Integration of ploidy, Orc1/Cdc6 homolog function, and lysine acetylation with phosphate limitation and UV stress responses of Haloferax volcanii.

Haloarchaea thrive in extreme environments where hypersalinity, DNA damage, and nutrient scarcity pose significant stresses. Here, we provide insight into how defined phosphate levels, UV stress, and Orc1/Cdc6 homolog type influence growth and genome dynamics (ploidy) in the model haloarchaeon Haloferax volcanii. During phosphate limitation, cells exhibited reduced growth, heightened UV sensitivity and dynamic changes in chromosome ploidy, with an early log-phase increase followed by a reduction at later growth stages. To uncover underlying mechanisms, we examined the sixteen Orc1/Cdc6 homologs of H. volcanii using bioinformatics, AI-based structural predictions, genetics, site-directed mutagenesis, and biochemical assays. When constitutively expressed, Orc10 and Orc14 were found to increase ploidy, while Orc1 and Orc10 impaired growth in a manner exacerbated by low phosphate and UV stress. Substitutions at the lysine acetylation site within the Walker A motif (Orc1 K165) and conserved DNA-binding residues (R498 in Orc1 and R304 in Orc14) were found to affect Orc/Cdc6 homolog functions. Moreover, a ∆pat2 lysine acetyltransferase mutant was found to exhibit reduced ploidy and a striking phosphate-dependent flocking phenotype during transition to stationary phase. These results highlight the interplay between phosphate availability, chromosomal regulation, and stress adaptation. Together, this study provides insight into mechanisms that promote H. volcanii survival in conditions that induce DNA damage and phosphate limitation.