RESUMEN
Transcription of the Escherichia coli hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (P(hyaA)) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase P(hyaA) expression under anaerobic conditions by antagonizing IscR binding at P(hyaA), since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of P(hyaA) depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both P(hyaA) and a constitutive promoter containing the IscR binding site from P(hyaA) than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of P(hyaA) than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased P(hyaA) expression under anaerobic conditions.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Hidrogenasas/biosíntesis , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Anaerobiosis , Sitios de Unión , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Hidrogenasas/genética , Hidrogenasas/metabolismo , Mutación , Oxígeno/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genéticaRESUMEN
IscR is an Fe-S protein that functions as a transcriptional regulator of Fe-S biogenesis and other Fe-S protein-encoding genes in Escherichia coli. In this study, we investigated the requirement for the ligation of the [2Fe-2S] cluster of IscR to regulate a subset of IscR target promoters (P(hyaA), P(ydiU), P(napF), and P(hybO)) and defined the requirements for sequence-specific binding to the IscR target site in the hyaA promoter region. In contrast to previous results with the iscR promoter, we found that the Fe-S cluster is dispensable for IscR regulation of P(hyaA), P(ydiU), P(napF), and P(hybO), since IscR mutants containing alanine substitutions of the cysteine Fe-S ligands retained IscR-dependent regulation of these promoters in vivo. In vitro assays showed that both [2Fe-2S]-IscR and an IscR mutant lacking the cluster (IscR-C92A/C98A/C104A) bound the hya site with similar affinity, explaining why the mutant protein retained its ability to repress P(hyaA) in vivo. Characterization of the oligomeric state of IscR showed that both apo-IscR and [2Fe-2S]-IscR were dimers in solution, and four protomers of either form bound to the hya site. Also, binding of either apo- or [2Fe-2S]-IscR to the hya site showed cooperativity, suggesting that both forms interact similarly with the target site. Analysis of mutations in the hya site using DNA competition assays showed that apo-IscR most likely recognizes an imperfect palindrome within the hya promoter. Furthermore, the strength of apo-IscR binding to P(sufA), P(ydiU), P(napF), and P(hybO) IscR sites correlated with the number of matches to the hya site bases shown to be important in the competition assay. Thus, our data indicated that, unexpectedly, apo-IscR is a site-specific DNA-binding protein, and the role of apo-IscR needs to be considered in developing models for how IscR globally regulates transcription.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Proteínas Hierro-Azufre/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Dimerización , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Hierro-Azufre/química , Datos de Secuencia Molecular , Mutación , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The goal in biomaterial surface modification is to retain a material's bulk properties while modifying only its surface to possess desired recognition and specificity. Here we develop a unique strategy for surface functionalization of an electrically conductive polymer, chlorine-doped polypyrrole (PPyCl), which has been widely researched for various electronic and biomedical applications. An M13 bacteriophage library was used to screen 10(9) different 12-mer peptide inserts against PPyCl. A binding phage (phiT59) was isolated, and its binding stability and specificity to PPyCl was assessed using fluorescence microscopy and titer count analysis. The relative binding strength and mechanism of the corresponding 12-mer peptide and its variants was studied using atomic force microscopy and fluorescamine assays. Further, the T59 peptide was joined to a cell adhesive sequence and used to promote cell attachment on PPyCl. This strategy can be extended to immobilize a variety of molecules to PPyCl for numerous applications. In addition, phage display can be applied to other polymers to develop bioactive materials without altering their bulk properties.
