Furlong C. E. 1977; Resolution of the multiplicity of the glutamate and aspartate transport systems of Escherichia coli . J Biol Chem 252:9055–9064
     [Google Scholar]
  28. Schneider E. 2001; ABC transporters catalyzing carbohydrate uptake. Res Microbiol 152:303–310
     [Google Scholar]
  29. Schneider E., Hunke S. 1998; ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol Rev 22:1–20
     [Google Scholar]
  30. Sonawane A., Klöppner U., Derst C., Röhm K. H. 2003a; Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase. Arch Microbiol 179:151–159
     [Google Scholar]
  31. Sonawane A., Klöppner U., Hövel S., Völker U., Röhm K. H. 2003b; Identification of Pseudomonas proteins coordinately induced by acidic amino acids and their amides: a two-dimensional electrophoresis study. Microbiology 149:2909–2918
     [Google Scholar]
  32. Sonawane A. M., Singh B., Röhm K. H. 2006; The AauR–AauS two-component system regulates uptake and metabolism of acidic amino acids in Pseudomonas putida . Appl Environ Microbiol 72:6569–6577
     [Google Scholar]
  33. Tolner B., Ubbink-Kok T., Poolman B., Konings W. N. 1995; Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli . J Bacteriol 177:2863–2869
     [Google Scholar]
  34. Tzou W. S., Hwang M. J. 1999; Modeling helix–turn–helix protein-induced DNA bending with knowledge-based distance restraints. Biophys J 77:1191–1205
     [Google Scholar]
  35. Urban A., Leipelt M., Eggert T., Jaeger K. E. 2001; DsbA and DsbC affect extracellular enzyme formation in Pseudomonas aeruginosa . J Bacteriol 183:587–596
     [Google Scholar]
  36. Willis R. C., Furlong C. E. 1975; Interactions of a glutamate-aspartate binding protein with the glutamate transport system of Escherichia coli . J Biol Chem 250:2581–2586
     [Google Scholar]
  37. Zwietering M. H., Jongenburger I., Rombouts F. M., van 't Riet K. 1990; Modeling of the bacterial growth curve. Appl Environ Microbiol 56:1875–1881
     [Google Scholar]
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Characterization of a Pseudomonas putida ABC transporter (AatJMQP) required for acidic amino acid uptake: biochemical properties and regulation by the Aau two-component system
Microbiology 154, 797 (2008); https://doi.org/10.1099/mic.0.2007/013185-0
/content/journal/micro/10.1099/mic.0.2007/013185-0
/content/journal/micro/10.1099/mic.0.2007/013185-0
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