Nucleotide sequence of a high copy number plasmid from<i>Halobacterium</i>strain GRB

Hackett, Neil R.; Krebs, Mark P.; DasSarma, Shiladitya; Goebel, Werner; RajBhandary, U L; Khorana, H. G.

doi:10.1093/nar/18.11.3408

Cited by 27 publications

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“…The best site for the insertion of foreign DNA into pGT5 might be in between ORF1 and -2 or in ORF2, which might not be required for plasmid replication, since RC plasmids such as pGRB1 and pRQ7 contain a single ORF (18,20). Recent experiments indicate that recombinant pGT5 containing foreign DNA inserted between ORF1 and -2 can be propagated into P. furiosus and related strains (1,53).…”

Section: Discussionmentioning

confidence: 99%

“…Several plasmids in extreme halophiles and methanogens have been described, and a few of them were completely sequenced: pHV2 (6.3 kb) from Haloferax volcanii (7), pGRB1 (1.8 kb) from Halobacterium sp. strain GRB and its relatives pGN101 and pHSB1 (18,19,26), and pME2001 (4.4 kb) from Methanobacterium thermoautotrophicum Marburg (4). The minimal replication regions for pHH1 (ϳ150 kb) and pNRC100 (ϳ200 kb) from Halobacterium salinarium (33,37) and pHK2 (10.5 kb) from Haloferax sp.…”

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Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile

et al. 1996

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The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the doublestranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.The isolation and characterization of plasmids are prerequisites for the development of genetic studies on new groups of microorganisms. Plasmids also are essential tools for studying in vivo and in vitro mechanisms such as DNA replication, recombination, and repair (28). Bacterial plasmids have been extensively analyzed and used in molecular and genetic work. In contrast, information about plasmids in the domain Archaea (the third domain of life sensu Woese et al. [49]) is much more limited. Several plasmids in extreme halophiles and methanogens have been described, and a few of them were completely sequenced: pHV2 (6.3 kb) from Haloferax volcanii (7), pGRB1 (1.8 kb) from Halobacterium sp. strain GRB and its relatives pGN101 and pHSB1 (18,19,26), and pME2001 (4.4 kb) from Methanobacterium thermoautotrophicum Marburg (4). The minimal replication regions for pHH1 (ϳ150 kb) and pNRC100 (ϳ200 kb) from Halobacterium salinarium (33, 37) and pHK2 (10.5 kb) from Haloferax sp. strain Aa2.2 (24) were defined and sequenced. The plasmid pGRB1 and its relatives, as well as pHK2, encode homologous proteins that exhibit similarities with Rep proteins from rolling-circle (RC) replicons of the X174 group (24, 25). In contrast, pNRC100 and pHV2 encode homologous Rep proteins (encoded by repH) unrelated to those of the RC replicons (33). Some of the haloarchaeal plasmids have been used successfully for the construction of shuttle vectors (23,29,30). The number of plasmids described for thermophilic archaea is restricted compared with those described for halophiles and methanogens. The first extrachromosomal element detected in a member of the order Sulfolobales, pSB12 (15.5 kb) (50), was in fact the genome of a lysogenic virus, Sulfolobus shibatae virus 1 (SSV1) (for a review, see refere...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile

et al. 1996

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“…Plasmid pNG11 (15.5 kb), which contains the origin of replication of the 200-kb H. halobium NRC-1 plasmid pNRC100, the Haloferax volcanii mev gene (22), and the pTZ19r Escherichia coli plasmid (Pharmacia-LKB, Piscataway, N.J.), has been previously described (28). Plasmid pCY1 was constructed by the ligation of two fragments, a 3.5-kb BamHI fragment containing the mev gene from pNGMEV100 (28) and the SalI fragment of 1.8-kb Halobacterium plasmid pGRB1 (16). Plasmid pCY33 (5.9 kb) was constructed by inserting into pCY1 by a multistep procedure a 601-bp fragment containing the bop promoter which had previously been amplified by PCR (32).…”

Section: Methodsmentioning

confidence: 99%

Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure

et al. 1996

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The bop gene of wild-type Halobacterium halobium NRC-1 is transcriptionally induced more than 20-fold under microaerobic conditions. bop transcription is inhibited by novobiocin, a DNA gyrase inhibitor, at concentrations subinhibitory for growth. The exposure of NRC-1 cultures to novobiocin concentrations inhibiting bop transcription was found to partially relax plasmid DNA supercoiling, indicating the requirement of high DNA supercoiling for bop transcription. Next, the bop promoter region was cloned on an H. halobium plasmid vector and introduced into NRC-1 and S9, a bop overproducer strain. The cloned promoter was active in both H. halobium strains, but at a higher level in the overproducer than in the wild type. Transcription from the bop promoter on the plasmid was found to be inhibited by novobiocin to a similar extent as was transcription from the chromosome. When the cloned promoter was introduced into S9 mutant strains with insertions in either of two putative regulatory genes, brp and bat, no transcription was detectable, indicating that these genes serve to activate transcription from the bop promoter in trans. Deletion analysis of the cloned bop promoter from a site ϳ480 bp upstream of bop showed that a 53-bp region 5 to the transcription start site is sufficient for transcription, but a 28-bp region is not. An 11-bp alternating purine-pyrimidine sequence within the functional promoter region, centered 23 bp 5 to the transcription start point, was found to display DNA supercoiling-dependent sensitivity to S1 nuclease and OsO 4 , which is consistent with a non-B-DNA conformation similar to that of left-handed Z-DNA and suggests the involvement of unusual DNA structure in supercoiling-stimulated bop gene transcription.The bop gene, encoding bacterio-opsin, the purple membrane protein of Halobacterium halobium, was one of the first archaeal genes to be cloned and sequenced (11). After cloning, several studies focused on the transcription of the bop gene and its regulation by oxygen and light. The start site for transcription was shown to map just 2 nucleotides upstream of the coding region by purification of the message, capping of its 5Ј end, and sequencing (6). Although this established bop mRNA as a primary transcript, no easily identifiable promoter could be found. Only a weak similarity to the TATA-like (box A) element was present (17). Interestingly, an 11-bp-long alternating purine-pyrimidine sequence centered 23 bp 5Ј to the transcription start site was observed and hypothesized to influence transcription by adopting left-handed Z-DNA conformation, which would be stabilized by high salt concentration and DNA supercoiling in H. halobium.The bop gene was shown to be transcriptionally induced at least 20-fold under microaerobic conditions in wild-type H. halobium strains (36, 43). Interestingly, bop gene transcription was inhibited by novobiocin, suggesting the involvement of DNA supercoiling in the modulation of promoter activity (42). Moreover, the involvement of two upstream genes in bop gene tr...

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“…GRB, four species of cccDNA (covalently-closed circular DNA) were identified: 1.6 kbp, 35 kbp, 65 kbp and an unsized but large 'minor cccDNA'. The small (actually 1.8 kbp) high-copy number plasmid has since been cloned and characterized (54) and shows remarkable heterogeneity in sequence (55,56).…”

Section: Resultsmentioning

confidence: 99%

Physical map and set of overlapping cosmid clones representing the genome of the archaeonHalobacteriumsp. GRB

1994

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Nucleotide sequence of a high copy number plasmid fromHalobacteriumstrain GRB

Cited by 27 publications

References 1 publication

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile

Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure

Physical map and set of overlapping cosmid clones representing the genome of the archaeonHalobacteriumsp. GRB

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