Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins

Abstract

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE in Escherichia coli by binding to the outer-membrane receptor protein FecA without entering the cell. Replete iron concentrations inhibit transcription of the fec transport system via the iron-loaded Fur repressor. Here we show that the Fur repressor activated by Mn2+ (used instead of Fe2+) binds to the promoter of the regulatory genes fecIR and to the promoter of fecABCDE. DNase I footprint analysis revealed that Mn2+-Fur (50 nM) protected 30 nucleotides of the coding strand and 24 nucleotides of the noncoding strand of the fecIR promoter. Higher amounts of Mn2+-Fur (100 nM) covered 41 nucleotides of the coding strand of the fecIR promoter and 38 nucleotides of the coding strand of the fecA promoter. The corresponding region of the noncoding strand of the fecA promoter was hypersensitive to DNase I. The results of a deletion analysis of the fecA promoter supported the previously assigned -35 and -10 regions and nucleotide position +11 for FecI-RNA polymerase interaction. Induction of fecIR transcription by iron limitation increased fecB-lacZ transcription 3.5-fold, whereas under constitutive fecIR transcription, iron limitation increased fecB-lacZ transcription twofold. The two iron-regulated sites of fec transport gene transcription suggest a fast response to sufficient intracellular iron concentrations by repression of fecABCDE transcription and a slower adaptation as the result of fecIR transcription inhibition.

Analysis of the Ferric Citrate Transport Gene Promoter of Escherichia coli

“…However, the finding that the crtL promoter of Myxococcus xanthus to which the CarQ ECF factor binds is critically dependent on a pentanucleotide sequence centered at the Ϫ31 position (19) indicates differences in the structural requirements of ECF factor promoters. Upstream deletions in the fecA promoter extending to nucleotides Ϫ28 and Ϫ19 reduced ␤-galactosidase activity of a plasmid-encoded fecA-lacZ operon fusion to 23 and 25% of wild-type activity, respectively, whereas a deletion covering the entire promoter region to ϩ1 completely abolished fecA-lacZ transcription (1). These data demonstrate that the Ϫ35 region can be partially replaced by other nucleotide sequences.…”

“…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).…”

The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the β′ Subunit of RNA Polymerase

“…Binding of ferric citrate to the FecA outer membrane protein initiates a signal transduction mechanism that induces transcription of the fecABCDE transport genes. In the FecA crystal structure, (Fe 3ϩ -citrate) 2 is bound to FecA (12). In addition, FecA transports (Fe 3ϩ -citrate) 2 across the outer membrane (6,18,25).…”

“…In addition, FecA transports (Fe 3ϩ -citrate) 2 across the outer membrane (6,18,25). FecA with bound ferric citrate transmits the signal to FecR (2), an inner membrane protein with the N-terminal domain (residues 1 to 84) located in the cytoplasm and the C-terminal domain (residues 101 to 317) located in the periplasm (46). Residues 85 to 100 form a transmembrane segment through which the signal is transmitted across the cytoplasmic membrane.…”

“…FecR interacts in the cytoplasm with the FecI sigma factor and activates FecI, which in turn directs the RNA polymerase core enzyme to the fecA promoter and initiates transcription of the fecABCDE transport genes (1,10). Transcription of the fecIR regulatory genes located upstream of fecA and of the fecABCDE genes is regulated by the intracellular concentration of iron (2,36). Under iron-replete conditions, the Fur repressor protein loaded with Fe 2ϩ binds to the fecI and fecA promoters and represses transcription.…”