SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation.

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning.

Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas surveillance complexes target double-stranded DNA, type III complexes target single-stranded RNA. Near-atomic resolution cryo-electron microscopy reconstructions of native type III Cmr (CRISPR RAMP module) complexes in the absence and presence of target RNA reveal a helical protein arrangement that positions the crRNA for substrate binding. Thumblike ß hairpins intercalate between segments of duplexed crRNA:target RNA to facilitate cleavage of the target at 6-nucleotide intervals. The Cmr complex is architecturally similar to the type I CRISPR-Cascade complex, suggesting divergent evolution of these immune systems from a common ancestor.

RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus.

CRISPR-Cas is a prokaryotic adaptive immune system that provides sequence-specific defense against foreign nucleic acids. Here we report the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). TtCsm is composed of five different protein subunits (Csm1-Csm5) with an uneven stoichiometry and a single crRNA of variable size (35-53 nt). The TtCsm crRNA content is similar to the Type III-B Cmr complex, indicating that crRNAs are shared among different subtypes. A negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes. crRNA-protein crosslinking studies show extensive contacts between the Csm3 backbone and the bound crRNA. We show that, like TtCmr, TtCsm cleaves complementary target RNAs at multiple sites. Unlike Type I complexes, interference by TtCsm does not proceed via initial base pairing by a seed sequence.

LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid.

Genome-wide comprehensive analysis of transcriptional regulation by ArgR in Thermus thermophilus.

ArgR is known to serve as a repressor/activator of the metabolism of arginine. To elucidate the role of ArgR in the metabolism of Thermus thermophilus cells, comparative genome-wide comprehensive analysis was conducted for wild-type T. thermophilus and its mutant lacking the argR gene. Transcriptome analysis and chromatin affinity precipitation coupled with high-density tiling chip (ChAP-chip) analysis identified 34 genetic loci that are directly regulated by ArgR and indicated that ArgR decreases the expression of arginine biosynthesis and also regulates several other genes involved in amino acid metabolism, including lysine biosynthetic genes, as suggested by our previous study. Among genes whose expression was regulated by ArgR, the largest effect of argR knockout was observed in a putative operon, including genes TTHA0284, TTHA0283, and TTHA0282 involved in arginine biosynthesis. The promoter of this operon, argG, was repressed approximately 21-fold by ArgR. DNase I footprint analysis coupled with electrophoretic mobility shift assay suggested that high arginine-dependent repression was attributed to the fact that the promoter contains three operators for ArgR binding and ArgR is bound to the binding sites cooperatively, possibly forming a DNA loop, in the hexameric form stabilized by arginine binding.

The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8.

BACKGROUND: RNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood. RESULTS: The genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H. CONCLUSION: This study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and YbeY proteins may act cooperatively in the stationary phase. This study also suggests that endo-RNases function mainly during the log phase, whereas exo-RNases function mainly during the stationary phase. RNase HI and RNase HII may have similar substrate selectivity.

Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus.

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Integrated database of information from structural genomics experiments.

Information from structural genomics experiments at the RIKEN SPring-8 Center, Japan has been compiled and published as an integrated database. The contents of the database are (i) experimental data from nine species of bacteria that cover a large variety of protein molecules in terms of both evolution and properties (http://database.riken.jp/db/bacpedia), (ii) experimental data from mutant proteins that were designed systematically to study the influence of mutations on the diffraction quality of protein crystals (http://database.riken.jp/db/bacpedia) and (iii) experimental data from heavy-atom-labelled proteins from the heavy-atom database HATODAS (http://database.riken.jp/db/hatodas). The database integration adopts the semantic web, which is suitable for data reuse and automatic processing, thereby allowing batch downloads of full data and data reconstruction to produce new databases. In addition, to enhance the use of data (i) and (ii) by general researchers in biosciences, a comprehensible user interface, Bacpedia (http://bacpedia.harima.riken.jp), has been developed.

Structure and function of a TetR family transcriptional regulator, SbtR, from thermus thermophilus HB8.

