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ATP synthases play a crucial role in energy production by utilizing the proton motive force (pmf) across the membrane to rotate their membrane-embedded rotor c-ring, and thus driving ATP synthesis in the hydrophilic catalytic hexamer. However, the mechanism of how pmf converts into c-ring rotation remains unclear. This study presents a 2.8 Å cryo-EM structure of the V o domain of V/A-ATPase from Thermus thermophilus, revealing precise orientations of glutamate (Glu) residues in the c 12 -ring. Three Glu residues face a water channel, with one forming a salt bridge with the Arginine in the stator (a/Arg). Molecular dynamics (MD) simulations show that protonation of specific Glu residues triggers unidirectional Brownian motion of the c 12 -ring towards ATP synthesis. When the key Glu remains unprotonated, the salt bridge persists, blocking rotation. These findings suggest that asymmetry in the protonation of c/Glu residues biases c 12 -ring movement, facilitating rotation and ATP synthesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The natural transformation system of the thermophilic bacterium Thermus thermophilus comprises at least 16 competence proteins. Recently we found that the outer membrane (OM) competence protein PilW interacts with the secretin channel, which guides type IV pili (T4P) and potential DNA transporter pseudopili through the OM. Here we have used biochemical techniques to study the interactions of cytoplasmic, inner membrane (IM) and OM components of the DNA transporter in T. thermophilus. We report that PilW is part of a heteropolymeric complex comprising of the cytoplasmic PilM protein, IM proteins PilN, PilO, PilC and the secretin PilQ. Co-purification studies revealed that PilO directly interacts with PilW. In vitro affinity co-purification studies using His-tagged PilC led to the detection of PilC-, PilW-, PilN- and PilO-containing complexes. PilO was identified as direct interaction partner of the polytopic IM protein PilC. PilC was also found to directly interact with the cytoplasmic T4P disassembly ATPase PilT1 thereby triggering PilT1 ATPase activity. This, together with the detection of heteropolymeric PilC complexes which contain PilT1 and the pilins PilA2, PilA4 and PilA5 is in line with the hypothesis that PilC connects the depolymerization ATPase to the base of the pili possibly allowing energy transduction for disassembly of the pilins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

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., (© 2024. The Author(s).)

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., (© 2024 The Authors. Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

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., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

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.

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 (=N 1 , N 11 -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.

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 c 552 and caa 3 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.

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.

Transcription regulation is a critical means by which microorganisms sense and adapt to their environments. Bacteria contain a wide range of highly conserved families of transcription factors that have evolved to regulate diverse sets of genes. It is increasingly apparent that structural similarities between transcription factors do not always equate to analogous transcription regulatory networks. For example, transcription factors within the copper-sensing operon repressor (CsoR)-resistance to cobalt and nickel repressor family have been found to repress a wide range of gene targets, including various metal efflux genes, as well as genes involved in sulfide and formaldehyde detoxification machinery. In this study, we identify the preferred DNA-binding sequence for the CsoR-like protein, TTHA1953, from the model extremophile Thermus thermophilus HB8 using the iterative selection approach, restriction endonuclease, protection, selection, and amplification. By mapping significant DNA motifs to the T. thermophilus HB8 genome, we identify potentially regulated genes that we validate with in vitro and in vivo methodologies. We establish TTHA1953 as a master regulator of the sulfur oxidation pathway, providing the first link between CsoR-like proteins and Sox regulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Discerning the determinants of protein thermostability is very important both from the theoretical and applied perspective. Different lines of evidence seem to indicate that a dynamical network of salt bridges/charged residues plays a fundamental role in the thermostability of enzymes. In this work, we applied measures of dynamic variance, like the Gini coefficients, Kullback-Leibler (KL) divergence and dynamic cross correlation (DCC) coefficients to compare the behavior of 3 pairs of homologous proteins from the thermophilic bacterium Thermus thermophilus and mesophilic Escherichia coli. Molecular dynamic (MD) simulations of these proteins were performed at 303 K and 363 K. In the characterization of their side chain rotamer distributions, the corresponding Gini coefficients and KL-divergence both revealed significant correlations with temperature. Similarly, a DCC analysis revealed a higher trend to de-correlate the movement of charged residues at higher temperatures in the thermophilic proteins, when compared with their mesophilic homologues. These results highlight the importance of dynamic electrostatic network interactions for the thermostability of enzymes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

