RESUMEN
The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/química , Methanocaldococcus/química , Sulfolobus/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/genética , Ligasas de Carbono-Nitrógeno con Glutamina como Donante de Amida-N/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Methanocaldococcus/enzimología , Modelos Moleculares , Simulación de Dinámica Molecular , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sulfolobus/enzimología , Thermus thermophilus/enzimologíaRESUMEN
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.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Luz , Plásmidos , Thermus thermophilus/genética , Thermus thermophilus/efectos de la radiación , Factores de Transcripción/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Transcripción Genética , Activación TranscripcionalRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Proteínas Represoras/metabolismo , Thermus thermophilus/genética , Transcripción Genética , Arginina/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Lisina/biosíntesis , Datos de Secuencia Molecular , Operón , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Thermus thermophilus/metabolismo , TranscriptomaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Thermus thermophilus/genética , Proteínas Bacterianas/genética , Análisis por Conglomerados , Genoma Bacteriano , Modelos Biológicos , Estabilidad del ARN , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Ribonucleasas/genética , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Thermus thermophilus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación de Nucleótidos de Alto Rendimiento , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , Ribonucleasas/química , Ribonucleasas/genética , Análisis de Secuencia de ARN , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Thermus thermophilus/genéticaRESUMEN
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.
Asunto(s)
Bases de Datos Factuales , Proteínas/química , Proteínas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalización , Genómica/métodos , Internet , Japón , Interfaz Usuario-ComputadorRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Resistencia a la Tetraciclina/genética , Thermus thermophilus/genética , Factores de Transcripción/química , Transcripción Genética , Secuencia de Bases , Secuencia de Consenso , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Thermus thermophilus/químicaRESUMEN
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.
Asunto(s)
Thermus thermophilus/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/genéticaAsunto(s)
Proteínas Bacterianas/química , Proteína Receptora de AMP Cíclico/química , Proteínas Hierro-Azufre/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas de Unión al ADN , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
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.
Asunto(s)
Acetilcoenzima A/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Represoras/metabolismo , Thermus thermophilus/genética , Acetilcoenzima A/genética , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Técnicas Biosensibles , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Conformación Molecular , Datos de Secuencia Molecular , Familia de Multigenes , Operón , Fenilacetatos/metabolismo , Proteínas Recombinantes , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Thermus thermophilus/enzimología , Transcripción GenéticaRESUMEN
Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B(1), which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system.
Asunto(s)
Proteínas Bacterianas/genética , Reparación del ADN/genética , Silenciador del Gen , Recombinación Genética/genética , Thermus thermophilus/genética , Tiamina/biosíntesis , Activación Transcripcional , Proteínas Bacterianas/metabolismo , Depuradores de Radicales Libres/metabolismo , Inmunoprecipitación , Mutagénesis , Mutación , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Thermus thermophilus/metabolismoRESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Thermus thermophilus/genética , Factores de Transcripción/metabolismo , Transferasas Alquil y Aril/genética , Alquilantes/farmacología , Alquilación/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Metilnitronitrosoguanidina/farmacología , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Fisiológico/genética , Thermus thermophilus/enzimología , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Ácidos Grasos/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/metabolismo , Thermus thermophilus/metabolismo , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas Biosensibles , Cristalografía por Rayos X , Medios de Cultivo , Ácidos Grasos/química , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Thermus thermophilus/genética , Thermus thermophilus/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Thermus thermophilus/fisiología , Transactivadores/metabolismo , Perfilación de la Expresión Génica , Análisis por Micromatrices , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Thermus thermophilus/genéticaRESUMEN
The TTHA1719 gene from Thermus thermophilus HB8 encodes an orthologue of the copper-sensing transcriptional repressor CsoR. X-ray crystal structure analysis of T. thermophilus CsoR indicated that it forms a homotetramer. The structures of the CsoR monomer and dimer are similar to those of Mycobacterium tuberculosis CsoR. In the absence of copper ions, T. thermophilus CsoR bound to the promoter region of the copper-sensitive operon copZ-csoR-copA, which encodes the copper chaperone CopZ, CsoR and the copper efflux P-type ATPase CopA, to repress their expression, while in the presence of approximately an equal amount of copper ion, CsoR was released from the DNA, to allow expression of the downstream genes. Both Cu(II) and Cu(I) ions could bind CsoR, and were effective for transcriptional derepression. Additionally, CsoR could also sense various other metal ions, such as Zn(II), Ag(I), Cd(II) and Ni(II), which led to transcriptional derepression. The copper-binding motif of T. thermophilus CsoR contains C-H-H, while those of most orthologues contain C-H-C. The X-ray crystal structure of T. thermophilus CsoR suggests that a histidine residue in the N-terminal domain is also involved in metal-ion binding; that is, the binding motif could be H-C-H-H, like that of Escherichia coli RcnR, which binds Ni(II)/Co(II). The non-conserved H70 residue in the metal-binding motif of T. thermophilus CsoR is important for its DNA-binding affinity and metal-ion responsiveness.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Thermus thermophilus/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Cobre/metabolismo , Cristalización , Regulación Bacteriana de la Expresión Génica , Conformación Molecular , Datos de Secuencia Molecular , Operón , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Thermus thermophilus/química , Thermus thermophilus/genéticaRESUMEN
The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage PhiYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)-regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection.
Asunto(s)
Thermus thermophilus/genética , Thermus thermophilus/virología , Proteínas Bacterianas/genética , Bacteriófagos/patogenicidad , Secuencia de Bases , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , ADN Bacteriano/genética , Perfilación de la Expresión Génica , Genes Bacterianos , Secuencias Invertidas Repetidas , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Thermus thermophilus/metabolismoRESUMEN
D-Alanine-D-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two D-Ala molecules using ATP to produce D-Ala-D-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 A resolution), TtDdl-adenylyl imidodiphosphate (2.6 A), TtDdl-ADP (2.2 A), TtDdl-ADP-D-Ala (1.9 A) and TtDdl-ATP-D-Ala-D-Ala (2.3 A). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdl-complex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism.
Asunto(s)
Péptido Sintasas/química , Thermus thermophilus/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Conformación ProteicaAsunto(s)
Proteínas Bacterianas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Escherichia coli/genética , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-beta-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 A resolution. The structure revealed that MjDHDPS forms a functional homotetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.