Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It was found to be able to grow in the presence of micromolar molecular oxygen (O₂). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O₂ to hydrogen peroxide (H₂O₂), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent V_max value of 37 mol NADH oxidized min^–1 mg^–1 protein. Apparent K_m values for NADH and O₂ were determined to be 7.5 M and 85 M, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85°C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H₂O₂ produced by the NADH oxidase to H₂O. It seems possible that O₂ can be reduced to H₂O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O₂.     

Alkali-tolerant high-activity catalase from a thermophilic bacterium and its overexpression in Escherichia coli

We have purified an alkali-tolerant catalase from the thermophilic bacterium Metallosphaera hakonensis. The catalase gene, which encodes 303 amino acids and has a calculated molecular mass of 33 kDa, including its putative signal peptide encoding sequence, was cloned. The deduced amino acid sequence exhibited a region-specific homology with the sequences of manganese catalases from thermophilic bacteria such as Thermus thermophilus and Thermus brockianus. When this gene was overexpressed in Escherichia coli, proteins of the expected size (33 kDa) were overproduced in the inactive form. We made several attempts to obtain active forms of or to activate these overproduced proteins. Upon their induction into E. coli, a 100-fold increase in the catalase activity was detected when high-concentration manganese was used as the medium. The catalase activity of the purified enzyme was optimal at a pH of 10.0. The alkali-tolerant property of this catalase makes it a promising enzyme in biotechnological applications such as H(2)O(2)-detoxifying systems.     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

A hexokinase with broad sugar specificity from a thermophilic bacterium

A recombinant thermophilic Thermus caldophilus GK24 hexokinase, one of the ROK-type (repressor protein, open reading frames, and sugar kinase) proteins, exists uniquely as a 120 kDa molecule with four subunits (31 kDa), in contrast to eukaryotic and bacterial sugar kinases which are monomers or dimers. The optimal temperature and pH for the enzyme reaction are 70-80 degrees C and 7.5, respectively. This enzyme shows broad specificity toward glucose, mannose, glucosamine, allose, 2-deoxyglucose, and fructose. To understand the sugar specificity at a structural level, the enzyme-ATP/Mg2+-sugar binding complex models have been constructed. It has been shown that the sugar specificity is probably dependent on the interaction energy occurred by the positional proximity of sugars bound in the active site of the enzyme, which exhibits a tolerance to modification at C2 or C3 of glucose.     

Occurrence of Sulfolobus acidocaldarius,an extremely thermophilic acidophilic bacterium,in New Zealand hot springs

Several hot springs in the Rotorua-Taupo regions, North Island, New Zealand, were tested for the presence of extremely thermophilic acidophilic bacteria. In the majority of the springs, ranging in temperature from 43–96°C and in pH from 2.1–6.9, direct microscopic observations revealed the presence of both rod-shaped and spherical bacteria. Isolations were attempted at 70°C and pH 2.0 and 7.0, with either yeast extract for heterotrophic growth, or elemental sulfur as the sole source of energy for autotrophic growth. Eight of the samples produced grwoth at pH 2.0 with either yeast extract or sulfur, but none of the samples grew at pH 7.0. All the isolates obtained, resembled Sulfolobus acidocaldarius, a thermophilic acidophilic bacterium which has previously been reported from various regions in the Northern Hemisphere. Immunofluorescence examination of six of these isolates revealed varying degrees of cross reactions with two already characterized Sulfolobus isolates from the Yellowstone National Park, U.S.A. This paper is the first published record of Sulfolobus from the Southern Hemisphere.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Characterization of three genes in the dam-containing operon of Escherichia coli

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.     

Expression of theunc genes inEscherichia coli

Theunc (or atp) operon ofEscherichia coli comprises eight genes encoding the known subunits of the proton-translocating ATP synthase (H⁺-ATPase) plus a ninth gene (uncI) of unknown function. The subunit stoichiometry of the H⁺-ATPase ( ₃₃₁₁₁a₁b₂c_10–15) requires that the respectiveunc genes be expressed at different rates. This review discusses the experimental methods applied to determining how differential synthesis is achieved, and evaluates the results obtained. It has been found that the primary level of control is translational initiation. The translational efficiencies of theunc genes are determined by primary and secondary mRNA structures within their respective translational initiation regions. The respective rates of translation are matched to the subunit requirements of H⁺-ATPase assembly. Finally, points of uncertainty remain and experimental strategies which will be important in future work are discussed.     

