RESUMEN

OBJECTIVE: To evaluate the utility of an automated insulated isothermal PCR (iiPCR) system for rapid and reliable on-site detection of African swine fever virus (ASFV) in swine biological samples. SAMPLE: Lymph node, tissue homogenate, whole blood, serum, spleen, and tonsil samples collected from swine in North and South Vietnam. PROCEDURES: Analytic sensitivity of the iiPCR system was determined by serial dilution and analysis of 2 samples (swine tissue homogenate and blood) predetermined to be positive for ASFV. Analytic specificity was assessed by analysis of 2 samples predetermined to be negative for ASFV and positive or negative for other swine pathogens (classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, and porcine circovirus type 2). Diagnostic performance of the iiPCR system for detection of ASFV was determined by analysis of the various tissue sample types. For all tests, a real-time PCR assay was used as the reference method. RESULTS: The iiPCR system was able to detect ASFV in swine blood or tissue homogenate at dilutions up to 106, whereas the real-time PCR assay was able to detect dilutions of up to 105 or 106. The iiPCR system had high analytic specificity for detection of ASFV versus other swine pathogens. Between 97% and 100% agreement was found between results of the iiPCR system for the various tissue samples and results of real-time PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: The evaluated iiPCR system was found to be a rapid, reliable, and sample-flexible method for ASFV detection and may be useful for disease surveillance and quarantine in national strategies for early ASF control.

Asunto(s)

Virus de la Fiebre Porcina Africana , Virus de la Fiebre Porcina Africana/genética , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sensibilidad y Especificidad , Porcinos

RESUMEN

Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress. Tk0537 was found to belong to a 1-Cys Prx6 subfamily based on sequence analysis and was named 1-Cys TkPrx. Using gel filtration chromatography, electron microscopy, and blue-native polyacrylamide gel electrophoresis, we observed that 1-Cys TkPrx exhibits oligomeric forms with reduced peroxide reductase activity as well as decameric and dodecameric forms that can act as molecular chaperones by protecting both proteins and DNA from oxidative stress. Mutational analysis showed that a cysteine residue at the N-terminus (Cys46) was responsible for the peroxide reductase activity, and cysteine residues at the C-terminus (Cys205 and Cys211) were important for oligomerization. Based on our results, we propose that interconversion between different oligomers is important for regulating the different functions of 1-Cys TkPrx.

Asunto(s)

Proteínas Arqueales/metabolismo , Daño del ADN , ADN Bacteriano/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Estrés Fisiológico , Thermococcus/metabolismo , Proteínas Arqueales/química , Clonación Molecular , Radical Hidroxilo/metabolismo , Chaperonas Moleculares/química , Proteínas Mutantes/química , Oxidación-Reducción , Peroxidasa/metabolismo , Peroxirredoxinas/química , Mutación Puntual , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Temperatura , Regulación hacia Arriba

RESUMEN

AAA(+) ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.

Asunto(s)

Proteínas Arqueales/metabolismo , ADN Helicasas/metabolismo , Thermococcus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , ADN Helicasas/química , ADN Helicasas/genética , Estabilidad de Enzimas , Calor , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Thermococcus/genética

RESUMEN

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K (m) value for p-nitrophenyl butyrate was 607.1 µM with V (max) values of 95.5 U/mg. The enzyme was active at a broad range of pH (5-8) and temperatures (70-95 °C) with the optimum pH and temperature being 8.0 and 85 °C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.

Asunto(s)

Proteínas Arqueales/biosíntesis , Proteínas Arqueales/química , Lisofosfolipasa/biosíntesis , Lisofosfolipasa/química , Thermococcus/enzimología , Secuencias de Aminoácidos , Proteínas Arqueales/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Calor , Concentración de Iones de Hidrógeno , Lisofosfolipasa/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato/fisiología , Thermococcus/genética

RESUMEN

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and ß subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; ß subunit was a tetramer that had sarcosine oxidase and L: -proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αß)(4) form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and ß subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αß)(4) complex may separately exist as a function enzyme in different conditions.

Asunto(s)

Sarcosina-Oxidasa/metabolismo , Thermococcus/enzimología , Secuencia de Bases , Biocatálisis , Cromatografía en Gel , Cartilla de ADN , Microscopía Electrónica , Reacción en Cadena de la Polimerasa , Sarcosina-Oxidasa/química

RESUMEN

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis. In this study, the TkDAP1 and TkDAP2 genes were cloned and expressed in Escherichia coli. The two proteins were purified homogeneity and analyzed by gel filtration chromatography and electron microscopy. TkDAP1 showed two oligomers, which were identified as an octodecimer and a dodecamer. TkDAP2 produced three native forms: octodecimer, dodecamer, and trimer. Dodecamer assembly was the main form in the two proteins. Finally, TkDAP1 was found to have higher deblocking aminopeptidase activity on the substrates of Ac-Leu-pNA and Ac-Ala-Ala-Ala, while TkDAP2 had higher aminopeptidase activity on the substrates of Leu-pNA and Ala-Ala-Ala-pNA.

Asunto(s)

Aminopeptidasas/metabolismo , Proteínas Arqueales/metabolismo , Isoenzimas/metabolismo , Proteínas Recombinantes/metabolismo , Thermococcus/enzimología , Secuencia de Aminoácidos , Aminopeptidasas/química , Aminopeptidasas/genética , Aminopeptidasas/ultraestructura , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/ultraestructura , Cromatografía en Gel , Clonación Molecular , Electroforesis en Gel Bidimensional , Escherichia coli , Expresión Génica , Calor , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Plásmidos , Polimerizacion , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Thermococcus/genética , Transformación Bacteriana

RESUMEN

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80-90°C. The apparent K (m) values for NAD(+) and D-G3P were 77.8 ± 7.5 µM and 49.3 ± 3.0 µM, respectively, with V (max) values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.

Asunto(s)

Proteínas Arqueales/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Thermococcus/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Dominio Catalítico , Clonación Molecular , Ácidos Difosfoglicéricos/metabolismo , Estabilidad de Enzimas , Gliceraldehído 3-Fosfato/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/aislamiento & purificación , Semivida , Calor , Cinética , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Peso Molecular , Mutación , NAD/metabolismo , Estrés Oxidativo , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Thermococcus/genética

RESUMEN

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.

Asunto(s)

Proteínas Arqueales/metabolismo , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Thermococcus/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Clonación Molecular , Escherichia coli , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/aislamiento & purificación , Oxidación-Reducción , Oxígeno/administración & dosificación , Dominios y Motivos de Interacción de Proteínas/genética , Alineación de Secuencia , Thermococcus/química , Thermococcus/genética

RESUMEN

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.

Asunto(s)

Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Thermococcus/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/ultraestructura , Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , Calor , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Presión Osmótica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Thermococcus/efectos de los fármacos , Thermococcus/enzimología , terc-Butilhidroperóxido/metabolismo