Thermus Thermophilus Research Articles

Comparative analysis of structural and functional aspects of phytoene synthase from Meiothermus taiwanensis strain RP

PurposeThe aim of this study is the in silico characterization of the structure and function of the phytoene synthase (PSY) of a red carotenoid producing thermophile Meiothermus taiwanensis strain RP with a comparative approach.MethodsPSYs from M. taiwanensis strain RP and other groups of thermophilic, mesophilic and psychrophilic bacteria, plants, protozoa, and algae were analyzed by ExPASy ProtParam, NCBI Conserved Domain Search, SOPMA, PSIPRED, Robetta server, ProQ, and QMEAN, with the superposition of 3-D structures in PyMOL.ResultsRP PSY shows the highest (97.5 %) similarity with M. ruber and the lowest with the psychrophile Gelidibacter algens (36.7 %). The amino acid sequence of RP PSY is one of the shortest, with 275 residues. The instability index of RP PSY is much lower compared with plant sequences. Alanine, arginine, glycine, and leucine residues are the highest in Meiothermus sp., and they have a high amount of alpha-helix. Most of the 32 active site residues are conserved in all the sequences. However, some residues are more prone to substitutions in other PSYs except M. ruber. The three-dimensional structures of M. taiwanensis strain RP, Gelidibacter algens, Thermus thermophiles, Meiothermus ruber, and Brassica napus PSYs were homology modeled, validated, and submitted to Protein Model Databank. The superposition of the 3-D structures shows that their active site region structure is identical.ConclusionRP PSY is one of the most stable PSYs and knowledge of its individual properties, similarities, and dissimilarities with other PSYs may be useful for genetic engineering and purification of the protein for improved carotenoid production.

A peroxiredoxin of Thermus thermophilus HB27: Biochemical characterization of a new player in the antioxidant defence

To fight oxidative damage due to reactive oxygen species (ROS), cells are equipped of different enzymes, among which Peroxiredoxins (Prxs) (EC 1.11.1.15) play a key role. Prxs are thiol-based enzymes containing one (1-Cys Prx) or two (2-Cys Prx) catalytic cysteine residues. In 2-Cys Prxs the cysteine residues form a disulfide bridge following reduction of peroxide which is in turn reduced by Thioredoxin reductase (Tr) /Thioredoxin (Trx) disulfide reducing system to regenerate the enzyme. In this paper we investigated on Prxs of Thermus thermophilus whose genome contains an ORF TT_C0933 encoding a putative Prx, belonging to the subfamily of Bacterioferritin comigratory protein (Bcp): the synthetic gene was produced and expressed in E. coli and the recombinant protein, TtBcp, was biochemically characterized. TtBcp was active on both organic and inorganic peroxides and showed stability at high temperatures. To get insight into disulfide reducing system involved in the recycling of the enzyme we showed that TtBcp catalically eliminates hydrogen peroxide using an unusual partner, the Protein Disulfide Oxidoreductase (TtPDO) that could replace regeneration of the enzyme. Altogether these results highlight not only a new anti-oxidative pathway but also a promising molecule for possible future biotechnological applications.

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the Thermus thermophilus 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating Escherichia coli 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.

Expression and regulation of the mer operon in Thermus thermophilus.

Mercury (Hg) is a highly toxic and widely distributed heavy metal, which some Bacteria and Archaea detoxify by the reduction of ionic Hg (Hg[II]) to the elemental volatile form, Hg(0). This activity is specified by the mer operon. The mer operon of the deeply branching thermophile Thermus thermophilus HB27 encodes for, an O-acetyl-l-homoacetylserine sulfhydrylase (Oah2), a transcriptional regulator (MerR), a hypothetical protein (hp) and a mercuric reductase (MerA). Here, we show that this operon has two convergently expressed and differentially regulated promoters. An upstream promoter, P oah , controls the constitutive transcription of the entire operon and a second promoter (P mer ), located within merR, is responsive to Hg(II). In the absence of Hg(II), the transcription of merA is basal and when Hg(II) is present, merA transcription is induced. This response to Hg(II) is controlled by MerR and genetic evidence suggests that MerR acts as a repressor and activator of P mer . When the whole merR, including P mer , is removed, merA is transcribed from P oah independently of Hg(II). These results suggest that the transcriptional regulation of mer in T. thermophilus is both similar to, and different from, the well-documented regulation of proteobacterial mer systems, possibly representing an early step in the evolution of mer-operon regulation.

Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase.

Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucyl-tRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.

