scholarly journals Residues Involved in FecR Binding Are Localized on One Side of the FecA Signaling Domain in Escherichia coli

2006 ◽  
Vol 188 (17) ◽  
pp. 6440-6442 ◽  
Author(s):  
Elena Breidenstein ◽  
Susanne Mahren ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Iron Transport ◽  
Ferric Citrate ◽  
Signaling Protein ◽  
Citrate Transport ◽  
Signaling Domain

ABSTRACT Ferric citrate transport in Escherichia coli involves proteins encoded by the fec genes, including the transport and signaling protein FecA and the signal transducing protein FecR. Randomly isolated FecA point mutants showed a reduced interaction with FecR and a reduced transcription initiation of the ferric citrate transport genes. The mutations were localized on one side of the FecA signaling domain, which might form the interface to FecR. Some of the mutants showed strongly reduced iron transport rates, which suggests that the signaling domain affects the structure of the FecA transporter domain.

scholarly journals Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli K-12

2003 ◽  
Vol 185 (13) ◽  
pp. 3745-3752 ◽  
Author(s):  
Sabine Enz ◽  
Heidi Brand ◽  
Claudia Orellana ◽  
Susanne Mahren ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Transcription Initiation ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Citrate Transport ◽  
Signal Transduction Proteins ◽  
K 12

ABSTRACT Transcription of the fecABCDE ferric citrate transport genes of Escherichia coli 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 FecR237-317 fragment interacts with the N-terminal FecA1-79 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 fecA 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 fec transport genes and identified sites in FecR and FecA that are important for signal transduction.

scholarly journals Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

2004 ◽  
Vol 186 (16) ◽  
pp. 5303-5310 ◽  
Author(s):  
Annette Sauter ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Transcription Initiation ◽  
Ferric Citrate ◽  
Globular Domain ◽  
Salt Bridges ◽  
The Individual ◽  
K 12

ABSTRACT The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the β-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the β-barrel and to contribute to the fixation of the globular domain inside the β-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.

scholarly journals Control of the Ferric Citrate Transport System ofEscherichia coli: Mutations in Region 2.1 of the FecI Extracytoplasmic-Function Sigma Factor Suppress Mutations in the FecR Transmembrane Regulatory Protein

2001 ◽  
Vol 183 (1) ◽  
pp. 162-170 ◽  
Author(s):  
Alfred Stiefel ◽  
Susanne Mahren ◽  
Martina Ochs ◽  
Petra T. Schindler ◽  
Sabine Enz ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Gene Transcription ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Caulobacter Crescentus ◽  
Ferric Citrate ◽  
Missense Mutations ◽  
Wild Type ◽  
Citrate Transport ◽  
K 12

ABSTRACT Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane ofEscherichia 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 thePseudomonas aeruginosa, Pseudomonas putida,Bordetella pertussis, Bordetella bronchiseptica, and Caulobacter crescentus genomes. The cytoplasmic portion of the FecR mutant proteins, FecR1–85, did not interact with wild-type FecI, in contrast to wild-type FecR1–85, 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 thefecR mutants by ferric citrate. Region 2.1 of ς70 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 thefecR mutants. The mutations reduced binding of the Fe2+ Fur repressor and as a consequence enhancedfecI transcription. The data reveal properties of the FecI ECF factor distinct from those of ς70 and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.

scholarly journals Ferric Citrate Transport of Escherichia coli: Functional Regions of the FecR Transmembrane Regulatory Protein

1998 ◽  
Vol 180 (9) ◽  
pp. 2387-2394 ◽  
Author(s):  
Dietrich Welz ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Cytoplasmic Membrane ◽  
Regulatory Protein ◽  
Ferric Citrate ◽  
Outer Membrane Receptor ◽  
Resistance Level ◽  
Ampicillin Resistance ◽  
Low Resistance ◽  
Citrate Transport

ABSTRACT Transcription of the ferric citrate transport genes ofEscherichia coli is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM β-lactamase. BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance. Cells that synthesized FecR′-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin. The ampicillin resistance of the low-resistance FecR′-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and indegP and ompT protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation. Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the β-lactamase activity of high-resistance FecR′-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence. All FecR′-BlaM proteins with at least 61 FecR residues complemented a fecR mutant such that it could grow on ferric citrate as the sole iron source and inducedfecA-lacZ transcription independent of ferric citrate. The low resistance mediated by two FecR′-BlaM proteins in afecA deletion mutant was increased 20-fold by transformation with a fecA-encoding plasmid. We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.

scholarly journals Analysis of the Ferric Citrate Transport Gene Promoter of Escherichia coli

2003 ◽  
Vol 185 (7) ◽  
pp. 2387-2391 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Claudia Menzel ◽  
Volkmar Braun
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Ferric Citrate ◽  
Nucleotide Substitutions ◽  
Citrate Transport ◽  
Σ Factor ◽  
Subsequent Step

ABSTRACT FecI, an extracytoplasmic-function σ 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.

scholarly journals Functional Interaction of Region 4 of the Extracytoplasmic Function Sigma Factor FecI with the Cytoplasmic Portion of the FecR Transmembrane Protein of the Escherichia coli Ferric Citrate Transport System

2002 ◽  
Vol 184 (13) ◽  
pp. 3704-3711 ◽  
Author(s):  
Susanne Mahren ◽  
Sabine Enz ◽  
Volkmar Braun
Keyword(s):  
Escherichia Coli ◽  
Sigma Factor ◽  
Transmembrane Protein ◽  
Ferric Citrate ◽  
Missense Mutations ◽  
E Coli ◽  
Citrate Transport ◽  
Extracytoplasmic Function Sigma Factor ◽  
Cognate Protein

ABSTRACT Transcriptional regulation of the ferric citrate transport genes of Escherichia coli is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the fecABCDE genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and fecB-lacZ transcription. The cloned region 4 of FecI interfered with fecB-lacZ transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of Pseudomonas aeruginosa was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the P. aeruginosa proteins and between a P. aeruginosa FecI homolog and E. coli FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.

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