Inteins are selfish genetic elements residing in open reading frames that can splice posttranslationally, resulting in the ligation of an uninterrupted, functional protein. Like other inteins, the DNA polymerase B (PolB) intein of the halophilic archaeon Haloferax volcanii has an active homing endonuclease (HEN) domain, facilitating its horizontal transmission. Previous work has shown that the presence of the PolB intein exerts a significant fitness cost on the organism compared to an intein-free isogenic H. volcanii. Here, we show that mutation of a conserved residue in the HEN domain not only reduces intein homing but also slows growth. Surprisingly, although this mutation is far from the protein splicing active site, it also significantly reduces in vitro protein splicing. Moreover, two additional HEN domain mutations, which could not be introduced to H. volcanii, presumably due to lethality, also eliminate protein splicing activity in vitro. These results suggest an interplay between HEN residues and the protein splicing domain, despite an over 35 Å separation in a PolB intein homology model. The combination of in vivo and in vitro evidence strongly supports a model of co-dependence between the self-splicing domain and the HEN domain that has been alluded to by previous in vitro studies of protein splicing with HEN domain-containing inteins.

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. We suggest that H. volcanii escapes Muller’s ratchet by means of an effective chromosome- equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.

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 Mullers 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. We suggest that H. volcanii escapes Mullers ratchet by means of an effective chromosome- equalizing gene-conversion mechanism facilitated by highly active homologous recombination, whereas H. mediterranei must elude the ratchet via a different, yet to be elucidated mechanism.