Polyploidy in halophilic Archaea: Evolutionary advantages and molecular regulation.

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Halophilic archaea are polyploid and the major chromosome contains several origins of replication. We will use the model species Haloferax volcanii to study the molecular details of copy number regulation using the two replication origins oriC1 and oriC2, which are very especially strong and weak, respectively. We have established Haloarchaeal Artificial Chromosomes (HACs) as an ideal tool to study the elements involved in the determination of replicon copy numbers and its growth phase-dependent regulation. Various HACs will be generated containing only the core origins C1 or C2, also adjacent genes, as well as mutated versions of core origins and extended origins. The effects on the HAC copy numbers will be quantified. In addition, tagged versions of the Origin Recognition Proteins (ORCs) and adjacent gene products will be used to unravel interaction networks at replication origins. Recently we have generated isogenic strains of H. volcanii with different ploidy levels. For the first time these strains will enable to quantify the evolutionary advantages of polyploidy using competitive growth and competitive survival experiments, respectively, of mixtures of color-coded cells. The advantages of many genome copies for the survival of stressful and mutagenic conditions, for long-term survival, for global gene regulation etc. will be addressed. Furthermore, the ploidy levels of several additional haloarchaeal species of until now uncharacterized genera will be determined.