Characterization and Reconstitute of a [Fe<Subscript>4</Subscript>S<Subscript>4</Subscript>] Adenosine 5′-Phosphosulfate Reductase from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Adenosine 5′-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron–sulfur cluster. The [Fe₄S₄] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> FtnA acts as an iron buffer for re-assembly of iron–sulfur clusters in response to hydrogen peroxide stress

Iron–sulfur clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron–sulfur clusters, the iron released from the disrupted iron–sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron–sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in FtnA can be retrieved by an iron chaperon IscA for the re-assembly of the iron–sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli FtnA may act as an iron buffer to sequester the iron released from the disrupted iron–sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron–sulfur clusters under normal physiological conditions.     

Cys92, Cys101, Cys197, and Cys203 Are Crucial Residues for Coordinating the Iron–Sulfur Cluster of RhdA from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron–sulfur clusters. But so far, there were no articles to confirm the existence of iron–sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV–Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron–sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron–sulfur cluster binding.     

Expression, purification and characterization of a high potential iron–sulfur protein from Acidithiobacillus ferrooxidans

The high potential iron–sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe₄S₄] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron–sulfur cluster.     

<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

Expression,purification and characterization of a cysteine desulfurase,IscS, from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

Iron–sulfur clusters are one of the most common types of redox center in nature. Three proteins of IscS (a cysteine desulfurase), IscU (a scaffold protein) and IscA (an iron chaperon) encoded by the operon iscSUA are involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans. In this study the gene of IscS from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The molecular mass of recombinant IscS was 46 kDa by SDS-PAGE. The IscS was a pyridoxal phosphate-containing protein, that catalyzed the elimination of S from l-cysteine to yield l-alanine and elemental sulfur or H₂S, depending on whether or not a reducing agent was added to the reaction mixture. Jia Zeng and Yanfei Zhang contributed equally to this work.     

Conjugative plasmid mediated inducible nickel resistance in<Emphasis Type="Italic"> Hafnia alvei</Emphasis> 5-5

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni²⁺ or 2 mM Co²⁺. The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni²⁺, and cobalt resistance to 2 mM Co²⁺. A 4.8-kb Sal–EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni²⁺) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni²⁺. By random TnphoA′-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A. Electronic Publication     

Effect of cobalt on <Emphasis Type="Italic">Escherichia coli</Emphasis> metabolism and metalloporphyrin formation

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.     

The RcnRA (YohLM) system of <Emphasis Type="Italic">Escherichia coli</Emphasis>: A connection between nickel,cobalt and iron homeostasis

The transporter RcnA has previously been implicated in Ni(II) and Co(II) detoxification in E. coli probably through efflux. Here we demonstrate that the divergently described rcnA and rcnR gene products constitute a link between nickel, cobalt and iron homeostasis. Deletion of the rcnA gene resulted in increased cellular nickel, cobalt and iron concentrations. Expression of rcnA was induced by Ni(II) or Co(II). Overproduction of rcnR inhibited induction of rcnA by metal cations but RcnR did not bind to the rcnA promoter in vitro. When rcnR or fur, the gene of the global repressor of iron homeostasis, was deleted, expression of rcnA was also induced by iron. The promoter region of rcnA was positive in a Fur titration (FURTA) in vivo assay indicative of Fur binding. Thus, rcnA is part of the Fur regulon of E.␣coli. The implications of a connection between the homoeostasis of closely related transition metals are discussed.     

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c′ and cytochrome c, are detected. A high potential iron–sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron–sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression and single-step purification of mercury transporter (merT) from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l⁻¹.     

Expression, Purification, and Characterization of a [Fe2S2] Cluster Containing Ferredoxin from Acidithiobacillus ferrooxidans

The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has an essential role in the maturation of various iron–sulfur (Fe-S) proteins and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from A. ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe₂S₂] cluster of the protein.     