An extreme thermophile, Thermus thermophilus , produces 16 different polyamines including long-chain and branched-chain polyamines. The composition and content of polyamines in the thermophile cells change not only with growth temperature but also with pH changes. In particular, cell growth decreased greatly at alkaline medium together with significant changes in the composition and content of polyamines. The amounts of tetraamines (spermine and its homologs) markedly decreased at alkaline pH. Thus, we knocked out the speE gene, which is involved in the biosynthesis of tetraamines, and changes of composition of polyamines with pH changes in the mutant cells were studied. Cell growth in the Δ speE strain was decreased compared with that of the wild-type strain for all pHs, suggesting that tetraamines are important for cell proliferation. Interestingly, the amount of spermidine decreased and that of putrescine increased in wild-type cells at elevated pH, although T. thermophilus lacks a putrescine synthesizing pathway. In addition, polyamines possessing a diaminobutane moiety, such as spermine, decreased greatly at high pH. We assessed whether the speB gene encoding aminopropylagmatine ureohydrolase (TtSpeB) is directly involved in the synthesis of putrescine. The catalytic assay of the purified enzyme indicated that TtSpeB accepts agmatine as its substrate and produces putrescine due to the change in substrate specificity at high pH. These results suggest that pH stress was exacerbated upon intracellular depletion of polyamines possessing a diaminobutane moiety induced by unusual changes in polyamine biosynthesis under high pH conditions.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB&#95;Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.

Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The natural transformation system of Thermus thermophilus has become a model system for studies of the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter in T. thermophilus is functionally linked to type IV pili (T4P) and the major pilin PilA4 plays an essential role in both systems. However, T4P are dispensable for natural transformation. In addition to pilA4, T. thermophilus has a gene cluster encoding the three additional pilins PilA1-PilA3; deletion of the cluster abolished natural transformation but retained T4P biogenesis. In this study, we investigated the roles of single pilins PilA1, PilA2 and PilA3 in natural transformation by mutant studies. These studies revealed that each of these pilins is essential for natural transformation. Two of the pilins, PilA1 and PilA2, were found to bind dsDNA. PilA1 and PilA3 were detected in the inner membrane (IM) but not in the outer membrane (OM) whereas PilA2 was present in both membranes. All three pilins where absent in pilus fractions. This suggests that the pilins form a short DNA binding pseudopilus anchored in the IM. PilA1 was found to bind to the IM assembly platform of the DNA transporter via PilM and PilO. These data are in line with the hypothesis that a DNA binding pseudopilus is connected via an IM platform to the cytosolic motor ATPase PilF., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Uptake of DNA from the environment into the bacterial cytoplasm is mediated by a macromolecular transport machinery that is similar in structure and function to type IV pili (T4P) and, indeed, DNA translocator and T4P share common components. One is the secretin PilQ which is assembled into homopolymeric complexes forming highly dynamic outer membrane (OM) channels mediating pilus extrusion and DNA uptake. How PilQ interacts with the motor is still enigmatic. Here, we have used biochemical and genetic techniques to study the interaction of PilQ with PilW, a unique protein which is essential for natural transformation and T4P extrusion of T. thermophilus. PilQ and PilW form high molecular mass complexes in the OM of T. thermophilus. When pilW was deleted, PilQ complexes were no longer observed but only PilQ monomers, accompanied by a loss of DNA uptake as well as a loss of T4P and twitching motility. Piliation of a ΔpilT2/ΔpilW double mutant suggests that PilW is important for stable assembly of PilQ complexes. To analyze the role of different regions of PilW, partial deletions (pilW ∆2 - 40 , pilW ∆50 - 150 , pilW ∆163 - 216 and pilW ∆216 - 292 ) were generated and the effect on DNA uptake, PilQ complex formation and T4P functions such as twitching motility, biofilm formation and cell-cell interaction was studied. These studies revealed that a central disordered region in PilW is required for pilus dynamics. We propose that PilW is part of a protein network that connects the transport ATPase to drive different functions of the DNA translocator and T4P., (Copyright © 2021. Published by Elsevier B.V.)

In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts (L8-P4 and L18-P8) help to orient the two independently folding S- and C-domains for concerted recognition of precursor tRNA substrates. Here, we analyze the effects of mutations in these tertiary contacts in P RNAs from three different species: (i) the psychrophilic bacterium Pseudoalteromonas translucida ( Ptr ), (ii) the mesophilic radiation-resistant bacterium Deinococcus radiodurans ( Dra ) , and (iii) the thermophilic bacterium Thermus thermophilus ( Tth ). We show by UV melting experiments that simultaneous disruption of these two interdomain contacts has a stabilizing effect on all three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures and a more concerted unfolding at higher temperatures. Thus, when the two domains tightly interact via the tertiary contacts, one domain facilitates structural transitions in the other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot structure between the P9 and P12 of its S-domain. This interaction was found to be particularly crucial for RNase P holoenzyme activity at near-physiological Mg 2+ concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the G,C-rich Tth P RNA., (© 2021 Marszalkowski et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism Thermus thermophilus HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5'-AWTGTRA(N) 6 TYACAWT-3', which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the T. thermophilus HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.

