An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Transcriptomic profiling and its implications for the H2 production of a non-methanogen deficient in the frhAGB-encoding hydrogenase.

The F420-reducing hydrogenase of methanogens functions in methanogenesis by providing reduced coenzyme F420 (F420H2) as an electron donor. In non-methanogens, however, their physiological function has not been identified yet. In this study, we constructed an ΔfrhA mutant, whose frhA gene encoding the hydrogenase α subunit was deleted, in the non-methanogenic Thermococcus onnurineus NA1 as a model organism. There was no significant difference in the formate-dependent growth between the mutant and the wild-type strains. Interestingly, the mutation in the frhA gene affected the expression of genes involved in various cellular functions such as H2 oxidation, chemotactic signal transduction, and carbon monoxide (CO) metabolism. Among these genes, the CO oxidation gene cluster, enabling CO-dependent growth and H2 production, showed a 2.8- to 7.0-fold upregulation by microarray-based whole transcriptome expression profiling. The levels of proteins produced by this gene cluster were also significantly increased not only under the formate condition but also under the CO condition. In a controlled bioreactor, where 100% CO was continuously fed, the ΔfrhA mutant exhibited significant increases in cell growth (2.8-fold) and H2 production (3.4-fold). These findings strongly imply that this hydrogenase is functional in non-methanogens and is related to various cellular metabolic processes through an unidentified mechanism. An understanding of the mechanism by which the frhA gene deletion affected the expression of other genes will provide insights that can be applied to the development of strategies for the enhancement of H2 production using CO as a substrate.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Visualization of an evaporating thin layer during the evaporation of a nanofluid droplet.

During the evaporation of a droplet, there exists an evaporating thin layer that is difficult to visualize because of optical restrictions. The present study visualized this thin layer by using a reflective-mode, confocal microscope that can provide improved signal-to-noise focal plane imaging over traditional optical microscopy while simultaneously serving as an interferometer when imaging thin liquid films. The spatial distribution of the evaporating thin layer thickness was determined from interferometric fringe analysis. Three distinct fringe patterns, or regions, were observed depending on the nanoparticle concentration. These regions are referred to as uniform, slow extension, and rapid extension. The formation of the three regions is closely associated with the variation of the evaporating thin layer thickness of a nanofluid droplet. The nanoparticle bank formed near the contact line region substantially affects the rate of change in the evaporating thin layer thickness that increases with the nanoparticle concentration.

Characterization of the frhAGB-encoding hydrogenase from a non-methanogenic hyperthermophilic archaeon.

The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and ß subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.

Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon.

A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 from carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, P TN0510 , was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase, and a Na(+)/H(+) antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of the wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of the wild-type strain. The P TN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and Western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine novel strong promoters of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable products such as enzymes and metabolites through metabolic engineering.

Development of a new contactless dielectrophoresis system for active particle manipulation using movable liquid electrodes.

This study presents a new DEP manipulation technique using a movable liquid electrode, which allows manipulation of particles by actively controlling the locations of electrodes and applying on-off electric input signals. This DEP system consists of mercury as a movable liquid electrode, indium tin oxide (ITO)-coated glass, SU-8-based microchannels for electrode passages, and a PDMS medium chamber. A simple squeezing method was introduced to build a thin PDMS layer at the bottom of the medium chamber to create a contactless DEP system. To determine the operating conditions, the DEP force and the friction force were analytically compared for a single cell. In addition, an appropriate frequency range for effective DEP manipulation was chosen based on an estimation of the Clausius-Mossotti factor and the effective complex permittivity of the yeast cell using the concentric shell model. With this system, we demonstrated the active manipulation of yeast cells, and measured the collection efficiency and the dielectrophoretic velocity of cells for different AC electric field strengths and applied frequencies. The experimental results showed that the maximum collection efficiency reached was approximately 90%, and the dielectrophoretic velocity increased with increasing frequency and attained the maximum value of 10.85 ± 0.95 µm/s at 100 kHz, above which it decreased.

Comparison of CO-dependent H2 production with strong promoters in Thermococcus onnurineus NA1.

