Abstract
Replication initiation in archaea involves a protein named ORC, Cdc6, or ORC1/Cdc6, which is homologous to the eukaryotic origin recognition complex (ORC) proteins and to the eukaryotic Cdc6. Archaeal replication origins are comprised of origin repeat regions and adjacent orc genes. Some archaea contain a single replication origin and a single orc gene, while others have more than one of each. Haloferax volcanii is exceptional because it contains, in total, six replication origins on three chromosomes and 16 orc genes. Phylogenetic trees were constructed that showed that orc gene duplications occurred at very different times in evolution. To unravel the influence of the ORC proteins on chromosome copy number and cellular fitness, it was attempted to generate deletion mutants of all 16 genes. A total of 12 single-gene deletion mutants could be generated, and only three orc gene turned out to be essential. For one gene, the deletion analysis failed. Growth analyses revealed that no deletion mutant had a growth defect, but some had a slight growth advantage compared to the wild type. Quantification of the chromosome copy numbers in the deletion mutants showed that all 12 ORC proteins influenced the copy numbers of one, two, or all three chromosomes. The lack of an ORC led to an increase or decrease of chromosome copy number. Therefore, chromosome copy numbers in Hfxvolcanii are regulated by an intricate network of ORC proteins. This is in contrast to other archaea, in which ORC proteins typically bind specifically to the adjacent origin.IMPORTANCE The core origins of archaea are comprised of a repeat region and an adjacent gene for an origin recognition complex (ORC) protein, which is homologous to eukaryotic ORC proteins. Haloferax volcanii is exceptional because it contains six replication origins on three chromosomes and an additional 10 orc genes that are not adjacent to an origin. This unique ORC protein repertoire was used to unravel the importance of core origin orc genes and of origin-remote orc genes. Remarkably, all ORC proteins influenced the copy number of at least one chromosome. Some of them influenced those of all three chromosomes, showing that cross-regulation in trans exists in Hfx. volcanii Furthermore, the evolution of the archaeal ORC protein family was analyzed.