Extreme Thermophilic Bacterium Research Articles

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Molecular Characterization of the Iron-Containing Alcohol Dehydrogenase from the Extremely Thermophilic Bacterium Pseudothermotoga hypogea.

Pseudothermotoga hypogea is an extremely thermophilic bacterium capable of growing at 90 °C and producing ethanol, which is catalyzed by an alcohol dehydrogenase (ADH). The gene encoding P. hypogea ADH (PhADH) was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained by sequencing all fragments of the gene, which were amplified from the genomic DNA. The deduced amino acid sequence showed high identity to iron-containing ADHs from other Thermotoga species and harbored typical iron- and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. Structural modeling showed that the N-terminal domain of PhADH contains an α/β-dinucleotide-binding motif and that its C-terminal domain is an α-helix-rich region containing the iron-binding motif. The recombinant PhADH was soluble, active, and thermostable, with a subunit size of 43 ± 1 kDa revealed by SDS-PAGE analyses. The recombinant PhADH (69 ± 2 U/mg) was shown to have similar properties to the native enzyme. The optimal pH values for alcohol oxidation and aldehyde reduction were 11.0 and 8.0, respectively. It was also thermostable, with a half-life of 5 h at 70 °C. The successful expression of the recombinant PhADH in E. coli significantly enhanced the yield of enzyme production and thus will facilitate further investigation of the catalytic mechanisms of iron-containing ADHs.

A 3.3 Å‐Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability

AbstractRespiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c1 was found to greatly enhance the interaction between cyt. b and cyt. c1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

A 3.3 Å-Resolution Structure of Hyperthermophilic Respiratory Complex III Reveals the Mechanism of Its Thermal Stability.

Respiratory chain complexes convert energy by coupling electron flow to transmembrane proton translocation. Owing to a lack of atomic structures of cytochrome bc 1 complex (Complex III) from thermophilic bacteria, little is known about the adaptations of this macromolecular machine to hyperthermophilic environments. In this study, we purified the cytochrome bc1 complex of Aquifex aeolicus, one of the most extreme thermophilic bacteria known, and determined its structure with and without an inhibitor at 3.3 Å resolution. Several residues unique for thermophilic bacteria were detected that provide additional stabilization for the structure. An extra transmembrane helix at the N‐terminus of cyt. c 1 was found to greatly enhance the interaction between cyt. b and cyt. c 1, and to bind a phospholipid molecule to stabilize the complex in the membrane. These results provide the structural basis for the hyperstability of the cytochrome bc1 complex in an extreme thermal environment.

Regulatory Factors for tRNA Modifications in Extreme- Thermophilic Bacterium Thermus thermophilus.

Thermus thermophilus is an extreme-thermophilic bacterium that can grow at a wide range of temperatures (50–83°C). To enable T. thermophilus to grow at high temperatures, several biomolecules including tRNA and tRNA modification enzymes show extreme heat-resistance. Therefore, the modified nucleosides in tRNA from T. thermophilus have been studied mainly from the view point of tRNA stabilization at high temperatures. Such studies have shown that several modifications stabilize the structure of tRNA and are essential for survival of the organism at high temperatures. Together with tRNA modification enzymes, the modified nucleosides form a network that regulates the extent of different tRNA modifications at various temperatures. In this review, I describe this network, as well as the tRNA recognition mechanism of individual tRNA modification enzymes. Furthermore, I summarize the roles of other tRNA stabilization factors such as polyamines and metal ions.

Integral caa3-Cytochrome c Oxidase from Thermus thermophilus: Purification and Crystallization.

Cytochrome c oxidase is a respiratory enzyme catalyzing the energy-conserving reduction of molecular oxygen to water-a fundamental biological process of cell respiration. The first crystal structures of the type A cytochrome c oxidases, bovine heart and Paracoccus denitrificans cytochrome c oxidases, were published in 1995 and contributed immensely to the understanding of the enzyme's mechanism of action. The senior author's research focus was directed toward understanding the structure and function of the type B cytochrome c oxidases, ba3-oxidase and type A2 caa3-oxidase, both from the extreme thermophilic bacterium Thermus thermophilus. While the ba3-oxidase structure was published in 2000 and functional characterization is well-documented in the literature, we recently successfully solved the structure of the caa3-nature made enzyme-substrate complex. This chapter is dedicated to the purification and crystallization process of caa3-cytochrome c oxidase.

