Isolation of Extrachromosomal Deoxyribonucleic Acids from Extremely Thermophilic Bacteria

Hishinuma, Fumio; Takano, Teruo; Sakaguchi, Kenji

doi:10.1099/00221287-104-2-193

Cited by 57 publications

(13 citation statements)

References 20 publications

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“…Agreement between the number of copies of the chromosome and of the pTT27 megaplasmid might imply that some cooperating mechanisms regulating both copy numbers exists in T. thermophilus HB8 cells. The HB8 strain also harbors the pTT8 plasmid at 8 copies per chromosome, as has already been reported (15). In consequence, the HB8 strain turns out to contain approximately 30 to 40 copies of the plasmid pTT8 per cell.…”

Section: Polyploidy Of T Thermophilusmentioning

confidence: 48%

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

2010

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An extremely thermophilic bacterium, Thermus thermophilus HB8, is one of the model organisms for systems biology. Its genome consists of a chromosome (1.85 Mb), a megaplasmid (0.26 Mb) designated pTT27, and a plasmid (9.3 kb) designated pTT8, and the complete sequence is available. We show here that T. thermophilus is a polyploid organism, harboring multiple genomic copies in a cell. In the case of the HB8 strain, the copy number of the chromosome was estimated to be four or five, and the copy number of the pTT27 megaplasmid seemed to be equal to that of the chromosome. It has never been discussed whether T. thermophilus is haploid or polyploid. However, the finding that it is polyploid is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be a polyploid organism. As is the case for D. radiodurans in the radiation environment, the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures. Polyploidy often complicates the recognition of an essential gene in T. thermophilus as a model organism for systems biology.

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Section: Polyploidy Of T Thermophilusmentioning

confidence: 48%

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

2010

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“…Judging from the intensity of the Northern blotting, the expression of total RNA in T. thermophilus cells was lower than that in E. coli cells. This can be accounted for by the likely low copy number of pEx-Asp* in T. thermophilus, because this expression vector has a replication origin derived from pTT8, whose copy number is about eight (26).…”

Section: Expression Of T Thermophilus Trnamentioning

confidence: 99%

Conserved Bases in the TΨC Loop of tRNA Are Determinants for Thermophile-specific 2-Thiouridylation at Position 54

Shigi

Suzuki

Tamakoshi

et al. 2002

Journal of Biological Chemistry

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2-Thioribothymidine (s 2 T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s 2 T54 is known to be responsible for the thermostability of tRNA. The s 2 T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s 2 T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNA Asp with an altered sequence and a host-vector for T. thermophilus. We were able to detect in vivo activity of s 2 T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNA Asp gene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction.A characteristic structural feature of tRNA is post-transcriptional modification. The roles of modified nucleosides in tRNA function are important and wide-ranging. They are known to include codon recognition, reading-frame maintenance, stabilization of the tertiary structure, and serving as identity determinants for amino acid specificity (1).The melting temperatures of tRNAs from the extreme thermophile Thermus thermophilus sp. are 3-10°C higher than those of corresponding tRNA species from the mesophilic bacterium Escherichia coli, a feature that cannot be explained solely by the higher G-C pair content in Thermus tRNAs (2). Analyses of modified nucleosides in tRNAs from T. thermophilus revealed a thermophile-specific sulfur-containing modified nucleoside that was identified as 2-thioribothymidine (s 2 T) 1 (2, 3), a 2-thiolated derivative of 5-methyluridine (ribothymidine (T)) located at position 54 in the T loop of almost all tRNAs (4). Because s 2 T54 is also present in tRNA from hyperthermophilic Archaea such as Pyrococcus furiosus, which contains about 0.77 mol % of s 2 T when cultured at 100°C (5), 2-thiolation of T54 is postulated to be a common modification responsible for the thermostabilization mechanism of tRNA in both thermophilic eubacteria and Archaea. The 2-thiolation of T54 increases along with elevation of the cultivation temperature without any changes in other modifications such as 1-methyladenosine at position 58 (m 1 A58) or 2Ј-O-methylguanosine at position 18 (Gm18) (6); more than half of the tRNAs in T. thermophilus HB8 cells grown at more than 80°C were found to contain s 2 T54 instead of T54, whereas at 50°C only a small proportion had s 2 T. In addition,...

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“…They were simple and fast compared with the time-consuming radioisotopic labelling method described by Hishinuma et al (1978) for the detection and isolation of plasmids present in T. flavus and T. thermophilus. They may be applied effectively to other bacterial species that are not amenable to existing plasmid detection and purification procedures.…”

Section: Resultsmentioning

confidence: 99%

Detection and purification of plasmids present inThermus strains from Icelandic hot springs

Koh

1985

World J Microbiol Biotechnol

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Isolation of Extrachromosomal Deoxyribonucleic Acids from Extremely Thermophilic Bacteria

Cited by 57 publications

References 20 publications

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

An Extreme Thermophile,Thermus thermophilus, Is a Polyploid Bacterium

Conserved Bases in the TΨC Loop of tRNA Are Determinants for Thermophile-specific 2-Thiouridylation at Position 54

Detection and purification of plasmids present inThermus strains from Icelandic hot springs

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