Cytochrome ba 3 from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba 3 . The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle., (Copyright © 2021 Elsevier B.V. All rights reserved.)

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE., Competing Interests: The authors have declared that no competing interests exist.

A comparative study of the possibilities of using ribokinase → phosphopentomutase → nucleoside phosphorylase cascades in the synthesis of modified nucleosides was carried out. Recombinant phosphopentomutase from Thermus thermophilus HB27 was obtained for the first time: a strain producing a soluble form of the enzyme was created, and a method for its isolation and chromatographic purification was developed. It was shown that cascade syntheses of modified nucleosides can be carried out both by the mesophilic and thermophilic routes from D-pentoses: ribose, 2-deoxyribose, arabinose, xylose, and 2-deoxy-2-fluoroarabinose. The efficiency of 2-chloradenine nucleoside synthesis decreases in the following order: Rib (92), dRib (74), Ara (66), F-Ara (8), and Xyl (2%) in 30 min for mesophilic enzymes. For thermophilic enzymes: Rib (76), dRib (62), Ara (32), F-Ara (<1), and Xyl (2%) in 30 min. Upon incubation of the reaction mixtures for a day, the amounts of 2-chloroadenine riboside (thermophilic cascade), 2-deoxyribosides (both cascades), and arabinoside (mesophilic cascade) decreased roughly by half. The conversion of the base to 2-fluoroarabinosides and xylosides continued to increase in both cases and reached 20-40%. Four nucleosides were quantitatively produced by a cascade of enzymes from D-ribose and D-arabinose. The ribosides of 8-azaguanine (thermophilic cascade) and allopurinol (mesophilic cascade) were synthesized. For the first time, D-arabinosides of 2-chloro-6-methoxypurine and 2-fluoro-6-methoxypurine were synthesized using the mesophilic cascade. Despite the relatively small difference in temperatures when performing the cascade reactions (50 and 80 °C), the rate of product formation in the reactions with Escherichia coli enzymes was significantly higher. E. coli enzymes also provided a higher content of the target products in the reaction mixture. Therefore, they are more appropriate for use in the polyenzymatic synthesis of modified nucleosides.

The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. coli fabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.

There is ongoing debate regarding the mechanism through which cation/proton antiporters (CPAs), like Thermus thermophilus NapA (TtNapA) and Escherichia coli NapA (EcNhaA), alternate between their outward- and inward-facing conformations in the membrane. CPAs comprise two domains, and it is unclear whether the transition is driven by their rocking-bundle or elevator motion with respect to each other. Here we address this question using metadynamics simulations of TtNapA, where we bias conformational sampling along two axes characterizing the two proposed mechanisms: angular and translational motions, respectively. By applying the bias potential for the two axes simultaneously, as well as to the angular, but not the translational, axis alone, we manage to reproduce each of the two known states of TtNapA when starting from the opposite state, in support of the rocking-bundle mechanism as the driver of conformational change. Next, starting from the inward-facing conformation of EcNhaA, we sample what could be its long-sought-after outward-facing conformation and verify it using cross-linking experiments., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.

The rotation of Paracoccus denitrificans F 1 -ATPase (PdF 1 ) was studied using single-molecule microscopy. At all concentrations of adenosine triphosphate (ATP) or a slowly hydrolyzable ATP analog (ATPγS), above or below K m , PdF 1 showed three dwells per turn, each separated by 120°. Analysis of dwell time between steps showed that PdF 1 executes binding, hydrolysis, and probably product release at the same dwell. The comparison of ATP binding and catalytic pauses in single PdF 1 molecules suggested that PdF 1 executes both elementary events at the same rotary position. This point was confirmed in an inhibition experiment with a nonhydrolyzable ATP analog (AMP-PNP). Rotation assays in the presence of adenosine diphosphate (ADP) or inorganic phosphate at physiological concentrations did not reveal any obvious substeps. Although the possibility of the existence of substeps remains, all of the datasets show that PdF 1 is principally a three-stepping motor similar to bacterial vacuolar (V 1 )-ATPase from Thermus thermophilus This contrasts with all other known F 1 -ATPases that show six or nine dwells per turn, conducting ATP binding and hydrolysis at different dwells. Pauses by persistent Mg-ADP inhibition or the inhibitory ζ-subunit were also found at the same angular position of the rotation dwell, supporting the simplified chemomechanical scheme of PdF 1 Comprehensive analysis of rotary catalysis of F 1 from different species, including PdF 1 , suggests a clear trend in the correlation between the numbers of rotary steps of F 1 and F o domains of F-ATP synthase. F 1 motors with more distinctive steps are coupled with proton-conducting F o rings with fewer proteolipid subunits, giving insight into the design principle the F 1 F o of ATP synthase., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus , but only in few strains of the species Thermus thermophilus has the pathway been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, in the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 bp, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O 2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

