An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1

Abstract

We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.

FIG. 1.

Chromosomal orc1-cdc6 loci and plasmid constructs for assaying ARS activity. The figure shows the orc6 (A), orc7 (B), and orc8 (C) genetic loci and schematics of DNA inserted into plasmid pNGMEV101 to assay for autonomous replication ability. The extent of regions cloned into pNGMEV101 is shown by lines below the genetic map with names of constructed plasmids indicated.

FIG. 2.

ARS assay for Halobacterium strain NRC-1 chromosomal elements. Halobacterium strain NRC-1 cultures were transformed with plasmid constructs, containing DNA sequences proximal to and including each of the chromosomal orc genes, and spotted in 50-μl aliquots onto CM⁺-mevinolin plates to test the replication abilities of those particular plasmids. Regions containing growth of transformants are seen as dense patches, and background is observed as individual colonies.

FIG. 3.

Replication assay for orc7 plasmids in Halobacterium strain NRC-1. (Left) Southern blot analysis of orc7-containing plasmids with a bla gene probe; (right) agarose gel of purified plasmids. Lanes 1 to 5 contain Halobacterium strain NRC-1 DNA transformed with plasmids (lane 1, pBBori7; lane 2, pBB7G1000; lane 3, pBB7G750; lane 4, pBB7G500; and lane 5, pBB7G250). Lanes 6 to 10 contain plasmid DNA purified from E. coli (lane 6, pBBori7; lane 7, pBB7G1000; lane 8, pBB7G750; lane 9, pBB7G500; and lane 10, pBB7G250) visualized with ethidium bromide. Lane M contains DNA ladder markers.

FIG. 4.

GC percentage plot of the 1 kb upstream of the orc7 gene. Plotted are GC contents of 70-mers in a 50-bp sliding window for the DNA sequence 1 kb 5′ to the orc7 gene translational start. Shown above is the 31-bp inverted repeat sequence flanking a very AT-rich segment of DNA. Indicated by arrows are the positions of the inverted repeats. The horizontal line represents 50% GC.

FIG. 5.

Multiple sequence alignment of large inverted repeats upstream of orc7 genes in Halobacterium strain NRC-1, H. marismortui, and H. volcanii. Shown are the aligned conserved ARS element sequences from three haloarchaea. In boldface italics and with large arrows above are the conserved large inverted repeats flanking the AT-rich region. Also shown in boldface, at position 121, is the endpoint for the insertion into pBB7G500.

FIG. 6.

Neighbor joining tree of Orc1-Cdc6 homologs found in sequenced archaeal genomes. Included in the tree are chromosomally encoded Orc1-Cdc6 homologs from all sequenced archaeal genomes to date and from the incomplete sequences of H. marismortui and H. volcanii. Homologs of Orc1, Cdc6, and Orp1 and Cdc18, from S. cerevisiae and S. pombe, respectively, are included as outgroups. Families of Orc1-Cdc6 genes are shown with the Halobacterium strain NRC-1 homolog name. Organism abbreviations are as follows: S.ce, S. cerevisiae; S.po, S. pombe; A.fu, Archaeoglobus fulgidus; M.th, M. thermoautotrophicus; M.ac, Methanosarcina acetivorans; NRC-1, Halobacterium strain NRC-1; H.vo, H. volcanii; H.ma, H. marismortui; T.ac, Thermoplasma acidophilum; T.vn, Thermoplasma volcanium; P.ae, Pyrobaculum aerophilum; S.so, S. solfataricus; and A.pe, Aeropyrum pernix.