Intermolecular Gene Conversion for the Equalization of Genome Copies in the Polyploid Haloarchaeon Haloferax volcanii: Identification of Important Proteins

Abstract

The model haloarchaeon Haloferax volcanii is polyploid with about 20 copies of its major chromosome. Recently it has been described that highly efficient intermolecular gene conversion operates in H. volcanii to equalize the chromosomal copies. In the current study, 24 genes were selected that encode proteins with orthologs involved in gene conversion or homologous recombination in archaea, bacteria, or eukaryotes. Single gene deletion strains of 22 genes and a control gene were constructed in two parent strains for a gene conversion assay; only radA and radB were shown to be essential. Protoplast fusions were used to generate strains that were heterozygous for the gene HVO_2528, encoding an enzyme for carotinoid biosynthesis. It was revealed that a lack of six of the proteins did not influence the efficiency of gene conversion, while sixteen mutants had severe gene conversion defects. Notably, lack of paralogous proteins of gene families had very different effects, e.g., mutant Δrad25b had no phenotype, while mutants Δrad25a, Δrad25c, and Δrad25d were highly compromised. Generation of a quadruple rad25 and a triple sph deletion strain also indicated that the paralogs have different functions, in contrast to sph2 and sph4, which cannot be deleted simultaneously. There was no correlation between the severity of the phenotypes and the respective transcript levels under non-stressed conditions, indicating that gene expression has to be induced at the onset of gene conversion. Phylogenetic trees of the protein families Rad3/25, MutL/S, and Sph/SMC/Rad50 were generated to unravel the history of the paralogous proteins of H. volcanii. Taken together, unselected intermolecular gene conversion in H. volcanii involves at least 16 different proteins, the molecular roles of which can be studied in detail in future projects.