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.

FIG. 1.

Map of pSCM201. The three open reading frames, named ORF1 (rep201), ORF2, and ORF3, are indicated with filled arrows. ori201 is indicated by a box, and the replication direction is indicated by an open arrow. The stem-loop represents the putative terminator of rep201. The restriction sites for appropriate enzymes are indicated, with their positions appearing in parentheses. Base 1 is arbitrarily defined by the first base of the HindIII recognition site.

FIG. 2.

Sequence analysis of the replicon of pSCM201. The AT-rich region of ori201 is boxed, and direct repeats within this AT-rich region are indicated with dashed arrows. The inverted repeats (IR-1 to IR-4) are shown with arrows and the two sets of direct repeats (DR-1 and DR-2) with half arrows. The putative iterons are surrounded by pentagons. The putative TATA box for rep201 is double underlined. The putative transcriptional terminator of rep201 is indicated with open arrows. The restriction sites (HindIII, NcoI, ClaI) are underlined. Primers' positions are indicated with bent arrows. The amino acid sequence of Rep201 (399 amino acids), encoded by the rep201 gene, is indicated below the DNA sequence. The numbers on the right indicate the nucleotide numbers of pSCM201, while the numbers on the left indicate amino acid numbers of Rep201. The putative LZ motif, the HTH motif, and the ATPase domain of Rep201 are also indicated.

FIG. 3.

Determination of the minimal replicon of pSCM201. A linear restriction map is shown on the top of the diagram. Putative ORFs (black arrows), ori201 (rectangle), and the terminator (stem-loop) shown on the restriction map were inferred from sequence analysis. Thick lines under the linear map represent the fragments cloned for autonomous-replication assays. The names of primers (indicated as gray block) are placed under the ends of each fragment. The names of recombinant plasmids containing these fragments are indicated on the left. The ability of the derivatives to replicate (repl.) in Haloarcula hispanica is indicated as “+” (yes) or “−” (no). Stability (stab.) indicates the ratio of cells (percentage) harboring the plasmids after 100 generations of growth without selective pressure.

FIG. 4.

Analyses of RIs of pSCM201 by Southern blotting (A) and electron microscopy (B). (A) Detection of the ssDNA RIs of pNB101 (left panel) and pSCM201 (right panel). (B) Uncut replication bubble-shaped molecule of pSCM201. The bar corresponds to 0.5 kb. dsDNA, double-stranded DNA.

FIG. 5.

Mapping of the RIP of pSCM201. (A) Outline of RIP mapping for the unidirectional theta replication of pSCM201 (not to scale). Nascent DNAs (leading strand and Okazaki fragments) initiated by a small RNA primer (black rectangles) were used as the templates in the primer extension reaction to determine the RIP. Ovals indicate the primers used in RIP mapping. The directions of pSCM201 replication, rep201 transcription, and primer extension are indicated by arrows. (B) Identification of the replication initiation point by RIP mapping. Primer extension was performed with the oligonucleotides RIPF for the top strand (left panel) and RIPR for the bottom strand (right panel). Lanes contain dideoxy sequencing reaction mixtures (GATC) and the products of the primer extension of pSCM201 RIs from Haloarcula sp. strain AS7094 (RH) and pSCM2011 RIs from E. coli (RE). The filled arrow indicates the RIP position of pSCM201. The open arrows indicate the positions of IR-1. The dashed lines indicate the positions of the AT-rich region. (C) Nucleotide sequence of a region containing IR-1, the AT-rich region, and the RIP of pSCM201. (D) Conserved inverted repeats in ori201 and the chromosomal replication origins of the four other haloarchaea. The filled arrows indicate the long inverted repeat flanking the AT-rich region in the four haloarchaeal chromosomal replication origins. The open arrows indicate IR-1 of ori201, which shares high homology with the inner sequence of those chromosomal long inverted repeats. NRC-1, Halobacterium sp. strain NRC-1; H. ma, Haloarcula marismotui; N. ph, Natronomonas pharaonis; H. vo, Haloferax volcanii.

FIG. 6.

Analysis of ClaI-cleaved RIs of pSCM201 by electron microscopy. (A and B) Replication bubble-shaped molecules of pSCM201. (C) Double-Y-shaped molecule of pSCM201. (D) X-shaped molecule of pSCM201. The bars correspond to 1.0 kb. The white arrows indicate the replication initiation point. (E) Analysis of the branch-point positions of the replication forks in the pSCM201 RIs. (Left) The pSCM201 RIs were schematically ordered by increasing extent of replication (one line indicates an unreplicated region; two lines indicate a replicated region). The positions of left-hand (L) and right-hand (R) branch points were measured relative to the same ClaI site. (Right) Positions of the left-hand (white circles) or right-hand (black circles) branch points are plotted versus the extent of replication.