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

2003 ◽  
Vol 185 (21) ◽  
pp. 6494-6494
Author(s):  
Sabine Enz ◽  
Heidi Brand ◽  
Claudia Orellana ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Ferric Citrate ◽  
Citrate Transport ◽  
Signal Transduction Proteins ◽  
K 12

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 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 Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

1998 ◽  
Vol 180 (20) ◽  
pp. 5313-5318 ◽  
Author(s):  
Keigo Shibayama ◽  
Shinji Ohsuka ◽  
Toshihiko Tanaka ◽  
Yoshichika Arakawa ◽  
Michio Ohta
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Glycosidic Linkage ◽  
Hydrophobic Cluster Analysis ◽  
E Coli ◽  
Catalytic Sites ◽  
K 12

ABSTRACT Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.

scholarly journals Overproduction of l-Cysteine andl-Cystine by Escherichia coli Strains with a Genetically Altered Serine Acetyltransferase

1998 ◽  
Vol 64 (5) ◽  
pp. 1607-1611 ◽  
Author(s):  
Shigeru Nakamori ◽  
Shin-ichiro Kobayashi ◽  
Chitose Kobayashi ◽  
Hiroshi Takagi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Feedback Inhibition ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Serine Acetyltransferase ◽  
Methionine Residue ◽  
Cysteine Desulfhydrase ◽  
K 12

ABSTRACT Organisms that overproduced l-cysteine andl-cystine from glucose were constructed by usingEscherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg ofl-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production ofl-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production ofl-cysteine plus l-cystine was markedly increased compared to production in JM39.

Sign in / Sign up

Export Citation Format

Share Document