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.     

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.     

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.     

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.     

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.     

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.     

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 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 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     

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⁺ .     

Ultrastructure of an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum

Calderobacterium hydrogenophilum is an extreme thermophilic, obligately chemoautotrophic, hydrogen-oxidizing bacterium. The cells were shown to be nonmotile straight rods of average size 0.4x2.5 m. After negative-staining of the whole cells, no flagella were observed. The multilayered cell wall was of type 1 and possessed a crystalline proteinaceous surface layer exhibiting p4 symmetry. The square unit cells had a lattice constant of approximately 11 nm. Cell division occurred by a constriction mechanism. C. hydrogenophilum differred from a similar hydrogen-oxidizing eubacterium, Hydrogenobacter thermophilus, by the absence of intracytoplasmic membrane structures in chemically fixed cells. However, an electron-dense intracytoplasmic hemispherical structure adhering to the inner membrane was frequently observed.     

Sequence divergence of the murB and rrfB genes from Escherichia coli and Salmonella typhimurium

The murB gene of Salmonella typhimurium was cloned and found to be 75% and 82% identical to the DNA and protein sequences, respectively, of the same gene in Escherichia coli. These identities are among the lowest recorded between the two bacteria. Nevertheless, wild-type S. typhimurium murB complemented the known temperature-sensitive E. coli mutant, and wild-type E. coli murB complemented three temperature-sensitive mutants of S. typhimurium. The 5S rRNA gene, rrfB, and the region between murB and rrfB were also cloned and sequenced. The rrfB gene of S. typhimurium differs from rrfB of E. coli in only 2 of 120 nt, but the region between murB and rrfB has diverged greatly and includes a sequence that elosely resembles a repetitive extragenic palindrome of the type normally associated with E. coli. Previous comparisons of gene divergence have suggested that the chromosomal mutation rate is lower in the vicinity of the origin of replication. However, the S. typhimurium murB gene, located 6 map minutes from the origin of replication, is highly substituted at synonymous sites and the sequence between murB and rrfB is significantly modified as well. Thus, murB is an exception to the general observation that genes near the origin of replication show less divergence than do genes elsewhere in the bacterial chromosome.Abbreviations CAI codon adaptation index - REP repetitive extragenic palindrome     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

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.     

Thermoanaerobium acetigenum spec. nov., a new anaerobic,extremely thermophilic,xylanolytic non-spore-forming bacterium isolated from an Icelandic hot spring

An anaerobic, extremely thermophilic, xylanolytic nonspore-forming bacterium, strain X6B, was isolated from a 70°C Icelandic hot spring sediment. The bacterium was rod-shaped, 3.6–5.9 m long and 0.7 to 1.0 m wide, and cells grew singly, in pairs, and occasionally formed chains. The bacterium was nonmotile with no flagella. Cells from mid-to late exponential gowth-phase cultures stained gram-negative but had a gram-positive like cell wall structure in transmission electron photomicrographs. The bacterium grew between 50°C and 78°C with an optimum temperature at about 65°C to 68°C. Growth occurred between pH 5.2 and 8.5 with an optimum pH close to 7. During growth on beech wood xylan, glucose and d-xylose, the isolate produced CO₂, acetate and H₂ as major fermentation products, and a small amounts of ethanol; lactate was not produced. X6B did not reduce acetone to isopropanol or sulphate or thiosulfate to sulfide. The base composition of X6B's cellular DNA was 35.7 mol% guanine + cytosine. The properties of this strain do not fit any previously described species. The name proposed for the isolated bacterium was Thermoanaerobium acetigenum, spec. nov.     

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.     

Alleviation of Type I Restriction in Escherichia coli K12 Carrying the arsR Gene from pKW301 of Acidiphilium multivorum AIU 301

A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is contained in pKW301. In Escherichia coli, arsR cloned under the control of P _lac in a multicopy vector alleviated restriction of nonmodified DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas in the Ard proteins the motif is in the C domain. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is –4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity.