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Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

ABSTRACT: We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

SUBMITTER: Blondal T

PROVIDER: S-EPMC546137 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

Blondal Thorarinn T Thorisdottir Audur A Unnsteinsdottir Unnur U Hjorleifsdottir Sigridur S Aevarsson Arnthor A Ernstsson Sveinn S Fridjonsson Olafur H OH Skirnisdottir Sigurlaug S Wheat Jon Oskar JO Hermannsdottir Anna Gudny AG Sigurdsson Snorri Th ST Hreggvidsson Gudmundur O GO Smith Albert Vernon AV Kristjansson Jakob K JK

Nucleic acids research 20050107 1

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic a ...[more]

PMID: 15642699

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Project description:BackgroundMany strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different Thermus thermophilus strains have been completed. This paper represents the completed genome from a second Thermus species - T. scotoductus.ResultsThe genome of Thermus scotoductus SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the Thermus thermophilus genomes. The T. thermophilus megaplasmid genes are part of the T. scotoductus chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of T. scotoductus and T. thermophilus. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from Meiothermus ruber. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of Thermus scotoductus was confirmed experimentally as expected as most of the proteins of the natural transformation system of Thermus thermophilus are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle.ConclusionsThe genome of Thermus scotoductus SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to Thermus thermophilus. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of Thermus scotoductus illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.

Dataset Information

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

Publications

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

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