RESUMEN
A sub-surface archaeal community at the Suiyo Seamount in the Western Pacific Ocean was investigated by 16S rRNA gene sequence and whole-cell in situ hybridization analyses. In this study, we drilled and cased holes at the hydrothermal area of the seamount to minimize contamination of the hydrothermal fluid in the sub-seafloor by penetrating seawater. PCR clone analysis of the hydrothermal fluid samples collected from a cased hole indicated the presence of chemolithoautotrophic primary biomass producers of Archaeoglobales and the Methanococcales-related archaeal HTE1 group, both of which can utilize hydrogen as an electron donor. We discuss the implication of the microbial community on the early history of life and on the search for extraterrestrial life.
Asunto(s)
Archaeoglobales/aislamiento & purificación , Ecosistema , Methanococcales/aislamiento & purificación , ARN Ribosómico 16S/genética , Agua de Mar/microbiología , Archaea/genética , Archaeoglobales/genética , Biomasa , Calor , Japón , Methanococcales/genética , Océano Pacífico , Filogenia , Reacción en Cadena de la Polimerasa , ARN de Archaea , Microbiología del AguaRESUMEN
Thermostabilization of enzymes is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces ancestral residues into a target enzyme, has previously been developed and used to improve the thermostabilities of thermophilic enzymes. Herein, we report a study that used the ancestral mutation method to improve the thermostability of Bacillus circulans beta-amylase, a mesophilic enzyme. A bacterial, common-ancestral beta-amylase sequence was inferred using a phylogenetic tree composed of higher plant and bacterial amylase sequences. Eighteen mutants containing ancestral residues were designed, expressed in Escherichia coli and purified. Several of these mutants were more thermostable than that of the wild-type amylase. Notably, one mutant had both greater activity and greater thermostability. The relationship between the extent to which the amino acid residues within 5 A of the mutation site were evolutionarily conserved and the extent to which thermostability was improved was examined. Apparently, it is necessary to conserve the residues surrounding an ancestral residue if thermostability is to be improved by the ancestral mutation method.
Asunto(s)
Bacillus/enzimología , Proteínas Bacterianas/metabolismo , beta-Amilasa/metabolismo , Bacillus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Semivida , Modelos Moleculares , Mutagénesis , Filogenia , Desnaturalización Proteica , Ingeniería de Proteínas/métodos , Temperatura , beta-Amilasa/química , beta-Amilasa/genéticaRESUMEN
Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.
Asunto(s)
Actinas/química , Células Eucariotas/química , Thermoplasma/química , Secuencia de Aminoácidos , Evolución Molecular , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Polímeros/química , Thermoplasma/clasificaciónRESUMEN
Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56 degrees C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.
Asunto(s)
Proteínas Arqueales/genética , Plásmidos/genética , Thermoplasma/genética , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Secuencia de Bases , Regulación de la Expresión Génica Arqueal , Datos de Secuencia Molecular , FilogeniaRESUMEN
Thermoplasma acidophilum is sensitive to the antibiotic drug novobiocin, which inhibits DNA gyrase. We characterized DNA gyrases from T. acidophilum strains in vitro. The DNA gyrase from a novobiocin-resistant strain and an engineered mutant were less sensitive to novobiocin. The novobiocin-resistant gyrase genes might serve as T. acidophilum genetic markers.