Differences in recombination between Haloferax volcanii and Haloferax mediterranei

Polyploidy, the phenomenon of having more than one copy of the genome in an organism, is common among haloarchaea. While providing short-term benefits for DNA repair, polyploidy is generally regarded as an 'evolutionary trap' that by the notion of the Muller's ratchet will inevitably conclude in the species' decline or even extinction due to a gradual reduction in fitness. In most reported cases of polyploidy in archaea, the genetic state of the organism is considered as homoploidy i.e. all copies of the genome are identical. Here we demonstrate that while this is indeed the prevalent genetic status in the halophilic archaeon H. volcanii, its close relative H. mediterranei maintains a prolonged heteroploidy state in a non-selective environment once a second allele is introduced. Moreover, a strong genetic linkage was observed between two distant loci in H. mediterranei indicating a low rate of homologous recombination while almost no such linkage was shown in H. volcanii indicating a high rate of recombination in the latter species. To determine whether a difference in RNA expression levels could explain the variations described above, a comparative analysis of DNA replication and homologous recombination factors in both Haloferax species was carried out. H. volcanii and H. mediterranei cultures were grown to an OD650 ˜ 0.2 and RNA was prepared using standard methods (PMID: 36125742), followed by DNAase digestion. Integrity of the RNA was checked using PCR, agarose gel electrophoresis, Qubit and Agilent TapeStation. RNA-Sequencing was carried out by the Deep Seq facility at the University of Nottingham. Sequencing and was performed using Illumina NextSeq500 instrument with a 150-cycle mid output sequencing kit to generate approx. 10 million paired end reads per sample (2 × 75bp). H. volcanii DNA repair and replication homologues were obtained from PMID: 33259746. H. mediterranei homologues were obtained using a BLASTN and SyntTax analysis (PMID: 23323735). Most genes involved in DNA replication and replication-restart showed no statistically-significant differences in expression levels between the two species. In terms of the homologous recombination pathway, although radA, mre11, rad50, and genes encoding DNA ligases had similar expression levels in both species, an increase in radB (PMID: 28501701) expression was noted in H. mediterranei stationary phase cells. This difference could result in divergent processing of DNA breaks and explain the decreased homologous recombination rate in H. mediterranei.