RESUMEN
Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch. Here, we report that adenosine monophosphate (AMP) activates both enzymatic activities of the bifunctional adenosine diphosphate (ADP)-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis (MmPFK/GK). To understand this phenomenon, we performed a comprehensive kinetic characterisation, including determination of the kinetics, substrate inhibition and AMP activation mechanism of this enzyme. We determined that MmPFK/GK has an ordered-sequential mechanism, in which MgADP is the first substrate to bind and AMP is the last product released. The enzyme also displays substrate inhibition by both sugar substrates; we determined that this inhibition occurs through the formation of catalytically nonproductive enzyme complexes caused by sugar binding. For both activities, the AMP activation mechanism occurs primarily through incremental changes in the affinity for the sugar substrate, with this effect being higher in the GK than in the PFK activity. Interestingly, due to the increase in the sugar substrate affinity caused by AMP, an enhancement in the sugar substrate inhibition effect was also observed for both activities, which can be explained by an increase in sugar binding leading to the formation of dead-end complexes. These results shed light on the regulatory mechanisms of methanogenic archaeal sugar metabolism, a phenomenon that has been largely unexplored.
Asunto(s)
Methanococcus , Fosfofructoquinasas , Adenosina Difosfato , Adenosina Monofosfato , Methanococcus/genética , AzúcaresRESUMEN
A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (â¼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.
Asunto(s)
Archaea/genética , ARN de Archaea/fisiología , ARN no Traducido/fisiología , Regulación de la Expresión Génica Arqueal , Halobacterium salinarum/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Methanococcus/genética , Pyrococcus furiosus/genética , Análisis de Secuencia de ARN , Sulfolobus solfataricus/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética , TranscriptomaRESUMEN
A plausible architecture of an ancient genetic code is derived from an extended base triplet vector space over the Galois field of the extended base alphabet {D,A,C,G,U}, where symbol D represents one or more hypothetical bases with unspecific pairings. We hypothesized that the high degeneration of a primeval genetic code with five bases and the gradual origin and improvement of a primeval DNA repair system could make possible the transition from ancient to modern genetic codes. Our results suggest that the Watson-Crick base pairing G identical with C and A=U and the non-specific base pairing of the hypothetical ancestral base D used to define the sum and product operations are enough features to determine the coding constraints of the primeval and the modern genetic code, as well as, the transition from the former to the latter. Geometrical and algebraic properties of this vector space reveal that the present codon assignment of the standard genetic code could be induced from a primeval codon assignment. Besides, the Fourier spectrum of the extended DNA genome sequences derived from the multiple sequence alignment suggests that the called period-3 property of the present coding DNA sequences could also exist in the ancient coding DNA sequences. The phylogenetic analyses achieved with metrics defined in the N-dimensional vector space (B(3))(N) of DNA sequences and with the new evolutionary model presented here also suggest that an ancient DNA coding sequence with five or more bases does not contradict the expected evolutionary history.
Asunto(s)
Evolución Molecular , Código Genético/genética , Modelos Genéticos , Acinetobacter baumannii/genética , Algoritmos , Aminoácidos/genética , Animales , Emparejamiento Base/genética , Análisis de Fourier , Genoma Mitocondrial/genética , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Methanococcus/genética , Mutación/genética , Filogenia , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Transcripción Genética/genéticaRESUMEN
Las bacterias metanogénicas obtienen su energía mediante la producción metabólica de gas metano, utilizando sustratos como dióxido de carbono, acetato y sustratos de metilo a través de procesos de hidrólisis y acetogenesis y son esenciales en la degradación anaerobia de la materia orgánica en la naturaleza. El propósito de esta investigación fue aislar bacterias metanogénicas para conservarlas en la colección de cultivos de la Universidad Colegio Mayor de Cundinamarca. El muestreo se realizó por duplicado de cuatro fuentes ubicadas en Bogotá D.C, Colombia, las cuales ofrecían las características ambientales para su desarrollo. El procedimiento incluyó la toma de la muestra en ambiente anaerobio, aislamiento en medios selectivos e identificación por observación de las características microscópicas con coloración de Gram y de características macroscópicas en los medios selectivos y verificación de producción de metano mediante prueba piloto. Los resultados permitieron evidenciar la presencia de bacterias de los géneros Methanococcus y Methanobacterium a partir de las fuentes seleccionadas para el estudio. Se concluyó que el mejor método para la conservación de estos géneros es la congelación con la adición de agentes reductores y glicerol como criopreservante.