Differential requirement for RecFOR pathway components in Thermus thermophilus.

Recombinational repair is an important mechanism that allows DNA replication to overcome damaged templates, so the DNA is duplicated timely and correctly. The RecFOR pathway is one of the common ways to load RecA, while the RuvABC complex operates in the resolution of DNA intermediates. We have generated deletions of recO, recR and ruvB genes in Thermus thermophilus, while a recF null mutant could not be obtained. The recO deletion was in all cases accompanied by spontaneous loss of function mutations in addA or addB genes, which encode a helicase-exonuclease also key for recombination. The mutants were moderately affected in viability and chromosome segregation. When we generated these mutations in a Δppol/addAB strain, we observed that the transformation efficiency was maintained at the typical level of Δppol/addAB, which is 100-fold higher than that of the wild type. Most mutants showed increased filamentation phenotypes, especially ruvB, which also had DNA repair defects. These results suggest that in T. thermophilus (i) the components of the RecFOR pathway have differential roles, (ii) there is an epistatic relationship of the AddAB complex over the RecFOR pathway and (iii) that neither of the two pathways or their combination is strictly required for viability although they are necessary for normal DNA repair and chromosome segregation.

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Atom-Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultra-Sensitive Nucleic Acid Testing.

The DNA-guided (gDNA) Argonaute from Thermus thermophilus (TtAgo) has little potential for nucleic acid detection and gene editing due to its poor dsDNA cleavage activity at relatively low temperature. Herein, the dsDNA cleavage activity of TtAgo is enhanced by using 2'-fluorine (2'F)-modified gDNA and developes a novel nucleic acid testing strategy. This study finds that the gDNA with 2'F-nucleotides at the 3'-end (2'F-gDNA) can promote the assembly of the TtAgo-guide-target ternary complex significantly by increasing its intermolecular force to target DNA and TtAgo, thereby providing ≈40-fold activity enhancement and decreasing minimum reaction temperature from 65 to 60 °C. Based on this outstanding advance, a novel nucleic acid testing strategy is proposed, termed FAST, which is performed by using the 2'F-gDNA/TtAgo for target recognition and combining it with Bst DNA polymerase for nucleic acid amplification. By integrating G-quadruplex and Thioflavin T, the FAST assay achieves one-pot real-time fluorescence analysis with ultra-sensitivity, providing a limit of detection up to 5 copies (20 µL reaction mixture) for miR-21 detection. In summary, an atom-modification-based strategy has been developed for enhancing the cleavage activity of TtAgo efficiently, thereby improving its practicability and establishing a TtAgo-based nucleic acid testing technology with ultra-sensitivity and high-specificity.

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate.

Prolyl-tRNA synthetase (ProRS), belonging to the family of aminoacyl-tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon-like type (E-type) and the prokaryote-like type (P-type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E-type ProRS but selectively charges the P-type tRNAPro, featuring the bacterium-specific acceptor-stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro-A76, resembling the characteristics of the P-type ProRS. Furthermore, akin to the P-type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor-stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P-type ProRS, which relies on a strictly conserved R residue within the bacterium-like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non-conserved sequence, RTR, within the otherwise non-interacting eukaryote-like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate.

Manganese-dependent transcription regulation by MntR and PerR in Thermus thermophilus HB8.

Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA-binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA-binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies in Thermus thermophilus HB8. By homology search, we classify the DtxR homolog as a manganese-specific, MntR (TtMntR), and the FUR homolog as a peroxide-sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes in vivo, and TtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We show TtPerR and TtMntR bind DNA in the presence of manganese in vitro and in vivo; however, TtPerR is unable to bind DNA in the presence of iron, likely due to iron-mediated histidine oxidation. Unlike canonical PerR homologs, TtPerR does not appear to contribute to peroxide detoxification. Instead, the TtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation.

Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus.

Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.

Isolation and Characterization of Thermus thermophilus Strain ET-1: An Extremely Thermophilic Bacterium with Extracellular Thermostable Proteolytic Activity Isolated from El Tatio Geothermal Field, Antofagasta, Chile.

The present study describes the isolation of an extremely thermophilic bacterium from El Tatio, a geyser field in the high planes of Northern Chile. The thermophile bacterium named Thermus thermophilus strain ET-1 showed 99% identity with T. thermophilus SGO.5JP 17-16 (GenBank accession No. CP002777) by 16S rDNA gene analysis. Morphologically, the cells were non-sporeforming Gram-negative rods that formed colonies with yellow pigmentation. This strain is able to proliferate between 55 and 80 °C with a pH range of 6-10, presenting an optimum growth rate at 80 °C and pH 8. The bacterium produces an extracellular protease activity. Characterization of this activity in a concentrated enzyme preparation revealed that extracellular protease had an optimal enzymatic activity at 80 °C at pH 10, a high thermostability with a half-life at 80 °C of 10 h, indicating that this enzyme can be classified as an alkaline protease. The proteolytic enzyme exhibits great stability towards chelators, divalent ions, organic solvents, and detergents. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF), implying that it was a serine protease. The high thermal and pH stability and the resistance to chelators/detergents suggest that the protease activity from this T. thermophilus. strain could be of interest in biotechnological applications.