The K428 residue from Thermus thermophilus SG0.5JP17-16 laccase plays the substantial role in substrate binding and oxidation

Laccases are multi-copper oxidases (MCOs) that catalyze the oxidation of various organic and inorganic compounds with concomitant reduction of dioxygen to water, and are considered to be green catalysts. Previous studies revealed that in MCOs, the structure of substrate-binding pocket was closely related to the substrate specificity. The amino acids on the pocket-constructing loops were involved in the identification of substrate and affected the catalytic properties of the enzyme. In the Thermus thermophilus SG0.5JP17-16 laccase (lacTT), the Lys428 residue is on pocket-constructing loops, to explore the role of the K428 residue and obtain mutants with enhanced catalytic activity, it was mutated to leucine, glutamic acid, arginine and methionine by site-directed mutagenesis. Structural data revealed that the K428 residue identified the conformation of the substrate by the steric hindrance and interactions with the substrate. Kinetic data indicated that the replacement of K428 by methionine resulted in a mutant with enhanced catalytic activity. The K428M mutant could decolorize the synthetic dyes more efficiently than the wild type enzyme. Altogether, this study provided a strategy to find the mutant with the enhanced catalytic activity. Communicated by Ramaswamy H. Sarma

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27

Deciphering the activity-conformation relationship of PTPase is of great interest to understand how PTPase activity is determined by its conformation. Here we studied the activity and conformational transitions of PTPase from thermus thermophilus HB27 in the presence of sodium dodecyl sulfate (SDS). Activity assays showed the inactivation of PTPase induced by SDS was in a concentration-dependent manner. Fluorescence and circular dichroism spectra suggested SDS induced significant conformational transitions of PTPase, which resulted in the inactivation of PTPase, and the changes of α-helical structure and tertiary structure of PTPase. Structural analysis revealed a number of hydrophobic and charged residues around the active sites of PTPase may be involved in the hydrophobic and ionic bonds interactions of PTPase and SDS, which are suggested to be the major driving force to result in PTPase inactivation and conformational transitions induced by SDS. Our results suggested the hydrophobic and charged residues around the active sites were essential for the activity and conformation of PTPase. Our study promotes a better understanding of the activity and conformation of PTPase.

Identifying heat shock response systems from the genomic assembly of Ureibacillus thermophilus LM102 using protein-protein interaction networks

Ureibacillus thermophilus strain LM102 is a facultative thermophile with growth in range 37 °C–60 °C. Upon identification using the 16S rRNA marker, it showed highest similarity of 99.8% with U. thermophilus strain HC148. A phylogenetic analysis revealed U. suwonensis strain 6 T19 to be the closest species to strain LM102. Our aim was to determine the unique thermotolerant properties of LM102 by identifying thermostablity of its proteins and the interactions existing in its heat shock response systems (HSRS). The strain was sequenced, assembled and the draft genome (3,017,325 bp) was analyzed. Post-annotation, we randomly selected a set of hundred proteins and computed the percentage distribution of 12 amino acids which have been substantially studied for their role in thermostability. The protein homologues were searched and the residues of LM102 were compared with Bacillus subtilis and Thermus thermophilus, a mesophile and hyperthermophile respectively. Within the 95% confidence limit, a Z-score of −0.61 was observed between LM102 and B. subtilis. However, a significantly lower value of −8.84 was observed for the pair LM102 and T. thermophilus. The amino acid distribution did not appear to influence the protein thermostability. Further, we investigated the role of Protein-Protein interactions by building networks for heat shock responses, namely DNA repair, transcriptional regulation, and activation of heat shock proteins. Interaction data retrieved from the STRING database for more than 50 species were used to build these networks. Highly clustered MCODE results notably revealed RNA 3′-5′ exonuclease, CshA and HemW previously unreported, in association with other proteins. Additionally, these and other proteins estimated from the HSRS networks were found in both mesophiles and thermophiles, suggesting a crucial role of gene regulatory networks in the cellular viability of LM102 at high temperatures.

Accretion history of large ribosomal subunits deduced from theoretical minimal RNA rings is congruent with histories derived from phylogenetic and structural methods

Accretions of tRNAs presumably formed the large complex ribosomal RNA structures. Similarities of tRNA secondary structures with rRNA secondary structures increase with the integration order of their cognate amino acid in the genetic code, indicating tRNA evolution towards rRNA-like structures. Here analyses rank secondary structure subelements of three large ribosomal RNAs (Prokaryota: Archaea: Thermus thermophilus; Bacteria: Escherichia coli; Eukaryota: Saccharomyces cerevisiae) in relation to their similarities with secondary structures formed by presumed proto-tRNAs, represented by 25 theoretical minimal RNA rings. These ranks are compared to those derived from two independent methods (ranks provide a relative evolutionary age to the rRNA substructure), (a) cladistic phylogenetic analyses and (b) 3D-crystallography where core subelements are presumed ancient and peripheral ones recent. Comparisons of rRNA secondary structure subelements with RNA ring secondary structures show congruence between ranks deduced by this method and both (a) and (b) (more with (a) than (b)), especially for RNA rings with predicted ancient cognate amino acid. Reconstruction of accretion histories of large rRNAs will gain from adequately integrating information from independent methods. Theoretical minimal RNA rings, sequences deterministically designed in silico according to specific coding constraints, might produce adequate scales for prebiotic and early life molecular evolution.

