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. 2012 Sep 14;13:478.
doi: 10.1186/1471-2164-13-478.

Diversity and Evolution of Multiple orc/cdc6-adjacent Replication Origins in Haloarchaea

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Free PMC article

Diversity and Evolution of Multiple orc/cdc6-adjacent Replication Origins in Haloarchaea

Zhenfang Wu et al. BMC Genomics. .
Free PMC article

Abstract

Background: While multiple replication origins have been observed in archaea, considerably less is known about their evolutionary processes. Here, we performed a comparative analysis of the predicted (proved in part) orc/cdc6-associated replication origins in 15 completely sequenced haloarchaeal genomes to investigate the diversity and evolution of replication origins in halophilic Archaea.

Results: Multiple orc/cdc6-associated replication origins were predicted in all of the analyzed haloarchaeal genomes following the identification of putative ORBs (origin recognition boxes) that are associated with orc/cdc6 genes. Five of these predicted replication origins in Haloarcula hispanica were experimentally confirmed via autonomous replication activities. Strikingly, several predicted replication origins in H. hispanica and Haloarcula marismortui are located in the distinct regions of their highly homologous chromosomes, suggesting that these replication origins might have been introduced as parts of new genomic content. A comparison of the origin-associated Orc/Cdc6 homologs and the corresponding predicted ORB elements revealed that the replication origins in a given haloarchaeon are quite diverse, while different haloarchaea can share a few conserved origins. Phylogenetic and genomic context analyses suggested that there is an original replication origin (oriC1) that was inherited from the ancestor of archaea, and several other origins were likely evolved and/or translocated within the haloarchaeal species.

Conclusion: This study provides detailed information about the diversity of multiple orc/cdc6-associated replication origins in haloarchaeal genomes, and provides novel insight into the evolution of multiple replication origins in Archaea.

