Ferric citrate transport of Escherichia coli: functional regions of the FecR transmembrane regulatory protein.

Transcription of the ferric citrate transport genes of Escherichia coli is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM beta-lactamase. BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance. Cells that synthesized FecR'-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin. The ampicillin resistance of the low-resistance FecR'-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in degP and ompT protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation. Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the beta-lactamase activity of high-resistance FecR'-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence. All FecR'-BlaM proteins with at least 61 FecR residues complemented a fecR mutant such that it could grow on ferric citrate as the sole iron source and induced fecA-lacZ transcription independent of ferric citrate. The low resistance mediated by two FecR'-BlaM proteins in a fecA deletion mutant was increased 20-fold by transformation with a fecA-encoding plasmid. We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.

Regulation of citrate-dependent iron transport of Escherichia coli: fecR is required for transcription activation by FecI.

Citrate-dependent Fe3+ transport into Escherichia coli K-12 is induced by iron and citrate. The inducer is probably ferric dicitrate which does not have to be taken up into the cytoplasm to induce transcription of the fec transport genes. Two regulatory genes, fecI and fecR, located upstream of the fecABCDE transport genes, are required for induction. We report that in vivo the chromosomally encoded FecI protein activates transcription of the fecA and fecB transport genes in response to ferric citrate and the FecR protein. Cells expressing chromosomally and plasmid-encoded truncated FecR derivatives no longer responded to ferric citrate and expressed the fec transport genes constitutively. The smallest active FecR derivative contained 59 amino acid residues as compared to the 317 residues of wild-type FecR. Constitutive induction was lower than induction of the FecR wild-type strain by ferric citrate. It is concluded that the N-terminal portion of FecR activates FecI and that the C-terminal portion of FecR responds to ferric citrate. Transcription of the fec transport genes is positively regulated by FecI and FecR and negatively regulated by the Fe2(+)-Fur repressor. Transcription activation and repression may occur independently of each other.