Reconstitution of Functionally Active Thermus thermophilus 30S Ribosomal Subunit from Ribosomal 16S RNA and Ribosomal Proteins.

In vitro reconstitution systems of ribosomal subunits from free ribosomal RNA and ribosomal proteins are helpful tool for studies on the structure, function and assembly of ribosome. Using this system mutant or modified ribosomal proteins or ribosomal RNA can be incorporated into ribosomal subunits for studying ribosome structure and function. Developing the protocol for reconstitution of 30S subunits from an extreme thermophilic bacterium Thermus thermophilus can be beneficial especially for structural studies, as proteins and nucleic acids from this organism are very stable and crystallize easier than those from mesophilic organisms.

Fast and reliable production, purification and characterization of heat-stable, bifunctional enzyme chimeras

Degradation of complex plant biomass demands a fine-regulated portfolio of glycoside hydrolases. The LE (LguI/Eco81I)-cloning approach was used to produce two enzyme chimeras CB and BC composed of an endoglucanase Cel5A (C) from the extreme thermophilic bacterium Fervidobacterium gondwanense and an archaeal β-glucosidase Bgl1 (B) derived from a hydrothermal spring metagenome. Recombinant chimeras and parental enzymes were produced in Escherichia coli and purified using a two-step affinity chromatography approach. Enzymatic properties revealed that both chimeras closely resemble the parental enzymes and physical mixtures, but Cel5A displayed lower temperature tolerance at 100°C when fused to Bgl1 independent of the conformational order. Moreover, the determination of enzymatic performances resulted in the detection of additive effects in case of BC fusion chimera. Kinetic measurements in combination with HPLC-mediated product analyses and site-directed mutation constructs indicated that Cel5A was strongly impaired when fused at the N-terminus, while activity was reduced to a slighter extend as C-terminal fusion partner. In contrast to these results, catalytic activity of Bgl1 at the N-terminus was improved 1.2-fold, effectively counteracting the slightly reduced activity of Cel5A by converting cellobiose into glucose. In addition, cellobiose exhibited inhibitory effects on Cel5A, resulting in a higher yield of cellobiose and glucose by application of an enzyme mixture (53.1%) compared to cellobiose produced from endoglucanase alone (10.9%). However, the overall release of cellobiose and glucose was even increased by catalytic action of BC (59.2%). These results indicate possible advantages of easily produced bifunctional fusion enzymes for the improved conversion of complex polysaccharide plant materials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0122-7) contains supplementary material, which is available to authorized users.

Biochemical characterization of a novel thermostable GH11 xylanase with CBM6 domain from Caldicellulosiruptor kronotskyensis

The extreme thermophilic bacterium Caldicellulosiruptor kronotskyensis can use hemicelluloses and cellulose as carbohydrate source. The gene Calkro_0081 encoded a novel GH11 xylanase (Xyn11A) with a catalytic domain (GH-CD) and a carbohydrate binding module (CBM6). The native Xyn11A and two corresponded truncations Xyn11A-CD (catalytic domain of Xyn11A) and Xyn11A-CBM (carbohydrate binding module of Xyn11A) were respectively cloned, heterologously expressed, and biochemically characterized. The recombinant Xyn11A is active in a wide temperature range from 40 to 95 degrees C with the highest activity at 75 degrees C. Xyn11A was completely stable at 70 degrees C for 6 h and more than 50% activity was retained after incubation for 6 h at 75 degrees C. The optimum pH of Xyn11A was 6.0, and it retained 100% activity after 15 h incubation in pH 5.5-7.5 at 70 degrees C. As for Xyn11A-CD, the optimal pH value and temperature were 6.0 and 65 degrees C, the residual activity retained 30% after 6h at 60 degrees C. The respective specific activities of Xyn11A, Xyn11A-CD, and Xyn11A-CBM were 1752.0, 986.8, and 0 IU/mg on beechwood xylan (BWX) at optimum conditions. The activity of Xyn11A is the highest among the reported thermostable xylanases at 75 C. Using BWX as substrate, the final products were xylose and xylobiose after hydrolysis with Xyn11A or Xyn11A-CD. No hydrolytic activity of CBM6 was found, while which shows crucial functions on thermostability and activity for Xyn11A. The characteristics of thermostability and high activity make Xyn11A a potential enzyme for industry application. (C) 2014 Elsevier B.V. All rights reserved.