Transcription factors (TFs) have been extensively researched in certain well-studied organisms, but far less so in others. Following the whole-genome sequencing of a new organism, TFs are typically identified through their homology with related proteins in other organisms. However, recent findings demonstrate that structurally similar TFs from distantly related bacteria are not usually evolutionary orthologs. Here we explore TTHB099, a cAMP receptor protein (CRP)-family TF from the extremophile Thermus thermophilus HB8. Using the in vitro iterative selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), we identified the preferred DNA-binding motif for TTHB099, 5'-TGT(A/g)NBSYRSVN(T/c)ACA-3', and mapped potential binding sites and regulated genes within the T. thermophilus HB8 genome. Comparisons with expression profile data in TTHB099-deficient and wild type strains suggested that, unlike E. coli CRP (CRP Ec ), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRP Ec .

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication., Competing Interests: Declaration of Interests P.D.Z., S.M.J., and H.Z. have submitted a patent application regarding novel uses of TtAgo (PCT/US2016/025724)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Eukaryotic Argonaute proteins strictly mediate RNA-guided RNA interference. In contrast, prokaryotic Argonautes can utilize DNA guides to target complementary DNA sequences to protect their hosts against invading DNA. In this issue of Cell, Jolly and colleagues demonstrate that Thermus thermophilus Argonaute additionally participates in DNA replication by unlinking catenated chromosomes., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the "parent" protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from T. thermophilus , exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria T. thermophilus to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of T. thermophilus cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact T. thermophilus cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in T. thermophilus cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the Escherichia coli and Thermus thermophilus ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading., Competing Interests: The authors declare no competing interest.

In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an α-glycoside-binding protein (αGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide α-glycosides namely trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6) and glucose within a resolution range of 1.6-2.0 Å. Despite transporting multiple types of sugars, αGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide α-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide α-glycosides) from the active site of the protein. Comparative assessment of open and closed conformations of αGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of αGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession number(s) 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE., (© 2019 Federation of European Biochemical Societies.)

Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5'-(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)-3' was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation., Competing Interests: The authors declare no conflict of interest. In addition, the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNA Phe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNA Val reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding., Competing Interests: The authors declare no competing interest.

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus' E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

Holliday junctions (HJs) are four-way DNA structures that occur in DNA repair by homologous recombination. Specialized nucleases, termed resolvases, remove (i.e., resolve) HJs. The bacterial protein RuvC is a canonical resolvase that introduces two symmetric cuts into the HJ. For complete resolution of the HJ, the two cuts need to be tightly coordinated. They are also specific for cognate DNA sequences. Using a combination of structural biology, biochemistry, and a computational approach, here we show that correct positioning of the substrate for cleavage requires conformational changes within the bound DNA. These changes involve rare high-energy states with protein-assisted base flipping that are readily accessible for the cognate DNA sequence but not for non-cognate sequences. These conformational changes and the relief of protein-induced structural tension of the DNA facilitate coordination between the two cuts. The unique DNA cleavage mechanism of RuvC demonstrates the importance of high-energy conformational states in nucleic acid readouts.

Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism's transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5'-AACnAACGTTnGTT-3' that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.

The ribosome, the largest RNA-containing macromolecular machinery in cells, requires metal ions not only to maintain its three-dimensional fold but also to perform protein synthesis. Despite the vast biochemical data regarding the importance of metal ions for efficient protein synthesis and the increasing number of ribosome structures solved by X-ray crystallography or cryo-electron microscopy, the assignment of metal ions within the ribosome remains elusive due to methodological limitations. Here we present extensive experimental data on the potassium composition and environment in two structures of functional ribosome complexes obtained by measurement of the potassium anomalous signal at the K-edge, derived from long-wavelength X-ray diffraction data. We elucidate the role of potassium ions in protein synthesis at the three-dimensional level, most notably, in the environment of the ribosome functional decoding and peptidyl transferase centers. Our data expand the fundamental knowledge of the mechanism of ribosome function and structural integrity.

