Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.
Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa.
Ankenbauer
R G
RG
Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010636:Phenols / Q000378:metabolism*; D010641:Phenotype; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013844:Thiazoles
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; C075523:FPTA protein, Pseudomonas; D007502:Iron Chelating Agents; D010636:Phenols; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D013844:Thiazoles; C025316:pyochelin; D007501:Iron