Expression and preparation of recombinant hepcidin in Escherichia coli

Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. In this work, hepcidin was expressed in Escherichia coli as a 10.5 kDa fusion protein (His-hepcidin) with a N-terminal hexahistidine tag. The expressed His-hepcidin existed in the form of inclusion bodies and was purified by IMAC under denaturation condition. Since the fusion partner for hepcidin did not contain other cysteine residues, the formation of disulfide bonds was performed before the His-tag was removed. Then, the oxidized His-hepcidin monomer was separated from protein multimers through gel filtration. Following monomer refolding, hepcidin was cleaved from fusion protein by enterokinase and purified with reverse-phase chromatography. The recombinant hepcidin exhibited obvious antibacterial activity against Bacillus subtilis.     

Expression and purification of His-tagged flavonol synthase of Camellia sinensis from Escherichia coli

Flavonols, a class of bioactive polyphenols present in plants, are the products of flavonol desaturation catalyzed by flavonol synthase (FLS). We cloned the cDNA coding for the enzyme FLS from Camellia sinensis (CsFLS) by end-to-end PCR followed by 5'- and 3'-RACE. The putative CsFLS had 333 amino acid residues, displayed identities to the FLSs of Arabidopsis and Ginkgo of 53% and 52.5%, respectively, and contained several conserved elements found in the 2-oxoglutarate-Fe(II)-dioxygenase superfamily. The cDNA of CsFLS was subcloned into pET28a(+) and introduced into Escherichia coli (BL21-CodonPlus-RIL). Induction with 0.1mM IPTG at low temperature (20 degrees C) led to higher amounts of CsFLS in the soluble fraction than induction at 30 degrees C. The enzyme aggregated into inclusion bodies could be rescued by denaturation with 6M urea and purification with a His.Bind purification kit. The purified protein was desalted by Amicon Ultra-15 centrifugal filter unit, and the His-tag was removed with thrombin. The finally purified protein was assayed with dihydroquercetin as substrate and the products were analyzed by HPLC. The addition of FeSO(4) to the buffers used in the CsFLS purification significantly increased the recovery of active enzyme. The CsFLS obtained in this study was found to have higher specific activity and lower K(m) than previously reported FLSs.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.     

Geobacillus galactosidasius sp. nov., a new thermophilic galactosidase-producing bacterium isolated from compost

Two thermophilic spore-forming strains, with optimum growth temperature at 70 °C, were isolated from compost of the “Experimental System of Composting” (Teora, Avellino, Italy). A phylogenetic analysis based on 16S rRNA gene sequences showed that these organisms represented a new species of the genus Geobacillus. Based on polyphasic taxonomic data the strains represented a novel species for which the name Geobacillus galactosidasius sp. nov. is proposed. The type strain is CF1B^T (= ATCC BAA-1450^T = DSM 18751^T).     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .     

Expression of subunits a and c of the sodium-dependent ATPase of Propionigenium modestum in Escherichia coli

The aim of the present study was to construct functional hybrid ATPases consisting of all Escherichia coli ATPase subunits excepts the F₀ subunits a or c which were replaced by the respective subunits of the Propionigenium modestum ATPase. This would give valuable information on the subunit(s) conferring the coupling ion specificity. Plasmids were constructed that carried the gene for subunit c (uncE) or subunit a (uncB) behind a tac promoter. These plasmids were transformed into E. coli strains which differed with respect to the unc operon and the expression of the P. modestum genes was verified biochemically. Enhanced expression of the P. modestum genes led to strong growth inhibition of all E. coli strains tested. However, the expressed P. modestum proteins could not functionally complement E. coli strains that lacked the homologous subunit.Abbreviations PCR Polymerase chain reaction - ACMA 9-amino-6-chloro-2-methoxyacridine - SDS sodium dodecyl sulfate - DCCD N,N-dicyclohexylcarbodiimide - PMSF pnenylmethyl sulfornyl fluoride - DFP dirsopropylfluorphosphat - TCA trichloroacetic acid     

Some properties of a beta-galactosidase from an extremely thermophilic bacterium

An inducible beta-galactosidase from an extremely thermophilic organism, Thermus strain 4-1A, has been isolated and partially purified. There were significant dissimilarities to T. aquaticus beta-galactosidase. It had a pl of 4.5, was inhibited by sulphydryl inhibitors and a number of transition metals, and was activated by EDTA and SH-containing reagents. The beta-galactosidase showed strong product inhibition, and weaker inhibition by some other mono- and disaccharides. It was very stable up to 90 degrees C at pH 8. On immobilization by diazonium linkage to porous glass, the pH optimum (6.0), the K(M) with ONPG (5mM) and the product inhibition were not altered.