Thermotoga neapolitana sp. nov. of the extremely thermophilic,eubacterial genus Thermotoga

A second species of the extremely thermophilic, eubacterial genus Thermotoga is described as clearly distinguished from the type species Thermotoga maritima by physiological and phylogenetic criteria. It is named Thermotoga neapolitana.     

Hydrogen production in anaerobic and microaerobic Thermotoga neapolitana

We have tested the hypothesis (Van Ooteghem et al. Appl Biochem Biotechnol 2002 98–100: 177–189) that microaerobic metabolism may increase the yield of H₂ from the thermophilic bacterium Thermotoga neapolitana. In anaerobic conditions, T. neapolitana converted glucose into acetic acid and lactic acid and yielded 2.4 ± 0.3 mol H₂ mol⁻¹ glucose. The bacterium tolerated low O₂ partial pressures but the H₂ yield was not improved under microaerobic conditions. Our results indicate that T. neapolitana only produces H₂ by anaerobic metabolism, and that the yield of H₂ can be maximised by minimising the production of lactic acid.     

Production of D-tagatose at high temperatures using immobilized Escherichia coli cells expressing L-arabinose isomerase from Thermotoga neapolitana

Escherichia coli cells expressing l-arabinose isomerase from Thermotoga neapolitana (TNAI) were immobilized in calcium alginate beads. The resulting cell reactor (2.4 U, t _1/2 = 43 days at 70°C) in a continuous recycling mode at 70°C produced 49 and 38 g d-tagatose/l from 180 and 90 g d-galactose/l, respectively, within 12 h.     

Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract

The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD⁺, Mn²⁺) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Membrane-associated redox activities in Thermotoga neapolitana

Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation. Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps. One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase. We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation. This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD⁺ from ferredoxin-dependent pyruvate oxidation. However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates. NMOR activity was also detected in aerobically prepared vesicles. By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles. During these studies, an extracytoplasmic hydrogenase activity was discovered. In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides. In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite. Received: 28 May 1998 / Accepted: 23 June 1998     

Isolation of auxotrophic and antimetabolite-resistant mutants of the hyperthermophilic bacterium Thermotoga neapolitana

Antimetabolite-resistant and auxotrophic mutants of the hyperthermophilic bacterium Thermotoga neapolitana were isolated to provide strains with genetic backgrounds amenable to genetic analyses. Norleucine, azaleucine, 4-nitropyridine-N-oxide, and 3-amino-1, 2, 4-triazole did not affect growth, while 5-fluorouracil (5 g/ml), 5-methyltryptophan (250g/ml), 6-azauracil (100 g/ml), and 4-fluorophenylalanine (30 g/ml) inhibited growth at the indicated minimum inhibitory concentrations. The effect of 5-fluorouracil was analyzed and found to be bacteriostatic. These inhibitors were used to select spontaneously arising resistant mutants. In addition, auxotrophic mutants requiring leucine, tryptophan, adenine, and histidine were isolated following mutagenesis with ethyl methanesulfonate. Six other auxotrophs with undefined growth requirements were also isolated. These strains will be useful for the development of genetic methods for T. neapolitana.     

<Emphasis Type="Italic">Thermotoga maritima</Emphasis> TM0298 is a highly thermostable mannitol dehydrogenase

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V _max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD⁺ than with NADP⁺. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from T. maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the Escherichia coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the -glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the T. maritima cofactor-dependent -glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.     

Fusion of carbohydrate binding modules from <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis> with a family 10 xylanase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> S7

Xylanase A of Thermotoga neapolitana contains binding domains both at the N- and C-terminal ends of the catalytic domain. In the N-terminal position it contains two carbohydrate-binding modules (CBM) which belong to family 22. These CBMs bind xylan but not to cellulose. The gene encoding the mature peptide of these CBMs was fused with an alkaline active GH10 xylanase from Bacillus halodurans S7 and expressed in Escherichia coli. The (His)₆ tagged hybrid protein was purified by immobilized metal affinity chromatography and characterized. Xylan binding by the chimeric protein was influenced by NaCl concentration and pH of the binding medium. Binding increased with increasing salt concentration up to 200 mM. Higher extent of binding was observed under acidic conditions. The fusion of the CBM structures enhanced the hydrolytic efficiency of the xylanase against insoluble xylan, but decreased the stability of the enzyme. The optimum temperature and pH for the activity of the xylanase did not change.     

