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Journal of Bacteriology, September 2003, p. 5508-5518, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5508-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vivo Reconstitution of the FhuA Transport Protein of Escherichia coli K-12

Michael Braun, Franziska Endriss, Helmut Killmann, and Volkmar Braun*

Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany

Received 4 April 2003/ Accepted 23 June 2003

The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and {phi}80 and for colicin M and microcin J25. The crystal structure reveals a ß-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a ß-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the ß-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type ß-barrel to form an active FhuA. Previous claims that the ß-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the ß-barrel, and they suggest that in wild-type FhuA the ß-barrel is formed prior to the insertion of the cork.

* Corresponding author. Mailing address: Mikrobiologie/Membranphysiologie, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Phone: (49) 7071 2972906. Fax: (49) 7071 5843. E-mail: volkmar.braun{at}mikrobio.uni-tuebingen.de.

Journal of Bacteriology, September 2003, p. 5508-5518, Vol. 185, No. 18
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.18.5508-5518.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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