Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

Mankin, Alexander S.; Zyrianova, Irina M.; Kagramanova, V. K.; Garrett, Roger A.

doi:10.1073/pnas.89.14.6535

Cited by 37 publications

(22 citation statements)

References 29 publications

(28 reference statements)

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“…It should be noted, however, that in the case of S. pneumoniae, due to the prolonged exposure of cells to the drug, the possibility remained that second site mutations could contribute to the resistance. Usage of an alternative selective marker (Ani), and the efficient transformation system available for H. halobium (18,41), allowed us to demonstrate that single nucleotide substitutions in the hairpins 89 and 91 are sufficient to confer Evn resistance. Both Evn footprints and resistance mutations indicate that two specific elements of 23S rRNA, hairpins 89 and 91, participate in the drug binding.…”

Section: Discussionmentioning

confidence: 99%

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Belova¹,

Tenson²,

Xiong³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Section: Discussionmentioning

confidence: 99%

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Belova¹,

Tenson²,

Xiong³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Stable shuttle vectors have been developed (29,37,45), and homologous recombination has been demonstrated (34,43). Selection methods include changes in phenotype from auxotrophy to prototrophy (17) and resistance against a variety of antibiotics such as mevinolin (34,37,45,63), novobiocin (29), thiostrepton, and anisomycin (43). Genetic systems have also been developed for Haloarcula strains (16) and other Halobacterium strains including Halobacterium sp.…”

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confidence: 99%

“…In the halophilic archaea, procedures allowing the uptake of bacteriophage (15), plasmid (14), and genomic DNA (17) have been established with Halobacterium halobium and Halobacterium (now Haloferax) volcanii. Stable shuttle vectors have been developed (29,37,45), and homologous recombination has been demonstrated (34,43). Selection methods include changes in phenotype from auxotrophy to prototrophy (17) and resistance against a variety of antibiotics such as mevinolin (34,37,45,63), novobiocin (29), thiostrepton, and anisomycin (43).…”

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Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

et al. 2007

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We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg Tk ) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu Tk ) or a gene cluster which includes apu Tk and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 M simvastatin were isolated. The transformants exhibited growth in the presence of 20 M simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg Tk locus when the endogenous hmg Tk gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg Pf ) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The ⌬apu Tk strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu Tk is a major polysaccharide-degrading enzyme in T. kodakaraensis.

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“…It is fairly easy to culture and, like many extreme halophilic and hyperthermophilic archaea, exhibits only one set of large rRNA genes per genome (15), such that the phenotypic effects of spontaneous mutations are not masked by transcripts from other gene copies as in bacteria and eucarya. A similar strategy was used earlier for an extreme halophile, by which two point mutations, conferring antibiotic resistance (17,22), were exploited to develop a plasmid vector system that could be used to introduce designed mutations into the single-copy rRNA operon of Halobacterium halobium (23). Like the extreme halophiles (22), the thermophilic archaea are relatively resistant to antibiotics (6).…”

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A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance

et al. 1994

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Development of transformable vectors for thermophilic archaea requires the characterization of appropriate selectable marker genes. Many antibiotic inhibitors of protein biosynthesis are known to bind to rRNA; therefore, we screened 14 for their capacity to inhibit growth of the thermophilic archaeon Sulfolobus acidocaldarius. Carbomycin, celesticetin, chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton all inhibited growth by different degrees. Spontaneous drug-resistant mutants were isolated from plates containing celesticetin or chloramphenicol. Six mutants from each plate exhibited a C-2585-to-U transition in the peptidyl transferase loop of 23S rRNA (corresponding to C-2452 in Escherichia coli 23S rRNA). The single-site mutation also conferred resistance to carbomycin. The mutated 23S rRNA gene provides a potentially useful and dominant marker for a thermophilic archaeal vector.The abundance of a special class of introns in the rRNA genes of thermophilic archaea, which sometimes encode "homing" endonucleases (9,19) chloramphenicol, puromycin, sparsomycin, tetracycline, and thiostrepton strongly inhibited growth of wild-type cells, while anisomycin, clindamycin, erythromycin, kanamycin, lincomycin, and spectinomycin had little or no effect on growth, even at the highest drug concentration tested (250 jig/ml). To ensure that the antibiotics were active at the high growth temperature (70'C), they were tested at the same temperature, and under similar conditions, on the thermophilic bacterium Thermus aquaticus (4) (

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Introducing mutations into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium halobium by using an rRNA operon-based transformation system.

Cited by 37 publications

References 29 publications

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

A novel site of antibiotic action in the ribosome: Interaction of evernimicin with the large ribosomal subunit

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

A spontaneous point mutation in the single 23S rRNA gene of the thermophilic arachaeon Sulfolobus acidocaldarius confers multiple drug resistance

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