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T Lien

Publications and source records attributed to T Lien.

At least 19 recordsLinked to original sources
PubMed2001

A novel organization of the dissimilatory sulfite reductase operon of Thermodesulforhabdus norvegica verified by RT-PCR.

The nucleotide sequence of the gene cluster for the dissimilatory sulfite reductase (Dsr) from the Gram-negative thermophilic sulfate reducer Thermodesulforhabdus norvegica was determined. The Dsr-encoding genes (dsrAB) were found to be located in an operon encompassing four other open reading frames in the following order: dsrD-dsrA-dsrB-dsrN-dsrC-fdx. Localization of these six genes in the same operon supports previous suggestions that dsrD, -C and -N play essential roles in the mechanism for reduction of sulfite to sulfide. Transcriptional analysis showed that these genes constitute a contiguous transcriptional unit of at least 5 kb. The phylogeny of Dsr is discussed.

Cloning, Molecular↗
PubMed2001

Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation.

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.

Amino Acid Sequence↗
PubMed2001

Differences in the oligomeric states of the LDH-like L-MalDH from the hyperthermophilic archaea Methanococcus jannaschii and Archaeoglobus fulgidus.

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. To gain insight into molecular relationships within this family, we studied two hyperthermophilic (LDH-like) L-MalDH (proteins with LDH-like structure and MalDH enzymatic activity) from the archaea Archaeoglobus fulgidus (Af) and Methanococcus jannaschii (Mj). The structural parameters of these enzymes determined by neutron scattering and analytical centrifugation showed that the Af (LDH-like) L-MalDH is a dimer whereas the Mj (LDH-like) L-MalDH is a tetramer. The effects of high temperature, cofactor binding, and high phosphate concentration were studied. They did not modify the oligomeric state of either enzyme. The enzymatic activity of the dimeric Af (LDH-like) L-MalDH is controlled by a pH-dependent transition at pH 7 without dissociation of the subunits. The data were analyzed in the light of the crystallographic structure of the LDH-like L-MalDH from Haloarcula marismortui. This showed that a specific loop at the dimer-dimer contact regions in these enzymes controls the tetramer formation.

Amino Acid Sequence↗
PubMed2000

Characterization of the desulforubidin operons from Desulfobacter vibrioformis and Desulfobulbus rhabdoformis.

The genes encoding the desulforubidin type of dissimilatory sulfite reductase (Dsr) from the sulfate-reducing bacteria Desulfobacter vibrioformis and Desulfobulbus rhabdoformis were cloned and sequenced. Similar to the genes for dissimilatory sulfite reductase from the genera Archaeoglobus, Desulfovibrio and Desulfotomaculum the dsr genes were found to form an operon, dsrABD, where dsrA and dsrB encode the structural subunits, alpha and beta, of Dsr, respectively. dsrD encodes a conserved unknown protein apparently restricted to sulfate-reducing species. In Desulfobacter vibrioformis a fourth gene, designated dsrN, was found downstream of dsrD forming a contiguous operon, dsrABDN. DsrN showed significant sequence homology to cobyrinic a,c-diamide synthase, which is involved in the biosynthesis of vitamin B12. A function for DsrN in amidation of siroheme is likely. Analysis of the dsrAB-encoded proteins confirmed that the high conservation observed for other types of dissimilatory sulfite reductase is also found in desulforubidin. The use of Dsr sequences in unravelling the phylogeny of sulfate-reducing bacteria is discussed.

Amino Acid Sequence↗
PubMed1999

Dissimilatory sulfite reductase from Archaeoglobus profundus and Desulfotomaculum thermocisternum: phylogenetic and structural implications from gene sequences.

The genes encoding the alpha- and beta-subunits of dissimilatory sulfite reductase, dsrAB, from the hyperthermophilic archaeon Archaeoglobus profundus and the thermophilic gram-positive bacterium Desulfotomaculum thermocisternum were cloned and sequenced. The dsrAB genes are contiguous, and most probably comprise an operon also including a dsrD homolog, a conserved gene of unknown function located downstream of dsrAB in all four sulfate reducers so far sequenced. Sequence comparison confirms that dissimilatory sulfite reductase, Dsr, is a highly conserved enzyme. A phylogenetic analysis using the available Dsr sequences, including Dsr-like proteins from nonsulfate reducers, suggests a paralogous origin of the alpha- and beta-subunits. Furthermore, the Dsr from sulfate reducers forms a separate cluster, with Dsr from the bacterial sulfate reducers Desulfotomaculum thermocisternum and Desulfovibrio vulgaris branching together, next to Dsr from Archaeoglobus profundus and Archaeoglobus fulgidus. Based on an alignment with the assimilatory sulfite reductase from Escherichia coli, the amino acid residues involved in binding of sulfite, siroheme, and [Fe4S4]-clusters have been tentatively identified, which is consistent with the binding of two sirohemes and four [Fe4S4]-clusters per alpha2beta2 structure. The evolution of Dsr and the structural basis for the binding of substrate and cofactors are discussed.

