Sites of interaction between the FecA and FecR signal transduction proteins of ferric citrate transport in escherichia coli K-12

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p> Transcription of the <jats:italic>fecABCDE</jats:italic> ferric citrate transport genes of <jats:italic>Escherichia coli</jats:italic> K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm. FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor. In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR <jats:sub>237-317</jats:sub> fragment interacts with the N-terminal FecA <jats:sub>1-79</jats:sub> fragment. In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis. They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins. The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium. Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E). One FecR mutant, FecR (D138E, V197A), induced <jats:italic>fecA</jats:italic> promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA. The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the <jats:italic>fec</jats:italic> transport genes and identified sites in FecR and FecA that are important for signal transduction. </jats:p>