SbtR is one of the four TetR family transcriptional regulators present in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified 10 genes controlled by four promoters with negative regulation by SbtR in vitro. The SbtR-regulated gene products include probable transporters, probable enzymes for sugar or amino acid metabolism, and nucleic acid-related enzymes. SbtR binds pseudopalindromic sequences, with the consensus sequence of 5'-TGACCCNNKGGTCA-3' surrounding the promoters, and has a proposed 1:1 dimer binding stoichiometry. The X-ray crystal structure analysis revealed that SbtR comprises either nine or 10 α-helices and forms a dimer, as in the typical TetR family proteins. Similar to many characterized TetR family regulators, SbtR has a predicted ligand-binding pocket at the center of each monomer. Interestingly, the SbtR dimer contains an intermolecular disulfide bridge, formed between the Cys164 residues at the entrance of the pocket. The Cys164Ser and Cys164Ala mutant SbtR proteins formed homodimers similar to that of the wild type, but their thermal stabilities were lower by about 8°C, indicating that the disulfide bridge contributes to the thermal stability of the protein. However, altered repression activity of the mutants was not observed in vitro. From these results, we propose that ligand-binding is essential for SbtR to disengage from DNA, in a similar manner to the other characterized TetR family regulators. The formation and reduction of the disulfide bond might function in controlling the ligand-binding affinity of this transcriptional regulator.

Transcriptional repression mediated by a TetR family protein, PfmR, from Thermus thermophilus HB8.

PfmR is one of four TetR family transcriptional regulators found in the extremely thermophilic bacterium, Thermus thermophilus HB8. We identified three promoters with strong negative regulation by PfmR, both in vivo and in vitro. PfmR binds pseudopalindromic sequences, with the consensus sequence of 5'-TACCGACCGNTNGGTN-3' surrounding the promoters. According to the amino acid sequence and three-dimensional structure analyses of the PfmR-regulated gene products, they are predicted to be involved in phenylacetic acid and fatty acid metabolism. In vitro analyses revealed that PfmR weakly cross-regulated with the TetR family repressor T. thermophilus PaaR, which controls the expression of the paa gene cluster putatively involved in phenylacetic acid degradation but not with another functionally identified TetR family repressor, T. thermophilus FadR, which is involved in fatty acid degradation. The X-ray crystal structure of the N-terminal DNA-binding domain of PfmR and the nucleotide sequence of the predicted PfmR-binding site are quite similar to those of the TetR family repressor QacR from Staphylococcus aureus. Similar to QacR, two PfmR dimers bound per target DNA. The bases recognized by QacR within the QacR-binding site are conserved in the predicted PfmR-binding site, and they were important for PfmR to recognize the binding site and properly assemble on it. The center of the PfmR molecule contains a tunnel-like pocket, which may be the ligand-binding site of this regulator.

A non-cold-inducible cold shock protein homolog mainly contributes to translational control under optimal growth conditions.

Cold shock proteins (Csps) include both cold-induced and non-cold-induced proteins, contrary to their name. Cold-induced Csps are well studied; they function in cold acclimation by controlling transcription and translation. Some Csps have been reported to contribute to other cellular processes. However, the functions of non-cold-induced Csps under optimal growth conditions remain unknown. To elucidate these functions, we used transcriptome and proteome analyses as comprehensive approaches and have compared the outputs of wild-type and non-cold-induced Csp-deletion mutant cells. As a model organism, we selected Thermus thermophilus HB8 because it has only two csp genes (ttcsp1 and ttcsp2); ttCsp1 is the only non-cold-induced Csp. Surprisingly, the amount of transcripts and proteins upon deletion of the ttcsp1 gene was quite different. DNA microarray analysis revealed that the deletion of ttcsp1 did not affect the amount of transcripts, although the ttcsp1 gene was constantly expressed in the wild-type cell. Nonetheless, proteomic analysis revealed that the expression levels of many proteins were significantly altered when ttcsp1 was deleted. These results suggest that ttCsp1 functions in translation independent of transcription. Furthermore, ttCsp1 is involved in both the stimulation and inhibition of translation of specific proteins. Here, we have determined the crystal structure of ttCsp1 at 1.65 Å. This is the first report to present the structure of a non-cold-inducible cold shock protein. We also report the nucleotide binding affinity of ttCsp1. Finally, we discuss the functions of non-cold-induced Csps and propose how they modulate the levels of specific proteins to suit the prevailing environmental conditions.

Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophilus HB8.

Phenylacetic acid (PAA) is a common intermediate in the catabolic pathways of several structurally related aromatic compounds. It is converted into phenylacetyl coenzyme A (PA-CoA), which is degraded to general metabolites by a set of enzymes. Within the genome of the extremely thermophilic bacterium Thermus thermophilus HB8, a cluster of genes, including a TetR family transcriptional regulator, may be involved in PAA degradation. The gene product, which we named T. thermophilus PaaR, negatively regulated the expression of the two operons composing the gene cluster in vitro. T. thermophilus PaaR repressed the target gene expression by binding pseudopalindromic sequences, with a consensus sequence of 5'-CNAACGNNCGTTNG-3', surrounding the promoters. PA-CoA is a ligand of PaaR, with a proposed binding stoichiometry of 1:1 protein monomer, and was effective for transcriptional derepression. Thus, PaaR is a functional homolog of PaaX, a GntR transcriptional repressor found in Escherichia coli and Pseudomonas strains. A three-dimensional structure of T. thermophilus PaaR was predicted by homology modeling. In the putative structure, PaaR adopts the typical three-dimensional structure of the TetR family proteins, with 10 α-helices. A positively charged surface at the center of the molecule is similar to the acyl-CoA-binding site of another TetR family transcriptional regulator, T. thermophilus FadR, which is involved in fatty acid degradation. The CoA moiety of PA-CoA may bind to the center of the PaaR molecule, in a manner similar to the binding of the CoA moiety of acyl-CoA to FadR.