Elucidating the preference of dimeric over monomeric form for thermal stability of Thermus thermophilus isopropylmalate dehydrogenase: A molecular dynamics perspective

An oligomer usually refers to a macromolecular complex formed by non-covalent interactions of monomers. Several thermophilic proteins are oligomers. The significance of oligomerization of individual proteins for stability at higher temperature is of prime importance for understanding evolution and increasing industrial productivity. The functional form of Thermus thermophilius isopropylmalate dehydrogenase (IPMDH), a widely studied protein to understand the factors affecting the thermal stability of a protein is a dimer, a simplest oligomer. To decipher the relationship between the effects of oligomerization on thermal stability of a protein, we have applied all-atom molecular mechanics approach by analyzing how temperature effects dynamics of a subunit in the presence and absence of another subunit in dimeric (SS) and monomeric forms (SA), respectively, before its denaturation begins. Comparing the difference in overall dynamic structural aspects at two different temperatures, 300 K and 337 K. Analysis of root mean square deviation (RMSD), root mean square fluctuations (RMSF) and Cα-Cα distance with an increase in temperature from 300 K to 337 K for a total of 0.2 μs reveals higher thermal stability of the dimer as compared to monomer. In contrast to dimeric form, the monomer is relatively stable at 300 K but cannot withstand the structural stability at 337 K leading to loosening of intramolecular interactions with maximum fluctuation at B23–B24 within a subunit. Energetic and structural properties indicate that B24–B24’ is the major contributor to maintaining subunit-subunit interaction at 337 K. Correlation between the favorable interaction energy (IE) with the minimal perturbance in Cα atoms of domain 2 in a subunit in the presence of another subunit enhances the rigidity of the domain with subunit-subunit interaction. Overall, the study indicates that the dimeric over monomeric form enhances the protein’s thermal stability and not all major subunit interacting regions contribute equally in maintaining the former.

DNA interference by a mesophilic Argonaute protein, CbcAgo.

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5'P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn 2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.

General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023.

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.

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.

DNA interference by a mesophilic Argonaute protein, CbcAgo

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn 2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.

Structural basis for ribosome recycling by RRF and tRNA.

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.

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction.

Multicopper oxidases (MCOs) catalyze the oxidation of a variety of substrates while reducing oxygen into water through four copper atoms. As an additional feature, some MCOs display an enhanced activity in solution in the presence of Cu2+. This is the case of the hyperthermophilic laccase HB27 from Thermus thermophilus, the physiologic role of which is unknown. As a particular feature, this enzyme presents a methionine rich domain proposed to be involved in copper interaction. In this work, laccase from T. thermophilus was produced in E. coli, and the effect of Cu2+ on its electroactivity at carbon nanotube modified electrodes was investigated. Direct O2 electroreduction is strongly dictated by carbon nanotube surface chemistry in accordance with the enzyme dipole moment. In the presence of Cu2+, an additional low potential cathodic wave occurs, which was never described earlier. Analysis of this wave as a function of Cu2+ availability allows us to attribute this wave to a cuprous oxidase activity displayed by the laccase and induced by copper binding close to the Cu T1 center. A mutant lacking the methionine-rich hairpin domain characteristic of this laccase conserves its copper activity suggesting a different site of copper binding. This study provides new insight into the copper effect in methionine rich MCOs and highlights the utility of the electrochemical method to investigate cuprous oxidase activity and to understand the physiological role of these MCOs.

Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

The Reactions of O2 and NO with Mixed-Valence ba3 Cytochrome c Oxidase from Thermus thermophilus

Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba3 (Tt ba3) cytochrome oxidase revealed that O2 binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010–21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the CuB+-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba3 (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380–1390) affects O2 and NO binding and the ability of CuB to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba3 with O2 were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O2 and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba3. The rate of O2 and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived CuB+-CO complex did not prevent O-O bond splitting and the resulting PM formation, which was significantly faster (5–10 times) than in the bovine heart enzyme. We propose that O2 binding to heme a3 in Tt ba3 causes CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects, thus allowing CuB+ to act as an electron donor in the mixed-valence enzyme. The significantly faster O2 binding and O-O bond cleavage in Tt ba3 compared to analogous steps in the aa3 oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.

An ancient type of MnmA protein is an iron-sulfur cluster-dependent sulfurtransferase for tRNA anticodons.

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe–S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe–S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter. Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe–4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe–S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe–S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.

ICETh1 and ICETh2, two interdependent mobile genetic elements in Thermus thermophilus transjugation.

Cell to cell DNA transfer between Thermus thermophilus, or transjugation, requires the natural competence apparatus (NCA) of the recipient cell and a DNA donation machinery in the donor. In T. thermophilus HB27, two mobile genetic elements with functional similarities to Integrative and Conjugative Elements (ICEs) coexist, ICETh1 encoding the DNA transfer apparatus and ICETh2, encoding a putative replication module. Here, we demonstrate that excision and integration of both elements depend on a single tyrosine recombinase encoded by ICETh2, and that excision is not required but improves the transfer of these elements to a recipient cell. These findings along with previous results suggest that ICETh1 and ICETh2 depend on each other for spreading among T. thermophilus by transjugation.