Control of the ferric citrate transport system of Escherichia coli: mutations in region 2.1 of the FecI extracytoplasmic-function sigma factor suppress mutations in the FecR transmembrane regulatory protein

Abstract

Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane of Escherichia coli K-12. Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active. In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of the Pseudomonas aeruginosa, Pseudomonas putida, Bordetella pertussis, Bordetella bronchiseptica, and Caulobacter crescentus genomes. The cytoplasmic portion of the FecR mutant proteins, FecR(1-85), did not interact with wild-type FecI, in contrast to wild-type FecR(1-85), which induced FecI-mediated fecB transport gene transcription. Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of the fecR mutants by ferric citrate. Region 2.1 of sigma(70) is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI. In addition, missense mutations in the fecI promoter region resulted in a twofold increased transcription in fecR wild-type cells and a partial restoration of fec transport gene transcription in the fecR mutants. The mutations reduced binding of the Fe(2+) Fur repressor and as a consequence enhanced fecI transcription. The data reveal properties of the FecI ECF factor distinct from those of sigma(70) and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.

FIG. 1

(A) Promoter sequence upstream of the fecI fecR operon. Positions of mutations are indicated by asterisks, positions of −35 and −10 promoter regions are illustrated in bold letters, and the transcription start point is indicated by an arrow. The consensus sequence of the Fur box is illustrated below. (B) Schematic map of ς^FecI illustrating regions homologous to ς⁷⁰. The sites of amino acid substitutions are indicated below ς^FecI.

FIG. 2

Alignment of the N termini of E. coli FecR homologs. Similarity search and sequence alignment were done as described in Materials and Methods. E. c., E. coli; P. a., P. aeruginosa; P. p., P. putida; B. b., B. bronchiseptica; B. p., B. pertussis; C. c., C. crescentus. Note the highly conserved tryptophan (W) residues corresponding to positions 19, 39, and 50 of E. coli FecR.