Analysis of the Ferric Citrate Transport Gene Promoter of Escherichia coli

Abstract

FecI, an extracytoplasmic-function factor, is required for initiation of transcription of the ferric citrate transport genes. A mutational analysis of the fecA promoter revealed that the nonconserved ؊10 region and a downstream regulatory element are important for fecA promoter activity. However, nucleotide substitutions in the well-conserved ؊35 region also have an effect on the fecA promoter activity. Titration of FecI suggests that the FecI-RNA polymerase holoenzyme does not bind strongly to the downstream regulatory element, which is therefore probably involved in a subsequent step of transcription initiation.In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a signal transduction mechanism that starts at the cell surface. (Fe 3ϩ citrate) 2 binds to the outer membrane protein FecA and without further transport into the cell induces transcription of the fec transport genes (9, 14). The signal transmitted by FecA loaded with (Fe 3ϩ citrate) 2 across the outer membrane is transmitted across the cytoplasmic membrane by FecR, a transmembrane protein (21, 28) that interacts with FecA in the periplasm and with the FecI extracytoplasmic-function (ECF) factor in the cytoplasm (7,8,18,24). FecR enables FecI to bind to the RNA polymerase core enzyme; this complex then binds to the fecA promoter to initiate transcription of the fec transport genes (1, 2). The only promoter known to be recognized by FecI is that of the fecABCDE operon; no other factor of E. coli is endowed with such a narrow promoter specificity (17).ECF factors belong to a subfamily of the 70 class, based on their sequence conservation and function across bacterial species (3,10,16,20). Comparisons of sequences indicate that the genomes of Caulobacter crescentus, Pseudomonas aeruginosa, Nitrosomonas europaea, and Streptomyces coelicolor are particularly rich in ECF factors and contain 13, 19, 22, and 50 predicted ECF factors, respectively. factors share four conserved regions which can be further subdivided. Region 4.2 recognizes the Ϫ35 element, and region 2.4 recognizes the Ϫ10 element of promoter DNA. A bacterial promoter consists of at least about 60 bp spanning the positions Ϫ40 to ϩ20 in relation to the ϩ1 start site of transcription (12). A comparison of ECF factors reveals that the Ϫ35 sequence and the spacing but not the sequence between the Ϫ35 and Ϫ10 regions are well conserved (Table 1) (4,5,10,11,19,20,23,29). The Ϫ10 sequences show less homology (Table 1), which is reflected by the low homology of regions 2.4 in the ECF factors (16). The characteristic feature of region 2.4 of 70 , a set of hydrophobic residues that form an amphipatic ␣-helix, is not present in the corresponding region of ECF factors (16). The diversity of the Ϫ10 sequences and region 2.4 probably accounts for the coexistence of multiple members of the ECF -factor subfamily in the same species.Previously, it was shown that transcription of the fec transport genes starts at nucleotide 2741 (7) of the fec sequence (22), resulti...

Transcriptional Response ofEscherichia colito TPEN

“…The genes (entB, entA, entD, entE, and fes) which encode proteins involved with enterobactin synthesis and uptake (26,29,54) and the fec genes (fecR and fecI), which encode proteins involved in Fe import (23), were significantly up-regulated. Several other genes (fhuA, exbB, exbD, fhuD, fhuC, and fhuF) associated with ferrichrome transport in E. coli were also up-regulated (19,56,62).…”

Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria

“…Previously, residues 247-268 within the periplasmic domain of the homologous sigma regulator, FecR, were named the LLLV region as this region includes conserved Leucine and Valine residues ( Fig S1) (7). Mutation of these conserved hydrophobic residues to proline was shown to abrogate binding to the FecA NTSD (7).…”

“…Sigma regulators are proteins of ~325 amino acids consisting of three domains, 1) an N-terminal antisigma domain (3,4) which regulates the sigma factor, 2) a single-pass transmembrane helix, and 3) a C-terminal periplasmic domain of ~200 residues, responsible for interacting with the transducer (5,6). The periplasmic domain of the sigma regulator FecR has been shown to interact with the N-terminal signaling domain (NTSD) of its cognate transporter/transducer, FecA (5,7), and mutation of conserved hydrophobic residues to proline within this periplasmic domain disrupted binding to the NTSD (7). The structure of the periplasmic domain of sigma regulators has not been described.…”

Cell-surface signaling inPseudomonas: stress responses, iron transport, and pathogenicity

“…σ FiuI mediates expression of the fiuA receptor gene in response to ferrichrome and σ FoxI that of the foxA receptor gene in response to ferrioxamine B (Llamas et al ., ). The σ FoxI recognition sequence −35 GAAAAT and −10 TGTCGG has been proposed (Enz et al ., ), although not experimentally confirmed.…”