Identification and characterization of base-multiplets, which are essentially mediated by base-pairing interactions, can provide insights into the diversity in the structure and dynamics of complex functional RNAs, and thus facilitate hypothesis driven biological research. The necessary nomenclature scheme, an extension of the geometric classification scheme for base-pairs by Leontis and Westhof, is however available only for base-triplets. In the absence of information on topology, this scheme is not applicable to quartets and higher order multiplets. Here we propose a topology-based classification scheme which, in conjunction with a graph-based algorithm, can be used for the automated identification and characterization of higher order base-multiplets in RNA structures. Here, the RNA structure is represented as a graph, where nodes represent nucleotides and edges represent base-pairing connectivity. Sets of connected components (of n nodes) within these graphs constitute subgraphs representing multiplets of "n" nucleotides. The different topological variants of the RNA multiplets thus correspond to different nonisomorphic forms of these subgraphs. To annotate RNA base-multiplets unambiguously, we propose a set of topology-based nomenclature rules for quartets, which are extendable to higher multiplets. We also demonstrate the utility of our approach toward the identification and annotation of higher order RNA multiplets, by investigating the occurrence contexts of selected examples in order to gain insights regarding their probable functional roles., (© 2019 Bhattacharya et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The 70S ribosome is a major target for antibacterial drugs. Two of the classical antibiotics, chloramphenicol (CHL) and erythromycin (ERY), competitively bind to adjacent but separate sites on the bacterial ribosome: the catalytic peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), respectively. The previously reported competitive binding of CHL and ERY might be due either to a direct collision of the two drugs on the ribosome or due to a drug-induced allosteric effect. Because of the resolution limitations, the available structures of these antibiotics in complex with bacterial ribosomes do not allow us to discriminate between these two possible mechanisms. In this work, we have obtained two crystal structures of CHL and ERY in complex with the Thermus thermophilus 70S ribosome at a higher resolution (2.65 and 2.89 Å, respectively) allowing unambiguous placement of the drugs in the electron density maps. Our structures provide evidence of the direct collision of CHL and ERY on the ribosome, which rationalizes the observed competition between the two drugs., (© 2019 Svetlov et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The elongation factor G (EF-G)-catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon-anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame., Competing Interests: The authors declare no conflict of interest.

Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The Δ bshA Δ sod and Δ bshA Δ pcat double mutant strains were as sensitive to Hg(II) as the Δ bshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δ sod and Δ pcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δ sod and Δ pcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments. IMPORTANCE Thermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus - Thermus phylum that, together with the Aquificae , constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes., (Copyright © 2019 Norambuena et al.)

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

Bacterium Thermus thermophilus Argonaute (Ago; Tt Ago) is a prokaryotic Ago (pAgo) that acts as the host defense against the uptake and propagation of foreign DNA by catalyzing the DNA cleavage reaction. The Tt Ago active site consists of a plugged-in glutamate finger with two arginine residues (R545 and R486) located symmetrically around it. An interesting challenge is to understand how they can collaboratively facilitate enzymatic catalysis. In Kluyveromyces polysporus Ago, a eukaryotic Ago, the evolutionarily symmetrical residues are arginine and histidine, both of which function to stabilize the plugged-in catalytic tetrad conformation. Surprisingly, our simulation results indicated that, in Tt Ago, only R545 is involved in the cleavage reaction by serving as a critical structural anchor to stabilize the catalytic tetrad Asp-Glu-Asp-Asp that is completed by the insertion of the glutamate finger, whereas R486 is not involved in target cleavage. The Tt Ago-mediated target DNA cleavage occurs in a substrate-assisted mechanism, in which the pro-Rp (Rp, a tetrahedral phosphorus center with "R-type" chirality) oxygen of scissile phosphate acts as a general base to activate the nucleophilic water. Our unexpected theoretical findings on distinct roles played by R545 and R486 in Tt Ago catalysis have been validated by single-point site-mutagenesis experiments, wherein the target cleavage is abolished for all mutants of R545. In sharp contrast, the cleavage activity is maintained for all mutants of R486. Our work provides mechanistic insights on the catalytic specificity of Ago proteins and could facilitate the design of new gene-editing tools in the long term., Competing Interests: The authors declare no conflict of interest.