Cytosine Methylation by the
            <i>Sua</i>
            I Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon

Grogan, Dennis W.

doi:10.1128/jb.185.15.4657-4661.2003

Cited by 49 publications

(52 citation statements)

References 29 publications

(28 reference statements)

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“…However, since motif IV in the ORF66 protein has the SPPY sequence, the protein is more likely a N 4 -cytosine MTase. Consistent with this prediction, ORF66 is 55% identical or 77% similar to an ORF in the S. acidocaldarius genome (Chen et al, unpublished data), which is presumably responsible for the GGCC-specific N4-cytosine methylating activity identified previously (24). The presence of viral encoded restriction-modification systems is consistent with the observed modification at specific sites in the STSV1 genome.…”

Section: Vol 79 2005supporting

confidence: 74%

Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

Xiang

Chen

Huang

et al. 2005

J Virol

118

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Section: Vol 79 2005supporting

confidence: 74%

Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

Xiang

Chen

Huang

et al. 2005

J Virol

118

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“…In previous studies (Kurosawa & Grogan, 2005;Grogan & Rockwood, 2010), pyrE-containing plasmids (Table 1) generated recombinants efficiently when electroporated into S. acidocaldarius without prior protection against the SuaI RM system, which cleaves unmethylated GGCC sites (Prangishvili et al, 1985;Grogan, 2003). The implications of this can be illustrated by plasmid pSaPEv3, which contains a complete pyrE gene in a standard bacterial cloning vector (Fig.…”

Section: Circularity and Orientation Of Donor Sequencesmentioning

confidence: 99%

“…Unmethylated plasmid was produced in E. coli strain DH5a, whereas methylated (SuaI-protected) was produced in E. coli strain ER1821 [pSaPEv3, pM.EsaBC4I] (Grogan, 2003). Plasmid DNA was purified by batch chromatography (Qiagen midi-prep kit, Qiatip 100 column); purity was confirmed by gel electrophoresis, and methylation of all GGCC sites was confirmed by complete resistance to excess HaeIII (Grogan, 2003).…”

Section: Methodsmentioning

confidence: 99%

“…To test these predictions, we protected pSaPEv3 DNA against the native restriction endonuclease SuaI (Prangishvili et al, 1985) by cognate methylation in E. coli (Grogan, 2003). The resulting preparation was divided into three portions, and each was treated to produce a distinct form of DNA: (i) uncut, (ii) EcoRI-cut, and (iii) HindIII-cut (Fig.…”

Section: Circularity and Orientation Of Donor Sequencesmentioning

confidence: 99%

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Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

2013

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Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotidemediated transformation of Sulfolobus and other micro-organisms.

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“…S1 in the supplemental material). To protect the plasmid DNA from the S. acidocaldarius restriction-modification (R-M) system, SuaI sites (GGCC) were methylated in vivo prior to transformation (13,14). Both vectors express the S. solfataricus ␤-D-glycosidase lacS reporter gene, but they use different promoters, i.e., the tf55␣ gene promoter in the case of pJlacS (15) and a maltose-inducible promoter in pClacS (16).…”

Section: Methodsmentioning

confidence: 99%

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

Sakofsky

Grogan

2013

J Bacteriol

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Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the ␤-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac ؊ mutants also grew faster than the Lac ؉ parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 ؋ 10 ؊4 mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G·C-to-A·T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.T he universal need of organisms to replicate genomes accurately, and in coordination with cell division, has special significance for "extreme" or "hyper-" thermophiles, which grow optimally at temperatures that kill mesophilic organisms and denature their macromolecules (1). The hyperthermophilic archaea which colonize terrestrial and marine hydrothermal systems represent diverse and ancient lineages, and all grow optimally at temperatures that accelerate DNA decomposition reactions by orders of magnitude relative to those in mesophiles (2). The idea that these archaea should have effective strategies of genome protection which compensate for diverse damaging effects of high temperature thus seems logical and is supported by the low rates of replication errors measured in several Sulfolobus isolates (3, 4). The extreme phylogenetic divergence separating archaea from bacteria and eukaryotes (5) further suggests that the strategies which preserve genome integrity in hyperthermophilic archaea may include molecular features not identified in model organisms. This idea remains consistent with the unusual gene inventories and certain biochemical features of DNA metabolism in these archaea (6). The mechanisms that maintain genomic integrity in hyperthermophilic archaea remain challenging to identify in vivo, however. Recent studies, for example, found that several putative ...

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Cytosine Methylation by the Sua I Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon

Cited by 49 publications

References 29 publications

Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

Sulfolobus tengchongensis Spindle-Shaped Virus STSV1: Virus-Host Interactions and Genomic Features

Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids

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