Optimal Preservation Method for Long-term Preservation in ddH2O and Starvation Adaptive Mechanisms of Flavobacterium columnare Revealed by Multi-omic Analysis

Aquatic bacterial pathogens cause recurrent diseases in aquaculture. However, their survival dynamics and adaptive mechanisms in nutrient-limited aquatic environments remain poorly understood. In this study, we used Flavobacterium columnare, the causative agent of columnare disease, as an example to study multi-level (morphological, genomic, transcriptomic, epigenomic) adaptive changes during prolonged starvation. Additionally, we proposed an alternative long-term preservation strategy for this pathogen. We stored F. columnare across four preservation water conditions and three temperatures for 12 months, and found that double-distilled water (ddH₂O) (at 22℃ and 28℃) and pond water (at 22℃) effectively maintained bacterial viability. Starvation induced a rhizoid-to-smooth colony morphological shift, with no significant genomic mutations detected. Epigenetically, the CAYNNNNNRTG motif exhibited significantly reduced DNA methylation in smooth morphotypes relative to rhizoid ones. Transcriptomic analysis identified 851 differentially expressed genes (DEGs; 418 upregulated, 433 downregulated) in smooth F. columnare, with upregulated DEGs enriched in cell wall/membrane biogenesis and replication/recombination/repair, and downregulated DEGs associated with translation/ribosomal biogenesis and energy production. Notably, denitrification-related genes (e.g., narT, CuNir, norD) and transposase genes (ISL3, IS1182 families) were significantly upregulated, whereas ribosomal protein and type VI secretion system genes were downregulated. Integrative methylome-transcriptome analysis revealed C⁶ᵐAYNNNNNRTG hypomethylation as a remarkable epigenetic signature associated with starvation adaptation in F. columnare, targeting gene regulatory regions to remodel expression networks. Our findings establish a practical F. columnare preservation method and elucidate key starvation-adaptive mechanisms, informing targeted interventions to mitigate this pathogen’s nutrient-deprivation tolerance in natural aquatic systems.