FERRIC CITRATE TRANSPORT OF ESCHERICHIA-COLI - FUNCTIONAL REGIONS OF THE FECR TRANSMEMBRANE REGULATORY PROTEIN

Transcription of the ferric citrate transport genes of Escherichia col i is induced by ferric citrate bound to the outer membrane receptor Fe cA. 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 ac ross 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 ext ending front residues 107 to 149 and residues 230 to 259 conferred hig h ampicillin resistance to cells, while BlaM fused to sites between re sidues 159 and 210 and between residues 265 and 301 conferred low resi stance. Cells that synthesized FecR'-BlaM with fusion joints between r esidues 8 and 81 of FecR were fully sensitive to ampicillin. The ampic illin resistance of the low-resistance FecR'-BlaM hybrids was increase d 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in degP and ompT protease mutants, which suggested tha t the decreased ampicillin resistance level of these hybrids was cause d by the formation of inclusion bodies and proteolytic degradation. Re placement of glycine by aspartate residues in the only hydrophobic Fec R sequence (residues 85 to 100) abolished the beta-lactamase activity of high-resistance FecR'-BlaM proteins, indicating that there are no o ther transmembrane regions in FecR that translocate BlaM I into the pe riplasm independent of the hydrophobic sequence. All FecR'-BlaM I prot eins with at least 61 FecR residues complemented a fecR mutant such th at it could grow on ferric citrate as the sole iron source and induced fecA-lacZ transcription independent of ferric citrate. The low resist ance 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 th e periplasm with its C-terminal region with FecA occupied by ferric ci trate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor .