Molecular cloning of the ferrichrome-iron receptor of Escherichia coli K-12.
A receptor protein in the outer membrane of Escherichia coli K-12 is required for the binding of ferrichrome-iron at the cell surface and for the transport of iron from this complex into the cell. This protein of Mr 78,000 is the product of the fhuA (previously called tonA) gene, located at 3.5 min on the E. coli chromosome. We cloned the fhuA gene into plasmid p343, a high-copy-number cosmid derived from pBR322. An 8.5-kilobase pair fragment of E. coli chromosomal DNA, generated by hydrolysis with the restriction endonuclease HindIII, was found to have conferred the FhuA+ phenotype to E. coli P8, which lacks the ferrichrome-iron receptor. A partial physical map of this recombinant plasmid pPM18 was established by determining the restriction endonuclease sites for BglII, EcoRI, PstI, PvuII, SmaI, and XhoI. The fhuA gene was localized to a 3.5-kilobase pair fragment of DNA whose extremities were defined by the restriction sites PstI-PvuII. A 7.5-fold enhancement of the rate of transport of iron from the ferrichrome complex was measured for cells which contained pPM18 as compared to wild-type E. coli K-12. Overproduction of the FhuA protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of the recombinant plasmid-containing strain. Proteins encoded by the subcloned DNA fragments were identified by [35S]methionine labeling of maxicells of E. coli CSR603, which contained recombinant plasmids; only one polypeptide chain, the presumptive fhuA gene product, was detected.
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001692:Biological Transport; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics*; D005290:Ferric Compounds / Q000378:metabolism; D005291:Ferrichrome / Q000378:metabolism; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics*; D010957:Plasmids
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D005290:Ferric Compounds; D008565:Membrane Proteins; D005291:Ferrichrome; D004262:DNA Restriction Enzymes