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.

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.

Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27.

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a C-terminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.

Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to 𝑃X  = 0.4 g L-1 h. The µð‘šð‘Žð‘¥ corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Searching for Frataxin Function: Exploring the Analogy with Nqo15, the Frataxin-like Protein of Respiratory Complex I from Thermus thermophilus.

Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.

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.

Crystal structure of adenylosuccinate lyase from the thermophilic bacterium Thermus thermophilus HB8.

Adenylosuccinate lyase (PurB) catalyzes two distinct reactions in the purine nucleotide biosynthetic pathway using the same active site. The ability to recognize two different sets of substrates is of structural and evolutionary interest. In the present study, the crystal structure of PurB from the thermophilic bacterium Thermus thermophilus HB8 (TtPurB) was determined at a resolution of 2.38 Šby molecular replacement using a structure predicted by AlphaFold2 as a template. The asymmetric unit of the TtPurB crystal contained two TtPurB molecules, and some regions were disordered in the crystal structure. The disordered regions were the substrate-binding site and domain 3. TtPurB forms a homotetramer and the monomer is composed of three domains (domains 1, 2 and 3), which is a typical structure for the aspartase/fumarase superfamily. Molecular dynamics simulations with and without substrate/product were performed using a full-length model of TtPurB which was obtained before deletion of the disordered regions. The substrates and products were bound to the model structures during the MD simulations. The fluctuations of amino-acid residues were greater in the disordered regions and became smaller upon the binding of substrate or product. These results demonstrate that the full-length model obtained using AlphaFold2 can be used to generate the coordinates of disordered regions within the crystal structure.

Resonance Raman spectra of blue copper proteins: Variable temperature spectra of Thermus thermophilus HB27 laccase.

The resonance Raman (rR) spectra of the oxidized type 1 copper active site (CuT1) in Thermus thermophilus HB27 laccase (Tth-lac) has been determined in the 20 to 80 °C temperature range using 633-nm excitation. The positions and relative intensities of rR peaks are virtually independent of temperature, indicating that CuT1 ligation is robust over the investigated range. The intensity-weighted average of Tth-lac Cu-SCys vibrations (<ν(Cu-SCys)>) = 423 cm-1) is higher than those of most cupredoxins but is comparable to those of other multicopper oxidases (MCOs). <ν(Cu-SCys)> values for Tth-lac and several CuT1 centers in cupredoxins and MCOs do not correlate well with Cu-SCys bond lengths but do exhibit systematic trends with redox thermodynamic properties. PROLOGUE: F. Ann Walker was a great scholar and dear friend. While at Columbia in the early 1960s, I (HBG) followed her graduate work at Brown on the effects of axial ligands on vanadyl ion EPR spectra. Dick Carlin, her thesis adviser, invited me to serve as external member of her thesis committee. I joined, made my way to Providence, met her just before the exam, and greatly admired (enjoyed!) her thoughtful responses to questions from physical chemists about metal-oxo electronic structures. Our friendship grew stronger over the years, enhanced by lively discussions of heme protein chemistry in San Francisco, Pasadena, Tucson, and at Gordon Research Conferences. Ann was a superstar in biological inorganic chemistry. She will be sorely missed but not forgotten.

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.

Structural analysis of a bacterial UDP-sugar 2-epimerase reveals the active site architecture before and after catalysis.

The sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, was first identified ∼40 years ago in the O-antigen of Pseudomonas aeruginosa O:3,a,d. Since then, it has been observed on the O-antigens of various pathogenic Gram-negative bacteria including Bordetella pertussis, Escherichia albertii, and Pseudomonas mediterranea. Previous studies have established that five enzymes are required for its biosynthesis beginning with uridine dinucleotide (UDP)-N-acetyl-d-glucosamine (UDP-GlcNAc). The final step in the pathway is catalyzed by a 2-epimerase, which utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate. Curious as to whether this biochemical pathway is found in extreme thermophiles, we examined the published genome sequence for Thermus thermophilus HB27 and identified five ORFs that could possibly encode for the required enzymes. The focus of this investigation is on the ORF WP_011172736, which we demonstrate encodes for a 2-epimerase. For this investigation, ten high resolution X-ray crystallographic structures were determined to resolutions of 2.3 Å or higher. The models have revealed the manner in which the 2-epimerase anchors its UDP-sugar substrate as well as its UDP-sugar product into the active site. In addition, this study reveals for the first time the manner in which any sugar 2-epimerase can simultaneously bind UDP-sugars in both the active site and the allosteric binding region. We have also demonstrated that the T. thermophilus enzyme is allosterically regulated by UDP-GlcNAc. Whereas the sugar 2-epimerases that function on UDP-GlcNAc have been the focus of past biochemical and structural analyses, this is the first detailed investigation of a 2-epimerase that specifically utilizes UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid as its substrate.

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.

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.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

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.

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.

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.