A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus.

BACKGROUND: Enzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited. RESULTS: In this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless ß-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol. CONCLUSIONS: Our results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

A new metabolic pathway for sym-homospermidine synthesis in an extreme thermophile, Thermus thermophilus.

In an extreme thermophile, Thermus thermophilus, sym-homospermidine is synthesized by the actions of two enzymes. The first enzyme coded by dhs gene (annotated to be deoxyhypusine synthase gene) catalyzes synthesis of an intermediate, supposed to be 1,9-bis(guanidino)-5-aza-nonane (=N1, N11-bis(amidino)-sym-homospermidine), from two molecules of agmatine in the presence of NAD. The second enzyme (aminopropylagmatinase) coded by speB gene catalyzes hydrolysis of the intermediate compound to sym-homospermidine releasing two molecules of urea.

Whole genome analyses for c-type cytochromes associated with respiratory chains in the extreme thermophile, Thermus thermophilus.

In thermophilic microorganisms, c-type cytochrome (cyt) proteins mainly function in the respiratory chain as electron carriers. Genome analyses at the beginning of this century revealed a variety of genes harboring the heme c motif. Here, we describe the results of surveying genes with the heme c motif, CxxCH, in a genome database comprising four strains of Thermus thermophilus, including strain HB8, and the confirmation of 19 c-type cytochromes among 27 selected genes. We analyzed the 19 genes, including the expression of four, by a bioinformatics approach to elucidate their individual attributes. One of the approaches included an analysis based on the secondary structure alignment pattern between the heme c motif and the 6th ligand. The predicted structures revealed many cyt c domains with fewer ß-strands, such as mitochondrial cyt c, in addition to the ß-strand unique to Thermus inserted in cyt c domains, as in T. thermophilus cyt c552 and caa3 cyt c oxidase subunit IIc. The surveyed thermophiles harbor potential proteins with a variety of cyt c folds. The gene analyses led to the development of an index for the classification of cyt c domains. Based on these results, we propose names for T. thermophilus genes harboring the cyt c fold.

Isolation and genomic analysis of a type IV pili-independent Thermus thermophilus phage, φMN1 from a Japanese hot spring.

A Thermus thermophilus lytic phage was isolated from a Japanese hot spring using a type IV pili-deficient strain as an indicator host, and designated as φMN1. Electron microscopic (EM) examination revealed that φMN1 had an icosahedral head and a contractile tail, suggesting that φMN1 belonged to Myoviridae. An EM analysis focused on φMN1 adsorption to the Thermus host cell showed that the receptor molecules for the phage were uniformly distributed on the outer surface of the cells. The circular double-stranded DNA of φMN1 was 76,659 base pairs in length, and the guanine and cytosine content was 61.8%. It was predicted to contain 99 open reading frames, and its putative distal tail fiber protein, which is essential for non-piliated host cell surface receptor recognition, was dissimilar in terms of sequence and length with its counterpart in the type IV pili-dependent φYS40. A phage proteomic tree revealed that φMN1 and φYS40 are in the same cluster, but many genes had low sequence similarities and some seemed to be derived from both mesophilic and thermophilic organisms. The gene organization suggested that φMN1 evolved from a non-Thermus phage through large-scale recombination events of the genes determining the host specificity, followed by gradual evolution by recombination of both the thermophilic and mesophilic DNAs assimilated by the host Thermus cells. This newly isolated phage will provide evolutionary insights into thermophilic phages.

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.

Ribosomal proteins can hold a more accurate record of bacterial thermal adaptation compared to rRNA.

Ribosomal genes are widely used as 'molecular clocks' to infer evolutionary relationships between species. However, their utility as 'molecular thermometers' for estimating optimal growth temperature of microorganisms remains uncertain. Previously, some estimations were made using the nucleotide composition of ribosomal RNA (rRNA), but the universal application of this approach was hindered by numerous outliers. In this study, we aimed to address this problem by identifying additional indicators of thermal adaptation within the sequences of ribosomal proteins. By comparing sequences from 2021 bacteria with known optimal growth temperature, we identified novel indicators among the metal-binding residues of ribosomal proteins. We found that these residues serve as conserved adaptive features for bacteria thriving above 40°C, but not at lower temperatures. Furthermore, the presence of these metal-binding residues exhibited a stronger correlation with the optimal growth temperature of bacteria compared to the commonly used correlation with the 16S rRNA GC content. And an even more accurate correlation was observed between the optimal growth temperature and the YVIWREL amino acid content within ribosomal proteins. Overall, our work suggests that ribosomal proteins contain a more accurate record of bacterial thermal adaptation compared to rRNA. This finding may simplify the analysis of unculturable and extinct species.

