M.FcoHsdM-dependent DNA methylation coordinates secretion-associated gene expression, interbacterial competition, and virulence in Flavobacterium columnare

Flavobacterium columnare causes columnaris disease in freshwater fish, but the contribution of DNA methylation to its physiology and pathogenicity remains poorly understood. Here, we characterized M. FcoHsdM, a type I restriction-modification methyltransferase previously identified by nanopore methylome analysis. Deletion of the gene encoding M. FcoHsdM abolished methylation at its associated recognition motifs without affecting planktonic growth. The mutant exhibited impaired biofilm formation, gliding motility, colony spreading, and gelatinolytic activity, together with increased outer-membrane vesicle production and complete attenuation in a zebrafish infection model. Transcriptomic and extracellular proteomic analyses revealed broad changes in secretion and cell surface associated functions. In particular, genes encoding components of a putative type VI secretion system were coordinately downregulated, and the mutant showed reduced antagonistic activity against Aeromonas hydrophila. Several predicted type IX secretion system substrates and extracellular enzymes were also reduced, potentially contributing to the motility and virulence defects. Two such motifs were identified in tssP, which encodes a predicted Bacteroidota-specific type VI secretion system membrane-complex component. One motif overlapped a computationally predicted binding site for the leucine-responsive regulatory protein Lrp, raising the hypothesis that intragenic methylation may influence regulator occupancy at the tssP locus. Collectively, these findings identify M.FcoHsdM-dependent methylation as an important contributor to secretion-associated gene expression, interbacterial antagonism, and virulence in F. columnare.