Asunto(s)
Bacteriófago M13/metabolismo , Compuestos de Cloro/química , Materiales Biocompatibles Revestidos/química , Ensayo de Materiales/métodos , Biblioteca de Péptidos , Péptidos/química , Polímeros/química , Mapeo de Interacción de Proteínas/métodos , Pirroles/química , Adsorción , Materiales Biocompatibles Revestidos/análisis , Conductividad Eléctrica , Electroquímica/métodos , Péptidos/análisis , Unión Proteica , Propiedades de SuperficieRESUMEN
IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA/SoxS/Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S](1+) cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Genes Bacterianos , Proteínas Hierro-Azufre/genética , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , ADN Bacteriano , Proteínas de Escherichia coli , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Represoras/química , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
The global anaerobic regulator FNR from Escherichia coli is a dimeric Fe-S protein that is inactivated by O(2) through disruption of its [4Fe-4S] cluster and conversion to a monomeric form. As a first step in elucidating the molecular interactions that control FNR dimerization, we have performed alanine-scanning mutagenesis of a potential dimerization domain. Replacement of many hydrophobic residues (Met-143, Met-144, Leu-146, Met-147, Ile-151, Met-157, and Ile-158) and two charged residues (Arg-140 and Arg-145) with Ala decreased FNR activity in vivo. Size exclusion chromatography and Fe-S cluster analysis of three representative mutant proteins, FNR-M147A, FNR-I151A, and FNR-I158A, showed that the Ala substitutions produced specific defects in dimerization. Because hydrophobic side chains are known to stabilize subunit-subunit interactions between alpha-helices, we propose that Met-147, Ile-151, and Ile-158 lie on the same face of an alpha-helix that constitutes a dimerization interface. This alignment would also position Arg-140, Met-144, and Asp-154 on the same helical face. In support of the unusual positioning of a negatively charged residue at the dimer interface, we found that replacing Asp-154 with Ala repaired the defects caused by Ala substitutions of other residues located on the same helical face. These data also suggest that Asp-154 has an inhibitory effect on dimerization, which may be a key element in the control of FNR dimerization by O(2) availability.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas Hierro-Azufre/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía en Gel , Dicroismo Circular , Dimerización , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Condroitín Liasas/genética , Condroitín Liasas/metabolismo , Flavobacterium/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Condroitín Liasas/aislamiento & purificación , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis Capilar , Flavobacterium/genética , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR. The ability of FNR to activate transcription initiation has been proposed to be dependent on protein-protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR-AR1 and FNR-AR3. To further our understanding of the role of FNR-AR1 and FNR-AR3 in transcription activation, we measured the effects of FNR-AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA. All the FNR-AR1 (FNR-S73F, FNR-T118A, FNR-S187P), FNR-AR3 (FNR-G85A) and FNR-AR1-AR3 (FNR-G85A-S187P) mutants that were tested decreased expression from PnarG and PdmsA in vivo. Transcription assays of PdmsA also showed that the FNR-AR mutant proteins impaired transcription activation in vitro. Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA-binding properties. The function of FNR-S187P and FNR-G85A was also measured in strains containing sigma70 mutants (sigma70-K593A, sigma70-R596A and sigma70-K597A) known to be impaired in FNR-dependent transcription activation. Of all of the combinations analysed, only FNR-G85 and sigma70-K597 showed a genetic interaction, supporting the notion that FNR-AR3 and sigma70 interact functionally in the process of transcription activation. Lastly, the transcription activation defect of the FNR-AR1 and FNR-AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate. As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR-AR1 and FNR-AR3 are altered in the presence of additional activators.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hierro-Azufre/genética , Oxidorreductasas/genética , Proteínas Bacterianas/metabolismo , Dimetilsulfóxido , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Nitrato Reductasas/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Liasas de Carbono-Azufre/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Prueba de Complementación Genética , Proteínas Hierro-Azufre/química , Mutación , Niacina/metabolismo , Fenotipo , Tiamina/metabolismoRESUMEN
To understand the role of the [4Fe-4S](2+) cluster in controlling the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) during changes in O(2) availability, we have characterized a mutant FNR protein containing a substitution of Leu-28 with His (FNR-L28H) which, unlike its wild type (WT) counterpart, is functional under aerobic growth conditions. The His-28 substitution appears to stabilize the [4Fe-4S](2+) cluster of FNR-L28H in the presence of O(2) because air-exposed FNR-L28H did not undergo the rapid [4Fe-4S](2+) to [2Fe-2S](2+) cluster conversion or concomitant loss in site-specific DNA binding and dimerization, which are characteristic of WT-FNR under these conditions. This increased cluster stability was not a result of His-28 replacing the WT-FNR cluster ligands because substitution of any of these four Cys residues (cysteine 20, 23, 29, or 122) with Ser resulted in [4Fe-4S](2+) cluster-deficient preparations of FNR-L28H. The Mössbauer spectra of FNR-L28H indicated that the coordination environment of the [4Fe-4S](2+) cluster did not differ from that of WT-FNR. Whole cell Mössbauer spectroscopy showed that aerobically grown cells overexpressing FNR-L28H had levels of the FNR species containing the [4Fe-4S](2+) cluster similar to those of cells grown under anaerobic conditions. Thus, the increase in cluster stability is sufficient to allow accumulation of the [4Fe-4S](2+) cluster form of FNR-L28H under aerobic conditions and provides a reasonable explanation for why this mutant protein is functional under aerobic growth conditions. From these results, we present a model to explain how WT-FNR is normally inactivated under aerobic growth conditions.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/genética , Oxígeno/farmacología , Aerobiosis , Anaerobiosis , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Proteínas Hierro-Azufre/química , Mutación , Espectrofotometría , Espectroscopía de Mossbauer , Sulfuros/análisis , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
In the past five to ten years, it has become increasingly apparent that the function of Fe-S clusters is not limited to electron transfer, a function implicit in their discovery. We now know that the vulnerability of these structures to oxidative destruction is used by nature in sensing O2, iron, and possibly also nitric oxide. Changes in the oxidation state of Fe-S clusters can also serve as a reversible switch.
Asunto(s)
Proteínas Hierro-Azufre/fisiología , Proteínas Bacterianas/fisiología , Escherichia coli/metabolismo , Estrés OxidativoRESUMEN
The global regulator FNR (for fumarate nitrate reduction) controls the transcription of >100 genes whose products facilitate adaptation of Escherichia coli to growth under O2-limiting conditions. Previous Mössbauer studies have shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster that, on exposure to oxygen, is converted into a [2Fe-2S]2+ cluster, a process that decreases DNA binding by FNR. Using 57Fe Mössbauer spectroscopy of E. coli cells containing overexpressed FNR, we show here that the same cluster conversion also occurs in vivo on exposure to O2. Furthermore, the data show that a significant amount of the [4Fe-4S]2+ cluster is regenerated when the cells are shifted back to an anaerobic environment. The present study also demonstrates that 57Fe Mössbauer spectroscopy can be employed to study the in vivo behavior of (overexpressed) proteins. The use of this technique to study other iron-containing cell components is discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/química , Escherichia coli/química , Proteínas Hierro-Azufre/química , Espectroscopía de Mossbauer , Transcripción Genética , Activación TranscripcionalRESUMEN
The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas Hierro-Azufre/metabolismo , Nitrato Reductasas/genética , Operón , Nitrato-Reductasa , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
FNR is a global regulator that controls transcription of genes whose functions facilitate adaptation to growth under O2 limiting conditions. It has long been appreciated that the activity of FNR must be regulated by O2 availability, since FNR dependent gene expression is observed in vivo only under anaerobic conditions, while similar levels of this protein are present in both aerobic and anaerobic grown cells. Recent progress in this field has shown that anaerobically purified FNR contains a [4Fe-4S]2+ cluster and that this [4Fe-4S]2+ cluster is sufficiently unstable toward O2 to make it suitable as an O2 sensor. The presence of the [4Fe-4S] cluster increases dimerization of FNR which is correlated with an increase in site-specific DNA binding of FNR, a property expected of transcription factors of the FNR/CRP family. According to Mössbauer spectroscopy on purified FNR and cells containing overexpressed FNR, the [4Fe-4S]2+ cluster of FNR is converted by O2 to a [2Fe-2S]2+ in high yield. The [2Fe-2S]2+ cluster can be reconverted to the [4Fe-4S]2+ cluster on reduction with dithionite in vitro raising the possibility that the [2Fe-2S]2+ cluster is a biologically inactive intermediate which may be more readily available for reconstitution into the [4Fe-4S]2+ form than the Fe-free apoform. The ability to observe, by Mössbauer spectroscopy, the Fe-S clusters of FNR in cells containing high levels of FNR should be of value in further unraveling how FNR functions in vivo. Attempts to reduce the [4Fe-4S]2+ cluster of FNR with dithionite indicated that the redox potential of the +1/+2 couple is < or = -650 mV and that the [4Fe-4S]+ cluster form is, therefore, not likely to occur in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Técnicas Biosensibles , Proteínas de Escherichia coli , Proteínas Hierro-Azufre/metabolismo , Oxígeno/análisis , Aerobiosis , Anaerobiosis , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/química , Oxidación-Reducción , Oxígeno/metabolismoRESUMEN
The sigma subunit of RNA polymerase orchestrates basal transcription by first binding to core RNA polymerase and then recognizing promoters. Using a series of 16 alanine-substitution mutations, we show that residues in a narrow region of Escherichia coli sigma70 (590 to 603) are involved in transcription activation by a mutationally altered CRP derivative, FNR and AraC. Homology modeling of region 4 of sigma70 to the closely related NarL or 434 Cro proteins, suggests that the five basic residues implicated in activation are either in the C terminus of a long recognition helix that includes residues recognizing the -35 hexamer region of the promoter, or in the subsequent loop, and are ideally positioned to permit interaction with activators. The only substitution that has a significant effect on activator-independent transcription is at R603, indicating that this residue of sigma70 may play a distinct role in transcription initiation.