A reporter gene system for the hyperthermophilic archaeon <i>Sulfolobus solfataricus</i> based on a selectable and integrative shuttle vector

Jonuscheit, Melanie; Martusewitsch, Erika; Stedman, Kenneth M.; Schleper, Christa

doi:10.1046/j.1365-2958.2003.03509.x

Cited by 139 publications

(149 citation statements)

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“…Nevertheless, single-base-pair changes, including 10 substitutions, 2 insertions, and 2 deletions, were observed, as well as a sequence duplication and two larger deletions ( Table 2). The deletion mutants, in particular, could prove useful for developing genetic systems for S. solfataricus P2 (15). Furthermore, about one-seventh of the mutations occurred elsewhere in the genome.…”

Section: Discussionmentioning

confidence: 99%

Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2

Redder

Garrett

2006

J Bacteriol

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The genome of Sulfolobus solfataricus P2 carries a larger number of transposable elements than any other sequenced genome from an archaeon or bacterium and, as a consequence, may be particularly susceptible to rearrangement and change. In order to gain more insight into the natures and frequencies of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS) elements and a single nonautonomous mobile element, SM2. For each of these, the element was identified and shown to be present at its original genomic position, consistent with a progressive increase in the copy numbers of the mobile elements. In addition, several base pair substitutions, as well as small deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries were defined using a specially developed "in vitro library" strategy. Moreover, while searching for the donor mobile elements, evidence was found for two major changes that had occurred in the genome of strain P2, one constituting a single deletion of about 4% of the total genome (124 kb), while the other involved the inversion of a 25-kb region. Both were bordered by IS elements and were inferred to have arisen through recombination events. The results underline the caution required in working experimentally with an organism such as S. solfataricus with a continually changing genome.

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Section: Discussionmentioning

confidence: 99%

Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2

Redder

Garrett

2006

J Bacteriol

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“…The salient features of these systems are that the uracil marker can be reused (see Figure 1b), and that transformation is carried out using circular DNA (transformation using linear DNA is inefficient in some species). Uracil-auxotrophic mutants have also been isolated in P. abyssi 48,81 , S. acidocaldarius 82 and S. solfataricus 83 , but gene knock-out systems have yet to be developed. A trivial problem is that Gelrite, the gelling agent used in solid media for hyperthermophiles, contains trace uracil 84 (see Box 3).…”

Section: Transformationmentioning

confidence: 99%

“…A trivial problem is that Gelrite, the gelling agent used in solid media for hyperthermophiles, contains trace uracil 84 (see Box 3). More seriously, in the widely used S. solfataricus P1 and P2 strains, gene-targeting constructs fail to recombine with the chromosome 83 . While it is possible that recombination is suppressed in S. solfataricus due to active transposable elements 84 , Halobacterium also suffers from active transposition but is proficient for recombination 85 .…”

Section: Transformationmentioning

confidence: 99%

Archaeal genetics — the third way

2005

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| For decades, archaea were misclassified as bacteria on account of their prokaryotic morphology. Molecular phylogeny eventually revealed that archaea, like bacteria and eukaryotes, are a fundamentally distinct domain of life. Genome analyses have confirmed that archaea share many features with eukaryotes, particularly in information processing, and therefore can serve as streamlined models for understanding eukaryotic biology. Biochemists and structural biologists have embraced the study of archaea but geneticists have been more wary, despite the fact that genetic techniques for archaea are quite sophisticated. It is high time for geneticists to start asking fundamental questions about our distant relatives.

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“…The fuselloviruses of Sulfolobus replicate as double-stranded circular DNA and have been used as self-spreading plasmids (Jonuscheit et al, 2003;Schleper et al, 1995;Stedman et al, 1999). However, with larger insertions these plasmids are not very stable (Jonuscheit et al, 2003). Their copy number control is also not well understood.…”

Section: Plasmid Copy Number and Stabilitymentioning

confidence: 99%

“…An impressive variety of mobile genetic elements has recently been discovered in Archaea in general, and in Sulfolobus in particular: viruses, autonomous insertion sequence (IS) elements, non-autonomous miniature inverted repeat transposable elements (MITEs), small nonconjugative plasmids and large conjugative plasmids (Brugger et al, 2002;Prangishvili et al, 2001;Rice et al, 2001;Zillig et al, 1998). Although there have been impressive recent developments in Sulfolobus genetics, this remains a bottleneck (Albers et al, 2006;Bartolucci et al, 2003;Jonuscheit et al, 2003;Stedman et al, 1999;Worthington et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Two novel conjugative plasmids from a single strain of Sulfolobus

et al. 2006

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Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4. The plasmids were separated from each other and transferred into Sulfolobus solfataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number and is stably maintained (pSOG2). Plasmid pSOG2 is the first Sulfolobus CP found to have these characteristics. The genomes of both pSOG plasmids have been sequenced and were compared to each other and the available Sulfolobus CPs. Interestingly, apart from a very well-conserved core, 70 % of the pSOG1 and pSOG2 genomes is largely different and composed of a mixture of genes that often resemble counterparts in previously described Sulfolobus CPs. However, about 20 % of the predicted genes do not have known homologues, not even in other CPs. Unlike pSOG1, pSOG2 does not contain a gene for the highly conserved PlrA protein nor for obvious homologues of partitioning proteins. Unlike pNOB8 and pKEF9, both pSOG plasmids lack the so-called clustered regularly interspaced short palindrome repeats (CRISPRs). The sites of recombination between the two genomes can be explained by the presence of recombination motifs previously identified in other Sulfolobus CPs. Like other Sulfolobus CPs, the pSOG plasmids possess a gene encoding an integrase of the tyrosine recombinase family. This integrase probably mediates plasmid site-specific integration into the host chromosome at the highly conserved tRNA Glu loci.

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A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector

Cited by 139 publications

References 46 publications

Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2

Mutations and Rearrangements in the Genome ofSulfolobus solfataricusP2

Archaeal genetics — the third way

Two novel conjugative plasmids from a single strain of Sulfolobus

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