Amino Acid Sequence↗
PubMed1998

Purification and characterization of a monomeric isocitrate dehydrogenase from the sulfate-reducing bacterium Desulfobacter vibrioformis and demonstration of the presence of a monomeric enzyme in other bacteria.

NADP(+)-specific isocitrate dehydrogenase (EC 1.1.1.42) was purified to homogeneity from the sulfate-reducing bacterium Desulfobacter vibrioformis, and shown to be a monomeric protein with a molecular mass of 80 kDa. The pH and temperature optima were 8.5 and 45 degrees C, respectively. The N-terminal amino acid sequence (Thr, Glu, Thr, Ile, Arg, Trp, Thr, X, Thr, Asp, Glu, Ala, Pro, Leu, Leu, Ala, Thr) showed similarity with that of other known monomeric isocitrate dehydrogenases. Catalytically active isocitrate dehydrogenase from D. vibrioformis was obtained by activity staining after SDS-PAGE and removal of SDS from the gel. This technique revealed a NADP(+)-dependent monomeric enzyme in other Desulfobacter spp., Desulfuromonas acetoxidans and Chlorobium tepidium. These findings imply that monomeric isocitrate dehydrogenases are present in distantly related bacteria and indicate an early evolution of monomeric isocitrate dehydrogenases in the bacterial lineage.

Bacterial Proteins↗
PubMed1998

Desulfobulbus rhabdoformis sp. nov., a sulfate reducer from a water-oil separation system.

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 degrees C, pH 6.8-7.2 and 1.5-2.0% (w/v) NaCl and 0.1-0.3% (w/v) MgCl2.6H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16T is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 mumol min-1 (mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1 (mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G + C. Strain M16T is equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1% 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T (= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.

Bacterial Proteins↗
PubMed1998

Petrotoga mobilis sp. nov., from a North Sea oil-production well.

Rod-shaped, thermophilic bacteria with a sheath-like outer structure (toga) were isolated from hot oilfield water of a North Sea oil reservoir. One of the isolates, designated SJ95(T), is an obligately anaerobic, sheathed, Gram-negative, fermentative bacterium capable of reducing elemental sulfur to hydrogen sulfide and tolerating high salt concentrations. The optimum growth conditions for this isolate are 58-60 degrees Celsius and pH 6.5-7.0 with 3-4% NaCl and 0.7% MgSO(4). 7H(2)O in the medium. Vitamins are required for growth. Growth is stimulated by yeast extract. Cells of strain SJ95(T) vary in size from 1-2 to 40-50 micron in length and are motile with a subpolar flagellation. Cels grown on xylan have xylanase activity, presumably associated with the toga, and glucose isomerase activity was detected in xylose-grown cells. The DNA G+C content is 31 and 34 mol%, determined by the thermal denaturation and HPLC methods, respectively. Phylogenetically, strain SJ95(T) is most closely related to Petrotoga miotherma with a 97.7% similarity level between their 165 rDNA sequences. The DNA-DNA reassociation value between the two DNAs was 35.6%. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain SJ95t (=DSM 10674t) is proposed as the type strain of a new species, Petrotoga mobilis. It can be readily distinguished from P. miotherma by its motility.

Base Composition↗
PubMed1997

Properties and primary structure of a thermostable L-malate dehydrogenase from Archaeoglobus fulgidus.

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus L-malate dehydrogenase was stable for 5 h at 90 degrees C, and the half-life at 101 degrees C was 80 min. Thus, A. fulgidus L-malate dehydrogenase is the most thermostable L-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to L-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to L-malate dehydrogenase from the archaeon Haloarcula marismortui and other L-lactate-dehydrogenase-like L-malate dehydrogenases.

Amino Acid Sequence↗
PubMed1997

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus.

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90 degrees C or above, and a half-life of 22 min was found for the enzyme when incubated at 90 degrees C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Amino Acid Sequence↗
PubMed1997

Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus.

NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95 degrees C. The enzyme was highly thermostable, having a half-life of 140 min at 100 degrees C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp.

Amino Acid Sequence↗
PubMed1997

Desulfobacter vibrioformis sp. nov., a sulfate reducer from a water-oil separation system.

A mesophilic, gram-negative, vibrio-shaped, marine, acetate-oxidizing sulfate reducer (strain B54) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 33 degrees C, pH 6.8 to 7.0, and concentrations of NaCl and MgCl2.6H2O of at least 1 and 0.3%, respectively. Of various organic acids tested, only acetate was used as an electron and carbon source. The presence of 2-oxoglutarate:dye oxidoreductase suggests acetate oxidation via an operative citric acid cycle. Even though growth of most Desulfobacter strains (including strain B54) did not occur on hydrogen, hydrogenase was detected at low activity. The growth yields were 4.6, 13.1, and 9.6 g of (dry weight) cells per mol of acetate oxidized with sulfate, sulfite, and thiosulfate, respectively, as electron acceptors. Strain B54 was able to fix dinitrogen. Desulforubidin and cytochromes of the c and b types were present. The G+C content of the DNA was 47 mol%. Strain B54 is most closely related to Desulfobacter latus, with a 16S rDNA sequence similarity of 98.1%. The DNA-DNA relatedness between them was 40.5%. On the basis of differences in genotypic, phenotypic, and immunological characteristics, we propose that strain B54 is a member of a new species, D. vibrioformis. It can be easily identified and distinguished from other Desulfobacter species by its large, vibrioshaped cells.