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Factor sigma/metabolismo , Transactivadores/metabolismo , Alanina , Secuencia de Aminoácidos , Factor de Transcripción de AraC , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Secuencias Hélice-Giro-Hélice , Proteínas Hierro-Azufre/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/metabolismo , Factor sigma/química , Factor sigma/genética , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (delta = 0.45 mm/s; DeltaEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow-green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (delta = 0.28 mm/s; DeltaEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Oxígeno/farmacología , Aire , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Hierro/análisis , Proteínas Hierro-Azufre/aislamiento & purificación , Cinética , Oxidación-Reducción , Espectrofotometría , Espectroscopía de Mossbauer , Sulfuros/análisisRESUMEN
Attention is drawn to a mechanism of redox control of gene expression involving Fe-S proteins which depends on the disassembly and reassembly of Fe-S clusters rather than a change in oxidation state. Iron Regulatory Protein (IRP)/aconitase and FNR are discussed as examples for such a mechanism.
Asunto(s)
Proteínas de Escherichia coli , Regulación de la Expresión Génica/fisiología , Proteínas Hierro-Azufre/metabolismo , Aconitato Hidratasa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Oxidación-Reducción , OxígenoRESUMEN
The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygen-stable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S2-/FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxígeno/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopolímeros , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Hierro-Azufre/química , Peso Molecular , Mutación , Unión Proteica , Análisis EspectralRESUMEN
In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR* is activated in response to anaerobiosis, we have analyzed FNR mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo. Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions. At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding. The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues. In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser. On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes. Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Hierro-Azufre , Oxígeno/farmacología , Aerobiosis , Secuencia de Aminoácidos , Anaerobiosis , Proteínas Bacterianas/genética , Cisteína/genética , Proteínas de Unión al ADN/genética , Escherichia coli/efectos de los fármacos , Operón Lac/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/genética , Unión Proteica , Serina/genética , Relación Estructura-ActividadRESUMEN
In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli , Proteínas Hierro-Azufre/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Proteínas de Unión al ADN/aislamiento & purificación , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/aislamiento & purificación , Cinética , Peso Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The Escherichia coli transcription factor FNR regulates expression of genes required for the metabolic switch between aerobic and anaerobic respiration. In order to investigate how FNR controls transcription of its target operons, DNA binding was examined for both the wild-type (WT) FNR protein and an altered function FNR* protein (DA154) that exhibits enhanced activity in the presence of oxygen both in vivo and in vitro, apparently due to the fact that DA154 is able to dimerize to a greater extent than WT FNR. Electrophoretic mobility shift assays, using a consensus symmetrical FNR target site, revealed that both DA154 and WT FNR gave rise to protein-DNA complexes of indistinguishable electrophoretic mobilities. In addition, an estimate of the molecular weight from the mobility of the DA154-DNA complex indicated that both mutant and WT FNR were dimeric when bound to DNA. Under the same binding conditions, DA154 showed an observed constant of approximately 3 x 10(8) M-1 for the consensus symmetrical target site. In addition, the results of DNA binding competition assays provided evidence that DA154 was a site-specific DNA binding protein, since this mutant protein bound to the consensus symmetrical target site with approximately 40-fold and 250-fold higher affinity than a natural target site from the nar promoter or a non-specific DNA target, respectively. Electrophoretic mobility shift DNA bending assays demonstrated protein-induced DNA bending by both DA154 and WT FNR. In addition, in vitro transcription assays using an FNR-dependent variant of the lac P1 promoter demonstrated levels of transcription activation by DA154 comparable to those observed in vivo. These results provide several new insights into how FNR functions to activate transcription of target genes.