Photoproduction of hydrogen by <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> from thermophilic fermentation effluent

Rhodobacter capsulatus was used for the phototrophic hydrogen production on effluent solution derived from the thermophilic fermentation of Miscanthus hydrolysate by Thermotoga neapolitana. Pretreatments such as centrifugation, dilution, buffer addition, pH adjustment and sterilization were suggested for the effluent before being fed to the photofermentation. Batch-wise experiments showed that R. capsulatus grows and produces hydrogen on the pretreated effluent solution. Moreover, it was found that the hydrogen yield increased from 0.3 to 1.0 L/L_culture by addition of iron to the effluent solution.     

Improved Methods for Cultivation of the Extremely Thermophilic Bacterium Thermotoga neapolitana

Growth medium components and cultivation conditions for the extremely thermophilic bacterium Thermotoga neapolitana were optimized. A defined marine salts medium was formulated. Trace amounts of iron stimulated growth of T. neapolitana, while zinc inhibited growth at concentrations exceeding 11.1 μM. Other trace metals had no effect on its growth. Of the vitamins tested, only biotin was required for optimal growth. A defined mineral medium containing 5 g of carbohydrates per liter as the carbon source and 0.5 g of cysteine per liter as the sulfur source and reductant supported growth. Growth was stimulated by inclusion of vitamin-free Casamino Acids. Elemental sulfur, cystine, and dimethyl disulfide in the growth medium enhanced growth. Elemental sulfur and cystine relieved growth inhibition by hydrogen. T. neapolitana formed colonies in 2 days on plates of complex medium solidified with gellan gum and in 4 days on defined medium. The efficiency of plating was determined when growing cultures were sampled both aerobically and anaerobically and plated under aerobic and anaerobic conditions. Mean plating efficiencies were improved by sampling the growing cultures under strictly anaerobic conditions. Little or no improvement was obtained by inoculating plates inside an anaerobic chamber. Plating efficiencies of approximately 80% were obtained. Polycarbonate jars with aluminum lids withstood repeated incubation at 77°C without significant deterioration of the anaerobic seal and provided the most consistent results.     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant

 The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-β-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U⋅mg^-1, 5.5 and 102^°C, respectively. The endoxylanase was stable at 90^°C and retained 50% activity when incubated for 2 h at 100^°C. Received: 19 May 1995/Received revision: 31 July 1995/Accepted: 7 September 1995     

Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

The xylA gene from Thermotoga neapolitana 5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.     

Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

The hexameric regulatory protein ArgR formed by arginine-mediated dimerization of identical trimers governs the expression of genes required for arginine metabolism and some other genes in mesophilic and moderately thermophilic bacteria. We have cloned the argR gene from two hyperthermophilic bacteria of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neapolitana and T. maritima share a low degree of sequence similarity with other bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore, the argR gene may be autoregulated. The protein has extremely high intrinsic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15° C. The formation of oligomeric ArgR-DNA complexes is a function of protein concentration, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its own operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of arginine. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine-bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host. Received: 19 July 1999 / Accepted: 4 November 1999     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

Characterization and Regulation of Sulfur Reductase Activity in Thermotoga neapolitana

The growth of the hyperthermophilic, anaerobic bacterium Thermotoga neapolitana is stimulated by elemental sulfur by an unknown mechanism. We detected hydrogen-dependent sulfur reductase (sulfhydrogenase) and polysulfide dehydrogenase activities in cell extracts of this organism, demonstrating that it has at least two pathways for sulfidogenesis. Hydrogen-dependent sulfur reductase and hydrogenase activities are catalyzed by the purified hydrogenase of Thermotoga maritima, and this enzyme was called the sulfhydrogenase (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). Cells grown without elemental sulfur or cystine had 1.3 to 3.3 times higher sulfhydrogenase activities than those grown with either of these sources of sulfane sulfur. Hydrogenase activity was 2 to 5 times higher. Polysulfide dehydrogenase was up to 48-fold more active in cell extracts than the sulfhydrogenase. The activity of polysulfide dehydrogenase was approximately twofold higher when cells were grown in the presence of elemental sulfur. Its activity was oxygen labile in crude extracts, and it appears to be a cytoplasmic enzyme. Polysulfide was preferred over elemental sulfur as an electron acceptor (K_m = 0.15 mM) and was more active with NADH (K_m = 0.03 mM) than NADPH (K_m = 0.41 mM). Growth in the presence of elemental sulfur appeared to slightly increase the activity of polysulfide dehydrogenase and slightly decrease both activities of sulfhydrogenase (hydrogenase and polysulfide reductase), while growth without elemental sulfur had the opposite effects. The greater activity of polysulfide dehydrogenase and its apparent regulation indicate that it is the more physiologically important means of polysulfide reduction.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).