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Genetic and physical mapping of DNA replication origins in Haloferax volcanii.

ABSTRACT: The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.

SUBMITTER: Norais C

PROVIDER: S-EPMC1868953 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Haloferax volcanii is polyploid and contains about 20 genome copies under optimal conditions. However, the chromosome copy number is highly regulated and ranges from two during phosphate starvation to more than 40 under conditions of phosphate surplus. The aim of this study was the characterization of the influence of two replication origins on the genome copy number. The origin repeats and the genes encoding origin recognition complex (ORC) proteins were deleted. The core origin oriC1-orc1 (ori1) deletion mutant had a lower genome copy number and a higher level of fitness than the wild type, in stark contrast to the oriC2-orc5 (ori2) deletion mutant. The genes adjacent to ori1 could not be deleted, and thus, at least two of them are probably essential, while deletion of the genes adjacent to ori2 was possible. Various fragments of and around the origins were cloned into a suicide plasmid to generate haloarchaeal artificial chromosomes (HACs). The copy number of the oriC1-orc1 HAC was much higher than that of the oriC2-orc5 HAC. The addition of adjacent genes influenced both the HAC copy number and the chromosome copy number. The results indicate that the origins of H. volcanii are not independent but that the copy number is regulated via a network of genes around the origins.IMPORTANCE Several species of archaea have more than one origin of replication on their major chromosome and are thus the only known prokaryotic species that allow the analysis of the evolution of multiorigin replication. The widely studied Haloferax volcanii H26 strain has a major chromosome with four origins of replication. Two origins, ori1 and ori2, were chosen for an in-depth analysis using deletion mutants and haloarchaeal artificial chromosomes. The analysis was not restricted to the core origin regions; origin-adjacent genes were also included. Because H. volcanii is polyploid, the effects on the chromosome copy number were of specific importance. The results revealed extreme differences between the two origins.

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Genetic and physical mapping of DNA replication origins in Haloferax volcanii.

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