Protein-protein interaction-mediated regulation of lysine biosynthesis of Thermus thermophilus through the function-unknown protein LysV.

Thermus thermophilus biosynthesizes lysine via α-aminoadipate as an intermediate using the amino-group carrier protein, LysW, to transfer the attached α-aminoadipate and its derivatives to biosynthetic enzymes. A gene named lysV, which encodes a hypothetical protein similar to LysW, is present in the lysine biosynthetic gene cluster. Although the knockout of lysV did not affect lysine auxotrophy, lysV homologs are conserved in the lysine biosynthetic gene clusters of microorganisms belonging to the phylum Deinococcus-Thermus, suggesting a functional role for LysV in lysine biosynthesis. Pulldown assays and crosslinking experiments detected interactions between LysV and all of the biosynthetic enzymes requiring LysW for reactions, and the activities of most of all these enzymes were affected by LysV. These results suggest that LysV modulates the lysine biosynthesis through protein-protein interactions.

Thermus thermophilus polyploid cells directly imaged by X-ray laser diffraction.

Thermus thermophilus is reportedly polyploid and carries four to five identical genome copies per cell, based on molecular biological experiments. To directly detect polyploidy in this bacterium, we performed live cell imaging by X-ray free-electron laser (XFEL) diffraction and observed its internal structures. The use of femtosecond XFEL pulses enables snapshots of live, undamaged cells. For successful XFEL imaging, we developed a bacterial culture method using a starch- and casein-rich medium that produces a predominance of rod-shaped cells shorter than the focused XFEL beam size, which is slightly smaller than 2 µm. When cultured in the developed medium, the length of T. thermophilus cells, which is typically ~4 µm, was less than half its usual length. We placed living cells in a micro-liquid enclosure array and successively exposed each enclosure to a single XFEL pulse. A cell image was successfully obtained by the coherent diffractive imaging technique with iterative phase retrieval calculations. The reconstructed cell image revealed five peaks, which are most likely to be nucleoids, arranged in a row in the polyploid cell without gaps. This study demonstrates that XFELs offer a novel approach for visualizing the internal nanostructures of living, micrometer-sized, polyploid bacterial cells.

Non-coding RNAs and functional RNA elements in Thermus thermophilus.

To complete the ThermusQ database, small non-coding RNAs (ncRNAs) and functional RNA elements found in Thermus thermophilus were summarized with annotations. The well-known three ncRNAs, M1 RNA, tmRNA and SRP RNA, were annotated as ttj8_nc001 to ttj8_nc003, and 10 novel RNAs were annotated as ttj8_nc004 to ttj8_nc013. Antisense RNAs for some ORFs were annotated as ttj8_EST00001 to ttj8_EST00006. In addition, a set of conserved sequences found in T. thermophilus HB27 were also described.

Functional characterization of a hypothetical protein (TTHA1873) from Thermus thermophilus.

Thermus thermophilus is an extremely thermophilic organism that thrives at a temperature of 65°C. T. thermophilus genome has ~2218 genes, out of which 66% (1482 genes) have been annotated, and the remaining 34% (736 genes) are assigned as hypothetical proteins. In this work, biochemical and biophysical experiments were performed to characterize the hypothetical protein TTHA1873 from T. thermophilus. The hypothetical protein TTHA1873 acts as a nuclease, which indiscreetly cuts methylated and non-methylated DNA in divalent metal ions and relaxes the plasmid DNA in the presence of ATP. The chelation of metal ions with EDTA inhibits its activity. These results suggest that the hypothetical protein TTHA1873 would be a CRISPR-associated protein with non-specific DNase activity and ATP-dependent DNA-relaxing activity.

Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection.

Recent advances in prokaryotic Argonaute proteins (pAgos) as potential genome-editing tools have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, pAgos-based isothermal detection remains challenging. Here, we report a true isothermal amplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), to detect RNA with ultrasensitivity and single-nucleotide resolution at a constant temperature of 66 °C. We demonstrate the reliable detection of lncRNA, mRNA, and virus RNA with attomolar sensitivity and that TtAgoEAR can be applied to detect RNA targets in in cell lines, saliva, and tissues. We utilize this assay to distinguish pancreatic cancer cells carrying the mutation from wild-type cells with as little as 2 ng of RNA material. We also show that TtAgoEAR is easily adaptable to a lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable and easy RNA detection in point-of-care diagnosis and field analysis.

Putrescine Biosynthesis from Agmatine by Arginase (TtARG) in Thermus thermophilus.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.