Figures

Figure 1
Figure 1
Bioinformatic and genetic identification of replication origins in H. hispanica. A. Seven replication origins, oriC1-cdc6A and oriC2-cdc6E in the main chromosome; oriC4-cdc6G, oriC5-cdc6H, oriC6-cdc6I and oriC7-cdc6J in the minichromosome; and oriP-cdc6K in the megaplasmid, were predicted by searching ORB motifs (indicated with small triangles) in the IRs located directly adjacent to orc/cdc6 genes (indicated with red arrowheads) using MEME software. Logo representations of ORB elements are presented on the right, and the spaces represent sequences that are not conserved. oriC3*: predicted deficient origin adjacent to cdc6D gene. B. Replication assay for plasmids containing the origins predicted in A. (Up) Southern blot analysis with a bla gene probe: lane T contains crude DNA extracted from the H. hispanica transformants, and lane P represents the purified plasmid as an input control; (down) summaries of the identification of origins in H. hispanica and the five origins with ARS activity (oriC1, 2, 6, 7, P) are indicated with filled ovals and are bolded in A.
Figure 2
Figure 2
Families of orc/cdc6-associated replication origins in the haloarchaeal genomes. A. Phylogenetic tree of origin-associated Orc/Cdc6 homologs (Hbo: Halogeometricum borinquense, Hhi: Haloarcula hispanica, Hje: Halalkalicoccus jeotgali B3, Hla: Halorubrum lacusprofundi, Hma: Haloarcula marismortui, Hmu: Halomicrobium mukohataei, HR1: Halobacterium salinarum R1, Htu: Haloterrigena turkmenica, Hut: Halorhabdus utahensis, Hvo: Haloferax volcanii DS2, Hwa: Haloquadratum walsbyi, Nma: Natrialba magadii, Nph: Natronomonas pharaonis, NRC-1: Halobacterium sp. NRC-1). * indicates the Orc/Cdc6 proteins encoded on extrachromosomal elements. Orc/Cdc6 proteins from other archaea (APE: Aeropyrum pernix, Pab: Pyrococcus abyssi, Sso: Sulfolobus solfataricus) are highlighted with yellow background. The origin conserved in all genomes was assigned the name oriC1, as in previous reports (in red), and the other two origin clusters with the top two members, excluding oriC1 in this study, were assigned the names oriCa (in blue) and oriCb (in pink). B. Logo representations of the putative ORB elements identified in the predicted replication origins adjacent to the orc/cdc6 genes. The ORB elements are not shown for origin families with only one member.
Figure 3
Figure 3
Two different candidate replication origins are adjacent to one orc/cdc6 gene (Hla_3512 or Hwa_HQ2959A). A. The sequence features of the two putative origins. The orc/cdc6 genes are indicated with black-boxed arrows, and their start site is numbered 1. The adjacent origins (I and II) were mapped with small arrowheads and rectangles indicating the ORB elements and AT-rich regions, respectively. Origin II, in both cases, contains conserved ORB elements, which are highlighted in blue. B. Logo representations of the ORB elements in the four candidate replication origins. The ORB elements (boxed) are highly conserved in Origin II in both cases.
Figure 4
Figure 4
Comparative analysis of the orc/cdc6-associated replication origins between the chromosomes of H. hispanica and H. marismortui. A. Distribution of the candidate orc/cdc6-associated replication origins in the chromosomes of H. hispanica (inside) and H. marismortui (outside). G + C content of the chromosome of H. hispanica was plotted, and significant variations in the two divergent regions are indicated with blue arrows. The predicted orc/cdc6-associated replication origins are indicated as ovals on the chromosome circle, and the shared orc/cdc6-associated replication origins in the two Haloarcula species, oriC1 and oriC2, are highlighted as filled ovals. B. Genome alignment of the chromosomes of H. hispanica and H. marismortui. Their shared orc/cdc6-associated replication origins are indicated as in A. Regions A and B represent discrepancies between the two chromosomes, which are exactly in accordance with the positions of their specific orc/cdc6-associated replication origins; oriC3-cdc6D* of H. hispanica and oriC3-cdc6i of H. marismortui are located in region A, and oriC4-cdc6g of H. marismortui is located in region B. The divergent regions and the edges of the similar regions were confirmed by BLASTN alignments of sequences, and shaded regions denote a similarity of over 70%. Linearized scaled bars are provided. C. A schematic representation of the two divergent regions (1 kb scale for Hhis_A, Hmar_A and Hhis_B; 2 kb scale for Hmar_B) between the two chromosomes. The orc/cdc6 genes are indicated. The polysaccharide biosynthesis genes are in yellow, transposase genes in purple, other genes with known functions in pink and hypothetical genes in gray. The species with the closest matches in the BLAST analysis is indicated on top of the gene: M, Methanobacterium; A, other non-halophilic archaea; B, eubacteria (the colors are designed to correspond to the marks in Additional file 6). The genes in clusters are also in clusters in other haloarchaea, as indicated at the top of the clusters.
Figure 5
Figure 5
Comparative genomic analysis of the extrachromosomal replicons of H. hispanica and H. marismortui. The orc/cdc6 genes (those from H. hispanica and H. marismortui are highlighted with a purple asterisk and a dark green round dot, respectively) that are associated with candidate replication origins are indicated, and the shared origins associated with cdc6G/cdc6a, cdc6K/cdc6k of the two strains are highlighted in bold. The homologous regions are boxed, and the lines in the box represent the regions that are continuous in H. marismortui.
Figure 6
Figure 6
Genome context analyses of the origins in the oriCa and oriCb families. A. Phylogenetic tree based on the 16S rRNA genes (Methanocaldococcus jannaschii was added as an outgroup) and the distribution of the oriCa and oriCb families in the haloarchaeal genomes. + indicates the presence of this family of origins in the chromosome, and (+) indicates its presence in an extrachromosomal element. B and C. The regions around oriCa (B) and oriCb (C). Shaded regions denote similarity greater than 70% by BLASTN analyses. The orc/cdc6 genes are highlighted in red; genes annotated with transposase (tnp) are highlighted in purple; rRNAs, including 16S rRNA, 23S rRNA and 5S rRNA, are highlighted in yellow; tRNAs are highlighted in green, with T, C and A representing the Thr-tRNA, Cys-tRNA and Ala-tRNA genes, respectively. oriCb origins of replication in H. volcanii, H. borinquense and H. lacusprofundi are indicated by teal rectangles.

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