To overproduce biotechnologically valuable products, the expression level of target genes has been modulated by using strong promoters. In a hyperthermophilic archaeon Thermococcus onnurineus NA1, two promoters, P(TN0413) and P(TN0157), which drive expression of the genes encoding the S-layer protein and glutamate dehydrogenase were inserted in front of a gene cluster encoding a carbon monoxide dehydrogenase, a hydrogenase and a Na⁺/H⁺ antiporter. Two promoters exhibited strong activity by increasing the transcription and translation levels of the gene cluster in the mutant strains by 2.5- to 49-folds and 1.4- to 3.3-folds, respectively, than the native promoter in the wild-type strain. While KS0413 with P(TN0413) promoter exhibited 2.7 to 4.7 times higher transcript level than KS0157 with P(TN0157) promoter, the levels of proteins were a little different between them. The biomass concentrations and H2 production rates of two mutants were 2- to 3-fold higher than those of the wild-type strain in a bioreactor where CO was supplied at a flow rate of 120 ml min⁻¹. Two mutants showed differential response to the higher CO flow rate, 240 ml min⁻¹, in terms of growth pattern and product formation, indicating two promoters were regulated by culture conditions. The results demonstrate that not only promoter strength but also product-forming conditions should be considered in promoter engineering.

CO-dependent H2 production by genetically engineered Thermococcus onnurineus NA1.

Hydrogenogenic CO oxidation (CO + H(2)O → CO(2) + H(2)) has the potential for H(2) production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H(2), has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H(2), the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na(+)/H(+) antiporter and a 1.8-fold-higher specific activity for CO-dependent H(2) production than did the wild-type strain. The H(2) production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H(2) production rate of the engineered strain was severalfold higher than those of any other CO-dependent H(2)-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H(2) production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

Genome sequence of an oligohaline hyperthermophilic archaeon, Thermococcus zilligii AN1, isolated from a terrestrial geothermal freshwater spring.

Thermococcus zilligii, a thermophilic anaerobe in freshwater, is useful for physiological research and biotechnological applications. Here we report the high-quality draft genome sequence of T. zilligii AN1(T). The genome contains a number of genes for an immune system and adaptation to a microbial biomass-rich environment as well as hydrogenase genes.

Modeling effective thermal conductivity enhanced by surface waves using the Boltzmann transport equation.

The thermal management of semiconductors at the device level has become a crucial issue owing to the high integration density and miniaturization of microelectronic systems. Because surface phonon polaritons (SPhPs) exhibit long propagation lengths, they are expected to contribute significantly to the heat dissipation in microelectronic systems. This study aims to numerically estimate the heat transfer due to SPhPs in a thin SiO2 film. The one-dimensional Boltzmann transport equation (BTE) is solved using the estimated propagation length based on the SPhP dispersion curves. The temperature profiles and heat fluxes are predicted and demonstrate the size effect of the film on the effective in-plane thermal conductivity of the SiO2 film. The results indicate that the temperature distribution was constant regardless of the film length and thickness because the propagation length was much longer than the film length. In addition, the heat flux increased with decreasing film thickness owing to the depth-averaged energy transfer. The effective thermal conductivities predicted using the BTE differed by ~ 16.5% from the values obtained from the analytical expression. The numerical results of this study can provide valuable data when studying the thermal behavior of SPhPs.

A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP+ as electron acceptors, respectively. While most FDHs exhibited NAD+-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (k cat /K m) of Fdh3 for NADP+ was measured to be 5,281 mM-1 s-1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 of Fdh3B may contribute to the stabilization of the 2'-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO2 reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.

Dynamic wetting and spreading characteristics of a liquid droplet impinging on hydrophobic textured surfaces.

We report on the wetting dynamics of a 4.3 µL deionized (DI) water droplet impinging on microtextured aluminum (Al 6061) surfaces, including microhole arrays (hole diameter 125 µm and hole depth 125 µm) fabricated using a conventional microcomputer numerically controlled (µ-CNC) milling machine. This study examines the influence of the texture area fraction ϕ(s) and drop impact velocity on the spreading characteristics from the measurement of the apparent equilibrium contact angle, dynamic contact angle, and maximum spreading diameter. We found that for textured surfaces the measured apparent contact angle (CA) takes on values of up to 125.83°, compared to a CA of approximately 80.59° for a nontextured bare surface, and that the spreading factor decreases with the increased texture area fraction because of increased hydrophobicity, partial penetration of the liquid, and viscous dissipation. In particular, on the basis of the model of Ukiwe and Kwok (Ukiwe, C.; Kwok, D. Y. Langmuir 2005, 21, 666), we suggest a modified equation for predicting the maximum spreading factor by considering various texturing effects and wetting states. Compared with predictions by using earlier published models, the present model shows better agreement with experimental measurements of the maximum spreading factor.

Wettability changes of TiO2 nanotube surfaces.