Characterization of a Thermostable Xylanase from the Extremely Thermophilic Bacterium Thermotoga hypogea

Thermotoga hypogea is an extremely thermophilic bacterium that can utilize both peptides and carbohydrates including xylan as growth substrates at 90 ºC. It was found that its xylanase activity was increased up to 9-fold in the presence of either xylan or xylose in growth media, which was independent of growth phases. More than 95% of xylanase activities were associated with cells of T. hypogea. Ultracentrifugation of the cell-free extract of the organism showed that most xylanase activities were in the soluble fraction. A xylanase was purified from the cell-free extract to homogeneity using an FPLC system. The purified enzyme had a molecular mass of 38 kDa as revealed by SDS-PAGE. Its native molecular mass was determined using gel-filtration to be 29 kDa, indicating it was a monomeric enzyme. The purified xylanase was stable with a half-life of 270 min at 90°C in the presence of xylan and BSA. Its optimal pH and temperature were determined to be 5.5 and 90°C, respectively. An apparent Km-value was determined to be 0.07% (w/v) of xylan. Hydrolytic products from xylan catalyzed by the purified enzyme were xylobiose and xylooligosaccharides (n>2). No cellulase activity was detected for the purified xylanase.

Characterization of a Thermostable Xylanase from the Extremely Thermophilic Bacterium Thermotoga hypogea

Characterization of a Thermostable Xylanase from the Extremely Thermophilic Bacterium Thermotoga hypogea

Effect of medium composition on biohydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus

Up to now, the analysis of the effects of medium composition on biohydrogen production of Caldicellulosiruptor saccharolyticus was focused mainly on salt concentrations and complex compounds. Within this work we studied the effects of the presence of organic and/or inorganic nitrogen in the medium composition aiming to induce metabolic changes in C. saccharolyticus to improve its hydrogen evolution rate (HER) and hydrogen specific productivity (qH2). Biohydrogen productivities and hydrogen to substrate yield (Y(H2/s)) of C. saccharolyticus on xylose in batch mode were higher working in a complex medium than in a defined one; but no significant difference could be settled according to hydrogen to carbon dioxide yields (Y(H2/CO2)). The specific growth rate of C. saccharolyticus on complex medium was settled at 0.1 h−1 operating in chemostat mode to achieve the highest H2-productivities under stable conditions. In chemostat mode on xylose, a reduction of the ammonium feed concentration in a defined medium until N-limiting conditions involved higher qH2 comparing with a straight C-limiting growth.In this contribution we present a methodology for quantitative process characterization and development based on physiological parameters, such as specific rates and yields.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Consolidated bioprocessing of untreated switchgrass to hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus DSM 8903

The abilities of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903 to ferment switchgrass (SWG), microcrystalline cellulose (MCC) and glucose to hydrogen (H2) in one-step were examined. Hydrogen production from glucose reached the theoretical maximum for dark fermentation of 4 mol H2/mol glucose. The H2 yield on MCC and SWG after 6 days of fermentation was 23.2 mmol H2/L or 9.4 mmol H2/g MCC and 14.3 mmol H2/L or 11.2 mmol H2/g SWG, respectively. The rate of H2 formation however was higher on MCC (0.7 mmol/Lh) than SWG (0.1 mmol/Lh). C. saccharolyticus DSM 8903 was able to produce H2 directly from mechanically-comminuted SWG without any physicochemical or biological pretreatment. Combining four processing steps (pretreatment, enzyme production, saccharification and fermentation) into a single biorefinery operation makes C. saccharolyticus DSM 8903 a promising candidate for consolidated bioprocessing (CBP) of lignocellulosic biomass.

Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T(1/2) of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.

