Surface signaling in ferric citrate transport gene induction: interaction of the FecA, FecR, and FecI regulatory proteins

Abstract

In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)(10)-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)(6)] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)(10)-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA(1-79) interacts with FecR(101-317) and FecR(1-85) interacts with FecI(1-173). These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

FIG. 1

Affinity chromatography of FecR-(His)₆ (A) and (His)₁₀-FecR (B) on a Ni-NTA agarose column. The eluted fractions were subjected to SDS-PAGE (with 15% acrylamide gels) and stained with Coomassie brilliant blue. (C) Western blotting of Ni-NTA agarose column eluant fractions with anti-FecR serum. FecR-(His)₆, (His)₁₀-FecR, and their proteolytic cleavage products are indicated by arrows. Numbers indicate molecular masses in kilodaltons.

FIG. 2

Binding of FecI to FecR-(His)₆ (A) and (His)₁₀-FecR (B) on a Ni-NTA agarose column. The eluted fractions were subjected to SDS-PAGE (with 15% acrylamide gels) and stained with Coomassie brilliant blue. FecI, FecR-(His)₆, (His)₁₀-FecR, and their proteolytic cleavage products are indicated by arrows. Numbers indicate molecular masses in kilodaltons.

FIG. 3

Binding of FecA to (His)₁₀-FecR (A) and FecR-(His)₆ (B) on a Ni-NTA agarose column and of FecAΔ47 to His₁₀-FecR (C) on a Ni-NTA agarose column. The eluted fractions were subjected to SDS-PAGE (with 15% acrylamide gels) and stained with Coomassie brilliant blue. FecA, FecAΔ47 (designated FecA47), FecR-(His)₆, (His)₁₀-FecR, and their proteolytic cleavage products are indicated by arrows. Numbers indicate molecular masses in kilodaltons.

FIG. 4

Diagrammatic representation of the cloning of FecA, FecR, and FecI regions into the plasmids pDP804 and pMS604. Domains of FecA and FecI were fused in frame to LexA_1–87WT, by which the Fos zipper domain originally present in pMS604 was deleted. Domains of FecR were all cloned into the XhoI-BglII restriction endonuclease sites present in pDP804. All fecR regions cloned are fused in frame to Lex_1–87408, which deleted the Jun zipper motif in pDP804.