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Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea.

ABSTRACT: A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon ( approximately 1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a putative replication initiation protein (Rep201). Electron microscopic observation and Southern blotting analysis demonstrated that pSCM201 replicates via a theta mechanism. Precise mapping of the putative origin suggested that the replication initiated from a fixed site close to the AT-rich region and proceeded unidirectionally toward the downstream rep201 gene, which was further confirmed by electron microscopic analysis of the ClaI-digested replication intermediates. To our knowledge, this is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. It provides a novel plasmid system to conduct research on archaeal DNA replication.

SUBMITTER: Sun C

PROVIDER: S-EPMC1698213 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea.

Sun Chaomin C Zhou Meixian M Li Yun Y Xiang Hua H

Journal of bacteriology 20060922 23

A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon ( approximately 1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a puta ...[more]

PMID: 16997958

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Project description:Organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. Cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. To improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersaline environments and routinely experience drastic changes in environmental salinity) and analyzed these new genomes in combination with those from 21 previously sequenced haloarchaeal isolates. We propose a generalized model for haloarchaeal management of cytoplasmic osmolarity in response to osmotic shifts, where potassium accumulation and sodium expulsion during osmotic upshock are accomplished via secondary transport using the proton gradient as an energy source, and potassium loss during downshock is via a combination of secondary transport and non-specific ion loss through mechanosensitive channels. We also propose new mechanisms for magnesium and chloride accumulation. We describe the expansion and differentiation of haloarchaeal general transcription factor families, including two novel expansions of the TATA-binding protein family, and discuss their potential for enabling rapid adaptation to environmental fluxes. We challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. The combination of broad (17 genera) and deep (≥5 species in four genera) sampling of a phenotypically unified clade has enabled us to uncover both highly conserved and specialized features of osmoadaptation. Finally, we demonstrate the broad utility of such datasets, for metagenomics, improvements to automated gene annotation and investigations of evolutionary processes.

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Dataset Information

Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea.

Publications

Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea.

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