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.

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 β-strands, such as mitochondrial cyt c, in addition to the β-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.

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 (GCｰ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.

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.

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.

Adenylosuccinate synthetase (PurA) is an enzyme responsible for the nitrogen addition to inosine monophosphate (IMP) by aspartate in the purine nucleotide biosynthetic pathway. And after which the fumarate is removed by adenylosuccinate lyase (PurB), leaving an amino group. There are two other enzymes that catalyze aspartate addition reactions similar to PurA, one in the purine nucleotide biosynthetic pathway (SAICAR synthetase, PurC) and the other in the arginine biosynthetic pathway (argininosuccinate sythetase, ArgG). To investigate the origin of these nitrogen-adding enzymes, PurA from Thermus thermophilus HB8 (TtPurA) was purified and crystallized, and crystal structure complexed with IMP was determined with a resolution of 2.10 Å. TtPurA has a homodimeric structure, and at the dimer interface, Arg135 of one subunit interacts with the IMP bound to the other subunit, suggesting that IMP binding contributes to dimer stability. The different conformation of His41 side chain in TtPurA and EcPurA suggests that side chain flipping of the His41 might play an important role in orienting γ-phosphate of GTP close to oxygen at position 6 of IMP, to receive the nucleophilic attack. Moreover, through comparison of the three-dimensional structures and active sites of PurA, PurC, and ArgG, it was suggested that the active sites of PurA and PurC converged to similar structures for performing similar reactions.

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.

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.

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.