An alkyltransferase-like protein from Thermus thermophilus HB8 affects the regulation of gene expression in alkylation response.

Alkylation is a type of stress that is fatal to cells. However, cells have various responses to alkylation. Alkyltransferase-like (ATL) protein is a novel protein involved in the repair of alkylated DNA; however, its repair mechanism at the molecular level is unclear. DNA microarray analysis revealed that the upregulation of 71 genes because of treatment with an alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine was related to the presence of TTHA1564, the ATL protein from Thermus thermophilus HB8. Affinity chromatography showed a direct interaction of purified TTHA1564 with purified RNA polymerase holoenzyme. The amino acid sequence of TTHA1564 is homologous to that of the C-terminal domain of Ada protein, which acts as a transcriptional activator. These results suggest that TTHA1564 might act as a transcriptional regulator. The results of DNA microarray analysis also implied that the alkylating agent induced oxidation stress in addition to alkylation stress.

Crystal structure of Sulfolobus tokodaii Sua5 complexed with L-threonine and AMPPNP.

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N(6)-threonylcarbamoyl moiety is composed of L-threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L-threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L-threonine more strongly than L-serine and glycine. The Kd values of Sua5 for L-threonine and L-serine are 9.3 µM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L-threonine, at 1.8 Å resolution. The L-threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of L-threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L-threonine is located in a suitable position to react together with ATP for the synthesis of N(6)-threonylcarbamoyladenosine.

TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation.

In the extremely thermophilic bacterium Thermus thermophilus HB8, one of the four TetR-family transcriptional regulators, which we named T. thermophilus FadR, negatively regulated the expression of several genes, including those involved in fatty acid degradation, both in vivo and in vitro. T. thermophilus FadR repressed the expression of the target genes by binding pseudopalindromic sequences covering the predicted -10 hexamers of their promoters, and medium-to-long straight-chain (C10-18) fatty acyl-CoA molecules were effective for transcriptional derepression. An X-ray crystal structure analysis revealed that T. thermophilus FadR bound one lauroyl (C12)-CoA molecule per FadR monomer, with its acyl chain moiety in the centre of the FadR molecule, enclosed within a tunnel-like substrate-binding pocket surrounded by hydrophobic residues, and the CoA moiety interacting with basic residues on the protein surface. The growth of T. thermophilus HB8, with palmitic acid as the sole carbon source, increased the expression of FadR-regulated genes. These results indicate that in T. thermophilus HB8, medium-to-long straight-chain fatty acids can be used for metabolic energy under the control of FadR, although the major fatty acids found in this strain are iso- and anteiso-branched-chain (C15 and 17) fatty acids.

Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms.

Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 Šresolution, space group P2(1)) and Aquifex aeolicus (AaMogA; 1.70 Šresolution, space group P2(1) and 1.90 Šresolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein-protein complex of MogA and MoeA has been proposed and the residues involved in protein-protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.

Identification of novel genes regulated by the oxidative stress-responsive transcriptional activator SdrP in Thermus thermophilus HB8.

The stationary phase-dependent regulatory protein (SdrP) from the extremely thermophilic bacterium, Thermus thermophilus HB8, a CRP/FNR family protein, is a transcription activator, whose expression increases in the stationary phase of growth. SdrP positively regulates the expression of several genes involved in nutrient and energy supply, redox control, and nucleic acid metabolism. We found that sdrP mRNA showed an increased response to various environmental or chemical stresses in the logarithmic growth phase, the most effective stress being oxidative stress. From genome-wide expression pattern analysis using 306 DNA microarray datasets from 117 experimental conditions, eight new SdrP-regulated genes were identified among the genes whose expression was highly correlated with that of sdrP. The gene products included manganese superoxide dismutase, catalase, and excinuclease ABC subunit B (UvrB), which plays a central role in the nucleotide excision repair of damaged DNA. Expression of these genes also tended to increase upon entry into stationary phase, as in the case of the previously identified SdrP-regulated genes. These results indicate that the main function of SdrP is in the oxidative stress response.