Biodiesel and biohydrogen production from cotton-seed cake in a biorefinery concept

Biodiesel production from cotton-seed cake (CSC) and the pretreatment of the remaining biomass for dark fermentative hydrogen production was investigated. The direct conversion to biodiesel with alkali free fatty acids neutralization pretreatment and alkali transesterification resulted in a biodiesel with high esters content and physicochemical properties fulfilling the EN-standards. Blends of cotton-seed oil methyl esters (CME) and diesel showed an improvement in lubricity and cetane number. Moreover, CME showed good compatibility with commercial biodiesel additives. On the basis of conversion of the remaining CSC to sugars fermentable towards hydrogen, the optimal conditions included removal of the oil of CSC and pretreatment at 10% NaOH (w/w dry matter). The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production, 84–112% of the control, from NaOH-pretreated CSC and low hydrogen production, 15–20% of the control, from the oil-rich and not chemically pretreated CSC, and from Ca(OH)2-pretreated CSC.

Integration of first and second generation biofuels: Fermentative hydrogen production from wheat grain and straw

Integrating of lignocellulose-based and starch-rich biomass-based hydrogen production was investigated by mixing wheat straw hydrolysate with a wheat grain hydrolysate for improved fermentation. Enzymatic pretreatment and hydrolysis of wheat grains led to a hydrolysate with a sugar concentration of 93.4g/L, while dilute-acid pretreatment and enzymatic hydrolysis of wheat straw led to a hydrolysate with sugar concentration 23.0g/L. Wheat grain hydrolysate was not suitable for hydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus at glucose concentrations of 10g/L or higher, and wheat straw hydrolysate showed good fermentability at total sugar concentrations of up to 10g/L. The mixed hydrolysates showed good fermentability at the highest tested sugar concentration of 20g/L, with a hydrogen production of 82–97% of that of the control with pure sugars. Mixing wheat grain hydrolysate with wheat straw hydrolysate would be beneficial for fermentative hydrogen production in a biorefinery.

Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii

Caldicellulosiruptor bescii is a cellulolytic/hemicellulolytic anaerobe, which extracellularly secretes various proteins, including multidomain cellulases with two-catalytic domains, for plant biomass degradation. Degradation by C.bescii cells has been well characterized, but degradation by the cell-free extracellular cellulase/hemicellulase system (CEC) of C.bescii has not been as well studied. In the present study, C.bescii CEC was prepared from cell-free culture supernatant, and the degradation properties for defined substrates and non-pretreated plant biomass were characterized. Four multidomain cellulases (Cbes_1857, Cbes_1859, Cbes_1865, and Cbes_1867), composed of the glycoside hydrolase families 5, 9, 10, 44, and 48, were the major enzymes identified in the CEC by mass spectrometry. The CEC degraded xylan, mannose-based substrates, β-1,4-linked glucans, including microcrystalline cellulose (Avicel), and non-pretreated timothy grass and rice straw. However, degradation of chitin, pectin, dextran, and wheat starch was not observed. The optimum temperatures for degradation activities were 75°C for timothy grass and Avicel, 85°C for carboxylmethyl cellulose, and >85°C for xylan. The optimum pH for these substrates was 5-6. The degradation activities were compared with a CEC derived from the fungus Trichoderma reesei, the most common enzyme used for plant biomass saccharification. The amounts of degraded Avicel, timothy grass, and rice straw by C.bescii CEC were 2.2-2.4-fold larger than those of T.reesei CEC. The high hydrolytic activity of C.bescii CEC might be attributed to the two-catalytic domain architecture of the cellulases.

Dilute-acid pretreatment of barley straw for biological hydrogen production using Caldicellulosiruptor saccharolyticus

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H3PO4 and H2SO4, and to a lesser extent, HNO3. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.

Growth and hydrogen production characteristics of Caldicellulosiruptor saccharolyticus on chemically defined minimal media

Caldicellulosiruptor saccharolyticus is an extreme thermophilic bacterium recognized for its saccharolytic ability and superior ability to produce high yields of hydrogen. However, most studies have been made using yeast extract (YE) as a rich but expensive nutrient source. For the first time, we show that C. saccharolyticus is able to grow on defined minimal media, including essential vitamins, provided that CO2 was allowed to accumulate sufficiently in the culture broth to activate growth. Growth and hydrogen production performance on minimal media was analyzed in both batch and continuous mode. Absence of YE resulted in similar or higher hydrogen yields and specific hydrogen productivities but lower volumetric hydrogen productivities than with YE. The results also indicate that YE is used as a carbon- and energy source thus affecting metabolic flux calculations. This study clarified that YE is not essential making C. saccharolyticus more attractive for fundamental studies on its metabolism and future industrial exploitation.