High-Level Resistance to Cobalt and Nickel but Probably No Transenvelope Efflux: Metal Resistance in the Cuban Serratia marcescens Strain C-1

Molecular mechanisms underlying inducible cobalt and nickel resistance of a bacterial strain isolated from a Cuban serpentine deposit were investigated. This strain C-1 was assigned to Serratia marcescens by 16S rDNA analysis and DNA/DNA hybridization. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for cobalt- and nickel-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The predicted NcrA product was a NreB ortholog of the major facilitator protein superfamily and central for cobalt/nickel resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in Escherichia coli and caused decreased accumulation of Co(II) and Ni(II) in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the nickel–cobalt transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni(II) and Co(II). Because the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.     

A thermostable hybrid cluster protein from Pyrococcus furiosus: effects of the loss of a three helix bundle subdomain

Pyrococcus furiosus hybrid cluster protein (HCP) was expressed in Escherichia coli, purified, and characterized. This is the first archaeal and thermostable HCP to be isolated. Compared with the protein sequences of previously characterized HCPs from mesophiles, the protein sequence of P. furiosus HCP exhibits a deletion of approximately 13 kDa as a single amino acid stretch just after the N-terminal cysteine motif, characteristic for class-III HCPs from (hyper)thermophilic archaea and bacteria. The protein was expressed as a thermostable, soluble homodimeric protein. Hydroxylamine reductase activity of P. furiosus HCP showed a K _m value of 0.40 mM and a k _cat value of 3.8 s⁻¹ at 70 °C and pH 9.0. Electron paramagnetic resonance spectroscopy showed evidence for the presence of a spin-admixed, S = 3/2 [4Fe–4S]⁺ cubane cluster and of the hybrid cluster. The cubane cluster of P. furiosus HCP is presumably coordinated by a CXXC–X₇–C–X₅–C motif close to the N-terminus, which is similar to the CXXC–X₈–C–X₅–C motif of the Desulfovibrio desulfuricans and Desulfovibrio vulgaris HCPs. Amino acid sequence alignment and homology modeling of P. furiosus HCP reveal that the deletion results in a loss of one of the two three-helix bundles of domain 1. Clearly the loss of one of the three-helix bundles of domain 1 does not diminish the hydroxylamine reduction activity and the incorporation of the iron–sulfur clusters.     

Immobilization of a recombinant strain producing glucose isomerase inside SiO<Subscript>2</Subscript>-xerogel and properties of prepared biocatalysts

An original method of immobilization of non-growing microorganism cells inside xerogel of silicium dioxide containing insoluble hydroxyl compounds of cobalt(II) has been developed. A recombinant strain producing glucose isomerase has been constructed on the basis of Escherichia coli with the use of a gene of Arthrobacter nicotianae. It was revealed that glucose isomerase activity and stability of biocatalysts prepared on the basis of the recombinant E. coli strain was 3–5 times greater compared with the biocatalysts prepared with the use of the donor strain A. nicotianae. Under conditions of continuous hydrolysis of 3 M fructose at 62–65°C in a fixed bed reactor, time of half-inactivation of the biocatalysts prepared from the recombinant strain and A. nicotianae was ∼60 and ∼25 days, respectively.     

Biorefining of precious metals from wastes: an answer to manufacturing of cheap nanocatalysts for fuel cells and power generation via an integrated biorefinery?

Bio-manufacturing of nano-scale palladium was achieved via enzymatically-mediated deposition of Pd from solution using Desulfovibrio desulfuricans, Escherichia coli and Cupriavidus metallidurans. Dried ‘Bio-Pd’ materials were sintered, applied onto carbon papers and tested as anodes in a proton exchange membrane (PEM) fuel cell for power production. At a Pd(0) loading of 25% by mass the fuel cell power using Bio-Pd _{D. desulfuricans} (positive control) and Bio-Pd _{E. coli} (negative control) was ~140 and ~30 mW respectively. Bio-Pd _{C. metallidurans} was intermediate between these with a power output of ~60 mW. An engineered strain of E. coli (IC007) was previously reported to give a Bio-Pd that was >3-fold more active than Bio-Pd of the parent E. coli MC4100 (i.e. a power output of >110 mW). Using this strain, a mixed metallic catalyst was manufactured from an industrial processing waste. This ‘Bio-precious metal’ (‘Bio-PM’) gave ~68% of the power output as commercial Pd(0) and ~50% of that of Bio-Pd _{D. desulfuricans} when used as fuel cell anodic material. The results are discussed in relation to integrated bioprocessing for clean energy.     

