Residues involved in FecR binding are localized on one side of the FecA signaling domain in Escherichia coli

Abstract

Ferric citrate transport in Escherichia coli involves proteins encoded by the fec genes, including the transport and signaling protein FecA and the signal transducing protein FecR. Randomly isolated FecA point mutants showed a reduced interaction with FecR and a reduced transcription initiation of the ferric citrate transport genes. The mutations were localized on one side of the FecA signaling domain, which might form the interface to FecR. Some of the mutants showed strongly reduced iron transport rates, which suggests that the signaling domain affects the structure of the FecA transporter domain.

FIG. 1.

Citrate-mediated ⁵⁵Fe³⁺ transport into E. coli AA93 Δfec expressing the fecBCDE transport genes on plasmid pUP40 (8) and the indicated FecA wild-type (WT) and mutant proteins. The fecA genes were cloned on pBAD18; transcription was induced by addition of 0.2% arabinose.

FIG. 2.

Immunoblot of wild-type (WT) FecA and mutant FecA proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arrow indicates the position of FecA. The protein identities of the immunoreactive upper band in the sample lacking FecA and all of the other samples are unknown. Aliquots of cells corresponding to an identical absorbance were loaded on the gel. Numbers mark the positions of the 70- and 100-kDa standard proteins.

FIG. 3.

NMR structure of FecA_1-74 (6). The sites of mutations isolated in this paper and the suppressor mutations (arrows with interrupted lines) (3) are approximately indicated.