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 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.

scholarly journals A new small regulatory protein, HmuP, modulates haemin acquisition in Sinorhizobium meliloti

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1873-1882 ◽  
Author(s):  
Vanesa Amarelle ◽  
Uriel Koziol ◽  
Federico Rosconi ◽  
Francisco Noya ◽  
Mark R. O'Brian ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
System Analysis ◽  
Cytoplasmic Membrane ◽  
Iron Acquisition ◽  
Rhodopseudomonas Palustris ◽  
Regulatory Protein ◽  
Main Function ◽  
Outer Membrane Receptor ◽  
Multiple Systems ◽  
Iron Restriction

Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted β-sheets are well conserved. Our data indicate that at least one of the β-sheets is important for HmuP activity.

scholarly journals Ferric Citrate Regulator FecR Is Translocated across the Bacterial Inner Membrane via a Unique Twin-Arginine Transport-Dependent Mechanism

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Ian J. Passmore ◽  
Jennifer M. Dow ◽  
Francesc Coll ◽  
Jon Cuccui ◽  
Tracy Palmer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Sigma Factor ◽  
Signal Sequence ◽  
Transmembrane Helix ◽  
Ferric Citrate ◽  
Inner Membrane ◽  
Content Type ◽  
Signal Cascade ◽  
Tat System

ABSTRACT In Escherichia coli, citrate-mediated iron transport is a key nonheme pathway for the acquisition of iron. Binding of ferric citrate to the outer membrane protein FecA induces a signal cascade that ultimately activates the cytoplasmic sigma factor FecI, resulting in transcription of the fecABCDE ferric citrate transport genes. Central to this process is signal transduction mediated by the inner membrane protein FecR. FecR spans the inner membrane through a single transmembrane helix, which is flanked by cytoplasm- and periplasm-orientated moieties at the N and C termini. The transmembrane helix of FecR resembles a twin-arginine signal sequence, and the substitution of the paired arginine residues of the consensus motif decouples the FecR-FecI signal cascade, rendering the cells unable to activate transcription of the fec operon when grown on ferric citrate. Furthermore, the fusion of beta-lactamase C-terminal to the FecR transmembrane helix results in translocation of the C-terminal domain that is dependent on the twin-arginine translocation (Tat) system. Our findings demonstrate that FecR belongs to a select group of bitopic inner membrane proteins that contain an internal twin-arginine signal sequence. IMPORTANCE Iron is essential for nearly all living organisms due to its role in metabolic processes and as a cofactor for many enzymes. The FecRI signal transduction pathway regulates citrate-mediated iron import in many Gram-negative bacteria, including Escherichia coli. The interactions of FecR with the outer membrane protein FecA and cytoplasmic anti-sigma factor FecI have been extensively studied. However, the mechanism by which FecR inserts into the membrane has not previously been reported. In this study, we demonstrate that the targeting of FecR to the cytoplasmic membrane is dependent on the Tat system. As such, FecR represents a new class of bitopic Tat-dependent membrane proteins with an internal twin-arginine signal sequence.

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 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 TonB Interacts with Nonreceptor Proteins in the Outer Membrane of Escherichia coli

2002 ◽  
Vol 184 (6) ◽  
pp. 1640-1648 ◽  
Author(s):  
Penelope I. Higgs ◽  
Tracy E. Letain ◽  
Kelley K. Merriam ◽  
Neal S. Burke ◽  
HaJeung Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Receptors ◽  
Strong Interactions ◽  
Cross Linking ◽  
Outer Membrane Receptor ◽  
Outer Membranes

ABSTRACT The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B12 across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.

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