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1.
Chunli Zheng Yanfei Zhang Yuandong Liu Anna Wu Lexian Xia Jia Zeng Jianshe Liu Guanzhou Qiu 《Current microbiology》2009,58(6):586-592
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. 相似文献
2.
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. 相似文献
3.
Yunjie Dai Jianshe Liu Chunli Zheng Anna Wu Jia Zeng Guanzhou Qiu 《Current microbiology》2009,59(5):559-564
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. 相似文献
4.
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. 相似文献
5.
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. 相似文献
6.
7.
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. 相似文献
8.
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 相似文献
9.
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. 相似文献
10.
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. 相似文献
11.
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. 相似文献
12.
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. 相似文献
13.
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. 相似文献
14.
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. 相似文献
15.
Marieke L. Overeijnder Wilfred R. Hagen Peter-Leon Hagedoorn 《Journal of biological inorganic chemistry》2009,14(5):703-710
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. 相似文献
16.
G. A. Kovalenko L. V. Perminova T. V. Chuenko L. I. Sapunova E. A. Shlyakhotko A. G. Lobanok 《Applied Biochemistry and Microbiology》2011,47(2):151-157
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. 相似文献
17.
Ping Yong Iryna P. Mikheenko Kevin Deplanche Mark D. Redwood Lynne E. Macaskie 《Biotechnology letters》2010,32(12):1821-1828
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. 相似文献
18.
Mandal S 《Current microbiology》2012,64(3):259-264
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. 相似文献
19.
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. 相似文献
20.
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 相似文献