Bacterial Proteins↗
PubMed1995

Thermodesulforhabdus norvegicus gen. nov., sp. nov., a novel thermophilic sulfate-reducing bacterium from oil field water.

A novel gram-negative, thermophilic, acetate-oxidizing, sulfate-reducing bacterium, strain A8444, isolated from hot North Sea oil field water, is described. The rod-shaped cells averaged 1 micron in width and 2.5 microns in length. They were motile by means of a single polar flagellum. Growth was observed between 44 and 74 degrees C, with an optimum at 60 degrees C. Spores were not produced. Sulfate and sulfite were used as electron acceptors. Sulfur, thiosulfate, nitrate, fumarate, and pyruvate were not reduced. In the presence of sulfate, growth was observed with acetate, lactate, pyruvate, butyrate, succinate, malate, fumarate, valerate, caproate, heptanoate, octanoate, nonadecanoate, decanoate, tridecanoate, pentadecanoate, palmitate, heptadecanoate, stearate, and ethanol. Pyruvate, lactate, and fumarate did not support fermentative growth. Cytochromes of the c-type were present. Desulfoviridin, desulforubidin, P582, and desulfofuscidin were not present. The G+C content of the DNA was 51 mol%. Sequence analysis of 16S rDNA showed that phylogenetically strain A8444 belongs to the delta subdivision of the Proteobacteria. The closest relatives are Desulfacinum infernum and Syntrophobacter wolinii: Strain A8444 is described as the type strain of the new taxon Thermodesulforhabdus norvegicus gen. nov., sp. nov.

Base Composition↗
PubMed1994

Archaeoglobus fulgidus Isolated from Hot North Sea Oil Field Waters.

A hyperthermophilic sulfate reducer, strain 7324, was isolated from hot (75 degrees C) oil field waters from an oil production platform in the Norwegian sector of the North Sea. It was enriched on a complex medium and isolated on lactate with sulfate. The cells were nonmotile, irregular coccoid to disc shaped, and 0.3 to 1.0 mum wide. The temperature for growth was between 60 and 85 degrees C with an optimum of 76 degrees C. Lactate, pyruvate, and valerate plus H(2) were utilized as carbon and energy sources with sulfate as electron acceptor. Lactate was completely oxidized to CO(2). The cells contained an active carbon monoxide dehydrogenase but no 2-oxoglutarate dehydrogenase activity, indicating that lactate was oxidized to CO(2) via the acetyl coenzyme A/carbon monoxide dehydrogenase pathway. The cells produced small amounts of methane simultaneously with sulfate reduction. F(420) was detected in the cells which showed a blue-green fluorescence at 420 nm. On the basis of morphological, physiological, and serological features, the isolate was classified as an Archaeoglobus sp. Strain 7324 showed 100% DNA-DNA homology with A. fulgidus Z, indicating that it belongs to the species A. fulgidus. Archaeoglobus sp. has been selectively enriched and immunomagnetically captured from oil field waters from three different platforms in the North Sea. Our results show that strain 7324 may grow in oil reservoirs at 70 to 85 degrees C and contribute to hydrogen sulfide formation in this environment.

Journal Article↗
PubMed1992

Immunomagnetically captured thermophilic sulfate-reducing bacteria from north sea oil field waters.

Immunomagnetic beads (IMB) were used to recover thermophilic sulfate-reducing bacteria from oil field waters from oil production platforms in the Norwegian sector of the North Sea. IMB coated with polyclonal antibodies against whole-cell antigens of the thermophilic Thermodesulfobacterium mobile captured strains GFA1, GFA2, and GFA3. GFA1 was serologically and morphologically identical to T. mobile. GFA2 and GFA3 were spore forming and similar to the Desulfotomaculum strains T90A and T93B previously isolated from North Sea oil field waters by a classical enrichment procedure. Western blots (immunoblots) of whole cells showed that GFA2, GFA3, T90A, and T93B are different serotypes of the same Desulfotomaculum species. Monoclonal antibodies (MAb) against T. mobile type strain cells were produced and used as capture agents on IMB. These MAb, named A4F4, were immunoglobulin M; they were specific to T. mobile and directed against lipopolysaccharides. The prevailing cells immunocaptured with MAb A4F4 were morphologically and serologically similar to T. mobile type strain cells. T. mobile was not detected in these oil field waters by classical enrichment procedures. Furthermore, extraction with antibody-coated IMB allowed pure strains to be isolated directly from primary enrichment cultures without prior time-consuming subculturing and consecutive transfers to selective media.

Journal Article↗