The Sulfur Oxidation Operon Repressor Function is Influenced by the Product of its Adjacent Upstream ORF in <Emphasis Type="Italic">Pseudaminobacter salicylatoxidans</Emphasis> KCT001

The repressor of sulfur-oxidizing (sox) operon regulates expression of genes encoding a multienzyme complex that governs the chemolithotrophic sulfur oxidation in Pseudaminobacter salycylatoxidans KCT001. The inducer of sox operon viz., thiosulfate and other sulfur anions had no impact on in vitro repressor–operator interaction which indicates an atypical derepression mechanism. The reduced repressor has higher affinity for its operator DNA. The sulfur oxidation repressor binds with operator regions and led to efficient repression in trans, however, increased repressor concentration resulted in higher gene expression. Using a reporter system in E. coli, the present study established that the thioredoxin-like protein, encoded in immediate upstream ORF, could nullify the observed reversal of the repression at higher repressor concentration. In this context, the involvement of the upstream gene product in the regulation of the sulfur oxidation gene expression has been reported.     

Iron–sulfur cluster biosynthesis: characterization of IscU–IscS complex formation and a structural model for sulfide delivery to the [2Fe–2S] assembly site

Recent work on the bacterial iron–sulfur cluster (isc) family of gene products, and eukaryotic homologs, has advanced the molecular understanding of cellular mechanisms of iron–sulfur cluster biosynthesis. Members of the IscS family are pyridoxyl-5′-phosophate dependent proteins that deliver inorganic sulfide during assembly of the [2Fe–2S] cluster on the IscU scaffold protein. Herein it is demonstrated through calorimetry, fluorescence, and protein stability measurements that Thermotoga maritima IscS forms a 1:1 complex with IscU in a concentration-dependent manner (K _D varying from 6 to 34 μM, over an IscS concentration range of approximately 2–50 μM). Docking simulations of representative IscU and IscS proteins reveal critical contact surfaces at the N-terminal helix of IscU and a C-terminal loop comprising a chaperone binding domain. Consistent with the isothermal titration calorimetry results described here, an overall dominant contribution of charged surfaces with a change in the molar heat capacity of binding, ΔC _p ~ 199.8 kcal K⁻¹ mol⁻¹, is observed that accounts for approximately 10% of the total accessible surface area at the binding interface. Both apo and holo IscUs and homologs were found to bind to IscS in an enthalpically driven reaction with comparable K _D values. Both helix and loop regions are highly conserved among phylogenetically diverse organisms from a pool of archael, bacterial, fungal, and mammalian representatives.     

A Novel Gene Cluster soxSRT Is Essential for the Chemolithotrophic Oxidation of Thiosulfate and Tetrathionate by Pseudaminobacter salicylatoxidans KCT001

Chemolithotrophic sulfur oxidation (Sox) in the α-proteobacterium Pseudaminobacter salicylatoxidans KCT001 was found to be governed by the gene cluster soxSRT–soxVWXYZABCD. Independent transposon-insertion mutations in the genes soxB, soxC, soxD, and also in a novel open reading frame (ORF), designated as soxT, afforded revelation of the entire sox locus of this bacterium. The deduced amino acid sequence of the novel ORF soxT comprised 362 residues and exhibited significant homology with hypothetical proteins of diverse origin, including a permease-like transport protein of Escherichia coli. Two contiguous ORFs, soxR and soxS, immediately preceded the soxT gene. The gene cluster soxSRT was located upstream of soxVWXYZABCD and was transcribed divergently with respect to the latter. Chemolithotrophic utilization of both thiosulfate and tetrathionate was observed to have been impaired in all of these Sox⁻ mutants, implicating the involvement of the gene cluster soxSRT–soxVWXYZABCD in the oxidation of both thiosulfate and tetrathionate. Pradosh Roy Deceased