[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

ThermusQ: Toward the cell simulation platform for Thermus thermophilus.

ThermusQ is a website (https://www.thermusq.net/) that aims to gather all the molecular information on Thermus thermophilus and to provide a platform to easily access the whole view of the bacterium. ThermusQ comprises the genome sequences of 22 strains from T. thermophilus and T. oshimai strains, plus the sequences of known Thermus phages. ThermusQ also contains information and map diagrams of pathways unique to Thermus strains. The website provides tools to retrieve sequence data in different ways. By gathering the whole data of T. thermophilus strains, the strainspecific characteristics was found. This bird's-eye view of the whole data will lead the research community to identify missing important data and the integration will provide a platform to conduct future biochemical simulations of the bacterium.

Incomplete denitrification phenotypes in diverse Thermus species from diverse geothermal spring sediments and adjacent soils in southwest China.

A few members of the bacterial genus Thermus have been shown to be incomplete denitrifiers, terminating with nitrite (NO2-) or nitrous oxide (N2O). However, the denitrification abilities of the genus as a whole remain poorly characterized. Here, we describe diverse denitrification phenotypes and genotypes of a collection of 24 strains representing ten species, all isolated from a variety of geothermal systems in China. Confirmed terminal products of nitrate reduction were nitrite or N2O, while nitric oxide (NO) was inferred as the terminal product in some strains. Most strains produced N2O; complete denitrification was not observed. Denitrification phenotypes were largely consistent with the presence of denitrification genes, and strains of the same species often had the same denitrification phenotypes and largely syntenous denitrification gene clusters. Genes for nirS and nirK coexisted in three Thermus brockianus and three Thermus oshimai genomes, which is a unique hallmark of some denitrifying Thermus strains and may be ecologically important. These results show that incomplete denitrification phenotypes are prominent, but variable, within and between Thermus species. The incomplete denitrification phenotypes described here suggest Thermus species may play important roles in consortial denitrification in high-temperature terrestrial biotopes where sufficient supply of oxidized inorganic nitrogen exists.

An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5' groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis ( TpsAgo) and analyzed the regulatory effect of 5'-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA targets at 65-90°C, which is mediated by a 5' hydroxyl or phosphate DNA guide. Notably, TpsAgo can utilize various 5'-modified DNA guides for catalysis, including 5'-NH 2C 6, 5'-Biotin, 5'-FAM and 5'-SHC 6 guides. Moreover, TpsAgo performs programmable cleavage of double-stranded DNA at temperatures over 80°C and strongly tolerates NaCl concentrations up to 3.2 M. These results provide insight into the catalytic performance of Agos by guide regulation, which may facilitate their biotechnological applications.

A Novel Trehalose Synthase for the Production of Trehalose and Trehalulose.

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.

Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile.

High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.

Broken symmetry between RNA enantiomers in a crystal lattice.

Explaining the origin of the homochirality of biological molecules requires a mechanism of disrupting the natural equilibrium between enantiomers and amplifying the initial imbalance to significant levels. Authors of existing models have sought an explanation in the parity-breaking weak nuclear force, in some selectively acting external factor, or in random fluctuations that subsequently became amplified by an autocatalytic process. We have obtained crystals in which l- and d-enantiomers of short RNA duplexes assemble in an asymmetric manner. These enantiomers make different lattice contacts and have different exposures to water and metal ions present in the crystal. Apparently, asymmetry between enantiomers can arise upon their mutual interactions and then propagate via crystallization. Asymmetric racemic compounds are worth considering as possible factors in symmetry breaking and enantioenrichment that took place in the early biosphere.

Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation.

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (MeAAs), is less efficient, especially when MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of MeAAs, achieving the incorporation of nine distinct MeAAs into both linear and thioether-macrocyclic peptide scaffolds.

A Novel Gene Cluster Is Involved in the Degradation of Lignin-Derived Monoaromatics in Thermus oshimai JL-2.

A novel gene cluster involved in the degradation of lignin-derived monoaromatics such as p-hydroxybenzoate, vanillate, and ferulate has been identified in the thermophilic nitrate reducer Thermus oshimai JL-2. Based on conserved domain analyses and metabolic pathway mapping, the cluster was classified into upper- and peripheral-pathway operons. The upper-pathway genes, responsible for the degradation of p-hydroxybenzoate and vanillate, are located on a 0.27-Mb plasmid, whereas the peripheral-pathway genes, responsible for the transformation of ferulate, are spread throughout the plasmid and the chromosome. In addition, a lower-pathway operon was also identified in the plasmid that corresponds to the meta-cleavage pathway of catechol. Spectrophotometric and gene induction data suggest that the upper and lower operons are induced by p-hydroxybenzoate, which the strain can degrade completely within 4 days of incubation, whereas the peripheral genes are expressed constitutively. The upper degradation pathway follows a less common route, proceeding via the decarboxylation of protocatechuate to form catechol, and involves a novel thermostable γ-carboxymuconolactone decarboxylase homolog, identified as protocatechuate decarboxylase based on gene deletion experiments. This gene cluster is conserved in only a few members of the Thermales and shows traces of vertical expansion of catabolic pathways in these organisms toward lignoaromatics.IMPORTANCE High-temperature steam treatment of lignocellulosic biomass during the extraction of cellulose and hemicellulose fractions leads to the release of a wide array of lignin-derived aromatics into the natural ecosystem, some of which can have detrimental effects on the environment. Not only will identifying organisms capable of using such aromatics aid in environmental cleanup, but thermostable enzymes, if characterized, can also be used for efficient lignin valorization. However, no thermophilic lignin degraders have been reported thus far. The present study reports T. oshimai JL-2 as a thermophilic bacterium with the potential to use lignin-derived aromatics. The identification of a novel thermostable protocatechuate decarboxylase gene in the strain further adds to its significance, as such an enzyme can be efficiently used in the biosynthesis of cis,cis-muconate, an important intermediate in the commercial production of plastics.

Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1.

Several native and engineered heat-stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72-74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3-5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half-life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high-temperature DNA polymerization.

MreB and MreC act as the geometric moderators of the cell wall synthetic machinery in Thermus thermophiles.

How cell morphology is maintained in thermophilic bacteria is unknown. In this study, the functions and mechanisms of the potential cell shape determinants (e.g. MreB, MreC, MreD and RodA homologues) of the model extremely thermophilic bacterium Thermus thermophilus were initially analyzed. Deletion of mreC, mreD or rodA only resulted in heterozygous mutants indicating that these genes are all essential. In the MreB-inhibited (by A22) strain and the heterozygous mreC, mreD or rodA mutant, cell morphologies were drastically changed, and enlarged spherical cells were eventually dead indicating that they are vital for cell shape maintenance. When fused to sGFP, MreB, MreC, MreD, RodA, and the enzymes involved in peptidoglycan synthesis (e.g. PBP2 and MurG) exhibited similar subcellular localization pattern, appearing as patches, or bands slightly angled to the cell length. The localizations and functions of all the 6 proteins required a natural peptidoglycan synthesis pattern, additionally those of MreD, RodA and MurG were dependent on MreB polymerization. Consistently, through comprehensive bacterial two-hybrid analyses, it was revealed that MreB could interact with itself, MreC, MreD, RodA and MurG, and MreC could associate with PBP2. In conclusion, in T. thermophilus, MreB, MreC, MreD, RodA and the peptidoglycan synthesis enzymes probably form a network of interactions centered with MreB and bridged with MreC, thereby maintaining cell morphology.

Quantification of purified endogenous miRNAs with high sensitivity and specificity.

MicroRNAs (miRNAs) are short (19-24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3'-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.

Distinct Expression of the Two NO-Forming Nitrite Reductases in Thermus antranikianii DSM 12462T Improved Environmental Adaptability.

Hot spring ecosystems are analogous to some thermal environments on the early Earth and represent ideal models to understand life forms and element cycling on the early Earth. Denitrification, an important component of biogeochemical nitrogen cycle, is highly active in hot springs. Nitrite (NO2-) reduction to nitric oxide (NO) is the significant and rate-limiting pathway in denitrification and is catalyzed by two types of nitrite reductases, encoded by nirS and nirK genes. NirS and NirK were originally considered incompatible in most denitrifying organisms, although a few strains have been reported to possess both genes. Herein, we report the functional division of nirS and nirK in Thermus, a thermophilic genus widespread in thermal ecosystems. Transcriptional levels of nirS and nirK coexisting in Thermus antranikianii DSM 12462T were measured to assess the effects of nitrite, oxygen, and stimulation time. Thirty-nine Thermus strains were used to analyze the phylogeny and distribution of nirS and nirK; six representative strains were used to assess the denitrification phenotype. The results showed that both genes were actively transcribed and expressed independently in T. antranikianii DSM 12462T. Strains with both nirS and nirK had a wider range of nitrite adaptation and revealed nir-related physiological adaptations in Thermus: nirK facilitated adaptation to rapid changes and extended the adaptation range of nitrite under oxygen-limited conditions, while nirS expression was higher under oxic and relatively stable conditions.

Chaperone client proteins evolve slower than non-client proteins.

Chaperones are important molecular machinery that assists proteins to attain their native three-dimensional structure crucial for function. Earlier studies using experimental evolution showed that chaperones impose a relaxation of sequence constraints on their "client" proteins, which may lead to the fixation of slightly deleterious mutations on the latter. However, we hypothesized that such a phenomenon might be harmful to the organism in a natural physiological condition. In this study, we investigated the evolutionary rates of chaperone client and non-client proteins in five model organisms from both prokaryotic and eukaryotic lineages. Our study reveals a slower evolutionary rate of chaperone client proteins in all five organisms. Additionally, the slower folding rate and lower aggregation propensity of chaperone client proteins reveal that the chaperone may play an essential role in rescuing the slightly disadvantageous effects due to random mutations and subsequent protein misfolding. However, the fixation of such mutations is less likely to be selected in the natural population.

Direct observation of helicase-topoisomerase coupling within reverse gyrase.

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.

Engineering novel S-glycosidase activity into extremo-adapted ß-glucosidase by rational design.

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted ß-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards ß-thioglycoside was observed in the single mutants TnoGH1-V287R (2500 M-1 s-1) and HorGH1-M229R (13,260 M-1 s-1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce ß-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of ß-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted ß-glycosidases. •ß-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.

Amylomaltase from Thermus filiformis: expression in Saccharomyces cerevisiae and its use in starch modification.

AIM: To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. METHODS AND RESULTS: TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5 U TfAM g-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. CONCLUSIONS: TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

Methods to Identify and Analyze Vesicle-Protected DNA Transfer.

Conjugation, transformation, and transduction constitute the three classical mechanisms involved in horizontal gene transfer (HGT) among prokaryotes. In addition, alternative HGT mechanisms exist in groups of organisms. Among them, the use of DNA-containing membrane vesicles as shuttle elements for HGT has been described for a number of microorganisms, including both thermophiles and mesophiles. Here we describe the methods followed to detect, purify, and analyze these vesicles.

Production and characterization of a recombinant thermophilic trehalose synthase from Thermus antranikianii.

Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co2+, Cu2+, Zn2+, sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (kcat/Km) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported.

Thermostable Amylosucrase from Calidithermus timidus DSM 17022: Insight into Its Characteristics and Tetrameric Conformation.

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.