Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron-sulfur proteins.

The iron-sulfur (Fe-S) cluster, the nonheme-iron cofactor essential for the activity of many proteins, is incorporated into target proteins with the aid of complex machinery. In bacteria, several proteins encoded by the iscRSUA-hscBA-fdx-ORF3 cluster (isc operon) have been proposed to execute crucial tasks in the assembly of Fe-S clusters. To elucidate the in vivo function, we have undertaken a systematic mutational analysis of the genes in the Escherichia coli isc operon. In all functional tests, i.e. growth rate, nutritional requirements and activities of Fe-S enzymes, the inactivation of the iscS gene elicited the most drastic alteration. Strains with mutations in the iscU, hscB, hscA, and fdx genes also exhibited conspicuous phenotypical consequences almost identical to one another. The effect of the inactivation of iscA was small but appreciable on Fe-S enzymes. In contrast, mutants with inactivated iscR or ORF3 showed virtually no differences from wild-type cells. The requirement of iscSUA-hscBA-fdx for the assembly of Fe-S clusters was further confirmed by complementation experiments using a mutant strain in which the entire isc operon was deleted. Our findings support the conclusion that IscS, via cysteine desulfurase activity, provides the sulfur that is subsequently incorporated into Fe-S clusters by assembler machinery comprising of the iscUA-hscBA-fdx gene products. The results presented here indicate crucial roles for IscU, HscB, HscA, and Fdx as central components of the assembler machinery and also provide evidence for interactions among them.     

The lipoate synthase from Escherichia coli is an iron-sulfur protein.

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.     

Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase

Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.     

The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.     

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.     

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.     

Repair of oxidized iron-sulfur clusters in Escherichia coli

The [4Fe-4S]2+ clusters of dehydratases are rapidly damaged by univalent oxidants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S]1+ form. Continued exposure to oxidants in vitro leads to further iron release. Experiments have shown that these clusters are repaired in vivo. We sought to determine whether repair is mediated by either the Isc or Suf cluster-assembly systems that have been identified in Escherichia coli. We found that all the proteins encoded by the isc operon were critical for de novo assembly, but most of these were unnecessary for cluster repair. IscS, a cysteine desulfurase, appeared to be an exception: although iscS mutants repaired damaged clusters, they did so substantially more slowly than did wild-type cells. Because sulfur mobilization should be required only if clusters degrade beyond the [3Fe-4S]1+ state, we used whole cell EPR to visualize the fate of oxidized enzymes in vivo. Fumarase A was overproduced. Brief exposure of cells to hydrogen peroxide resulted in the appearance of the characteristic [3Fe-4S]1+ signal of the oxidized enzyme. When hydrogen peroxide was then scavenged, the enzyme activity reappeared within minutes, in concert with the disappearance of the EPR signal. Thus it is unclear why IscS is required for efficient repair. The iscS mutants grew poorly, allowing the possibility that metabolic defects indirectly slow the repair process. Our data did indicate that damaged clusters decompose beyond the [3Fe-4S]1+ state in vivo when stress is prolonged. Under the conditions of our experiments, mutants that lacked other repair candidates--Suf proteins, glutathione, and NADPH: ferredoxin reductase--all repaired clusters at normal rates. We conclude that the mechanism of cluster repair is distinct from that of de novo assembly and that this is true because mild oxidative stress does not degrade clusters in vivo to the point of presenting an apoenzyme to the de novo cluster-assembly systems.     

The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.     

The role of the iron-sulfur cluster in Escherichia coli endonuclease III. A resonance Raman study.

Resonance Raman spectroscopy has been used to investigate the function and properties of the iron-sulfur cluster in Escherichia coli endonuclease III. Resonance Raman spectra in the Fe-S stretching region are indicative of a [4Fe-4S]2+ cluster with complete cysteinyl sulfur coordination, and vibrational assignments are made by analogy with bacterial ferredoxins. Minor changes in the vibrational frequencies of the modes primarily involving Fe-S(Cys) stretching accompany the binding of the inhibitor thymine glycol or an oligonucleotide containing a reduced apyrimidinic site. These changes are consistent with perturbation of the orientation of the ligating cysteinyl residues and rule out the possibility that the [4Fe-4S] cluster is directly involved with substrate or inhibitor binding. It is concluded that a structural role is most likely for the [4Fe-4S] cluster in endonuclease III.     

Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical.

Two-dimensional gel analyses were made of proteins synthesized in Escherichia coli during various O2- -generating conditions. Nine proteins were constitutively synthesized over wild-type levels in superoxide dismutase (sodA sodB) double mutants. Addition of redox cycling agents such as paraquat and plumbagin at various concentrations induced up to 13 proteins in wild-type cells. Among these 13 were 5 of the 9 constitutively synthesized in the sodA sodB double mutants. Addition of these agents to the superoxide dismutase mutants in low micromolar concentrations induced an additional set of 14 proteins. The proteins induced included only five proteins that have been previously associated with stress responses, consisting of endonuclease IV (Nfo), three oxyR-regulated proteins, and one heat shock protein. O2- -mediated induction of the superoxide inducible (Soi) proteins in the wild type was independent of the oxyR+ gene for all but the three oxyR-regulated proteins. Analyses of proteins from three soi::lacZ gene fusions previously isolated (T. Kogoma, S. B. Farr, K. M. Joyce, and D. O. Natvig, Proc. Natl. Acad. Sci. USA 85:4799-4803, 1988) indicated the specific loss of one of these induced proteins in each fusion strain and the constitutive expression of some Soi proteins.     

High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe-2S] and one Fe (II)-mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe-2S] center, are expressed by Burkholderia cepacia at approximately 30mg of crude PDO and approximately 1mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E. coli cell line, growth parameters, time of induction, media composition, and iron-sulfur additives resulted in yields of about 1g/L for PDO and about 0.2g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5-9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was <8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.     

Mutant MotB proteins in Escherichia coli.

The MotB protein of Escherichia coli is an essential component in each of eight torque generators in the flagellar rotary motor. Based on its membrane topology, it has been suggested that MotB might be a linker that fastens the torque-generating machinery to the cell wall. Here, we report the isolation and characterization of a number of motB mutants. As found previously for motA, many alleles of motB were dominant, as expected if MotB is a component of the motor. In other respects, however, the motB mutants differed from the motA mutants. Most of the mutations mapped to a hydrophilic, periplasmic domain of the protein, and nothing comparable to the slow-swimming alleles of motA, which show normal torque when tethered, was found. Some motB mutants retained partial function, but when tethered they produced subnormal torque, indicating that their motors contained only one or two functional torque generators. These results support the hypothesis that MotB is a linker.     

The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli

The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ^S-dependent genes when compared to the wild-type rpoS ⁺ strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ⁷⁰-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.     

The effect of magnesium starvation on the dissociation of ribosomal proteins from Escherichia coli K-12 ribosomes.

The effect of magnesium starvation upon the fate of individual ribosomal proteins was studied in Escherichia coli. During a 21 h incubation in the absence of Mg2+ the 30 S subunit was more susceptible to degradation, retaining an average 31.9% of its ribosomal proteins as compared to 40.0% for the 50 S subunit. An examination of those 50-S proteins dissociated to a lesser extent than the average value (L1, L2, L3, L7, L10, L13, L16, L17, L19, L21, L22, L23, and L29) revealed that, with the exception of L16, all were classified by Dohme and Nierhaus [5] as tightly bound. Of the ribosomal proteins dissocated during magnesium starvation only five were reincorporated (and these to a minimal degree) during recovery of cells in a medium containing Mg2+. These studies suggest that ribosomal proteins once released from the ribosome particles during magnesium starvation are not reutilized in the assembly of new subunits.     

Genetic studies of the ribosomal proteins in Escherichia coli. 3. Compositions of ribosomal proteins in various strains of Escherichia coli

Summary The comparative chromatographic investigations into the ribosomal proteins of various strains of E. coli have demonstrated that most of the strains including three strains of E. coli subsp. communior had ribosomes with the same protein compositions (C-type). The ribosomes from strain B are different from the C-type ribosomes in having the specific 30-4 (B) component in place of 30-4 (B-type), while those from strains K 12 may be distinguished from the type-C ribosomes by the presence of the specific 30-7 (K) component in place of 30-7 (K-type) or, in addition to 30-7 (K), the presence of 30-9 (W3637) in place of 30-9 (K-3637 type). Two strains, IAM 1132 and IAM 1182, have R-type ribosomes, in which at least six 50s proteins and four 30s protein components are distinct from the corresponding protein components in the C-type ribosomes.