Refining Salinivibrio pangenome dynamics and biotechnological potential through comparative analysis

Current understanding of genomic diversity within the halophilic genus Salinivibrio relies predominantly on draft genomes, with only seven complete genomes among the 62 publicly available. Previous pangenome analysis suggested a closed genomic structure while concluding that Salinivibrio lacks polyhydroxyalkanoate (PHA) degradation capacity despite possessing biosynthesis genes. Here, we present eight complete Salinivibrio genomes from Pearse Lakes (Rottnest Island, Western Australia) generated using Oxford Nanopore long-read sequencing, alongside re-analysis of 38 high-quality public genomes (≥90% completeness and ≤5% contamination cut-off). Pangenome analysis revealed a more open structure than previously reported, with a core genome comprising 25% of total gene clusters and an accessory genome accounting for 71%. Panstripe analysis demonstrated significant temporal signal in gene gain and loss events associated with phylogenetic branch length (core: P=1.72×10⁻⁴; tip: P=2.64×10⁻¹⁴). All 46 genomes contained complete PHA biosynthesis operons (phaB-phaA-phaP-phaC) with high sequence conservation under strong purifying selection (Z=30.30, P<0.001). In a genome that readily gains and loses genes, this conservation indicates that PHA synthesis is a maintained pathway, which is difficult to reconcile with a previous report that Salinivibrio lacks PHA degradation capacity. We therefore searched the genomes by Hidden Markov Model-based homology rather than standard annotation and identified seven putative depolymerases that form a single accessory cluster in 15% of strains, all previously annotated as 3-oxoadipate enol-lactonase-2. These candidates retained all catalytic residues characteristic of active depolymerases but are divergent from reference PHA depolymerases which could explain why annotation missed them. They remain putative and require biochemical confirmation. Both the expanded pangenome and these candidates emerged from standardized homology-based re-analysis, showing that annotation-dependent approaches can overlook genomic diversity and divergent enzyme families in non-model organisms. Together, these results establish Salinivibrio as a genomically dynamic genus with potential for halophilic bioplastic production.