This study examines the effect of environmental and experimental conditions, such as temperature and time, on the wettability properties of titania nanotube (TNT) surfaces fabricated by anodization. The fabricated TNTs are 60-130 nm inner diameter and 7-10 µm height. One-microliter water droplets were used to define the wettability of the TNT surfaces by measuring the contact angles. A digital image analysis algorithm was developed to obtain contact angles, contact radii and center heights of the droplets on the TNT surfaces. Bare titanium foil is inherently less hydrophilic with approximately 60°-80° contact angle. The as-anodized TNT surfaces are more hydrophilic and annealing further increases this hydrophilic property. Furthermore, it was found that the TNT surface became more hydrophobic when aged in air over a period of three months. It is believed that the surface wettability can be changed due to alkane contamination and organic contaminants in an ambient atmosphere. This work can provide guidelines to better specify the environmental conditions that changes surface properties of TNT surfaces and therefore affect their desirable function in specific applications such as orthopedic implants.

Biological process for coproduction of hydrogen and thermophilic enzymes during CO fermentation.

To develop a thermophilic cell factory system that uses CO gas, we attempted to engineer a hyperthermophilic carboxydotrophic hydrogenic archaeon Thermococcus onnurineus NA1 to be capable of producing thermophilic enzymes along with hydrogen (H2). The mutant strains 156T-AM and 156T-POL were constructed to have another copy of a gene encoding α-amylase or DNA polymerase, respectively, and exhibited growth rates and H2 production rates distinct from those of the parental strain, 156T, in gas fermentation using 100% CO or coal-gasified syngas. Purified α-amylase displayed starch-hydrolyzing activity, and whole-cell extracts of 156T-AM showed saccharifying activity for potato peel waste. PCR amplification was used to demonstrate that purified DNA polymerase was free from bacterial DNA contamination, in contrast to commercial bacteria-made enzymes. This study demonstrated that this archaeal strain could coproduce enzymes and H2 using CO-containing gas, providing a basis for cell factories to upcycle industrial waste gas.

Biohydrogen production of obligate anaerobic archaeon Thermococcus onnurineus NA1 under oxic conditions via overexpression of frhAGB-encoding hydrogenase genes.

BACKGROUND: The production of biohydrogen (H2) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H2 formation is more thermodynamically feasible at elevated temperatures and fewer undesired side products are produced. However, these microbes require anoxic culture conditions for growth and H2 production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O2). Therefore, the development of an O2-tolerant strain would be useful for industrial applications. RESULTS: In this study, we found that the overexpression of frhAGB-encoding hydrogenase genes in Thermococcus onnurineus NA1, an obligate anaerobic archaeon and robust H2 producer, enhanced O2 tolerance. When the recombinant FO strain was exposed to levels of O2 up to 20% in the headspace of a sealed bottle, it showed significant growth. Whole transcriptome analysis of the FO strain revealed that several genes involved in the stress response such as chaperonin ß subunit, universal stress protein, peroxiredoxin, and alkyl hydroperoxide reductase subunit C, were significantly up-regulated. The O2 tolerance of the FO strain enabled it to grow on formate and produce H2 under oxic conditions, where prior O2-removing steps were omitted, such as the addition of reducing agent Na2S, autoclaving, and inert gas purging. CONCLUSIONS: Via the overexpression of frhAGB genes, the obligate anaerobic archaeon T. onnurineus NA1 gained the ability to overcome the inhibitory effect of O2. This O2-tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H2 production.

Oxygen-mediated growth enhancement of an obligate anaerobic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

Mechanical model of an arched basilar membrane in the gerbil cochlea.

The frequency selectivity of a gerbil cochlea, unlike other mammals, does not depend on varying thickness and width of its basilar membrane from the basal to the apical end. We model the gerbil arched basilar membrane focusing on the radial tension, embedded fiber thickness, and the membrane arch, which replace the functionality of the variation in thickness and width. The model is verified with the previous gerbil cochlea model which estimated the equivalent basilar membrane thickness and is shown to be more accurate than the flat sandwiched basilar membrane model. The simple sinusoidal-shaped bending mode assumption in previous models is found to be valid in the present model with <12% error. Parametric study on the present model shows that fiber thickness contribution to the membrane stiffness is close to the 3rd order, higher than the 1st order estimation of previous models. We found that the effective Young's modulus of the fiber bundle is at least 6 orders higher than the shear modulus of the soft-cells and the membrane radial bending stiffness is more sensitive to the membrane arch and the shear modulus of the soft-cells near the apical end.

Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate.

Previously, we reported that the hyperthermophilic archaeon Thermococcus onnurineus NA1 could grow on formate and produce H2. Formate conversion to hydrogen was mediated by a formate-hydrogen lyase complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecular changes. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genome sequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found among the genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formate transporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage, carried out by culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning.

Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.