Abstract 2801 Exploring FcoP: A Potential Virulence Factor in Flavobacterium columnare

Flavobacterium columnare is a Gram-negative, rod-shaped bacterium responsible for columnaris disease, an acute zoonotic infection affecting both wild and farmed freshwater fish.Under virulent conditions, F. columnare forms anaerobic biofilms and relies on nitrous oxide reductase (NosZ), encoded by the nosZ gene, as one of its key metabolic enzymes.NosZ activity depends on associated nos genes, which facilitate copper transport (nosL), sulfur transport (nosD), ABC transporters (nosF), and redox activity (nosXR).Active NosZ functions as a homodimer, requiring two CuA sites (each containing two copper ions) and two CuZ sites (each containing four copper ions), amounting to 12 copper ions per active complex.Additionally, the other Nos enzymes essential for NosZ activity are also copper-dependent.Given this reliance on copper, we hypothesize that F.columnare might have a nutritional requirement for copper only under anaerobic, biofilm conditions.Genomic neighborhood analysis of the nos operon revealed a pair of proteins, termed FcoP and FcoH, downstream of nosZ.FcoP belongs to the MbnP-like family (PF20243) and FcoH is a member of the bacterial cytochrome c peroxidase (bCcP)/MauG superfamily (PF03150).Members of the PF03150 family are known to modify PF20243 proteins.Given the genomic association with identified copper handling proteins and the presence of a PF03150 superfamily member, we hypothesize that FcoH may modify FcoP in a way that enables copper binding.Here, we present size exclusion chromatography (SEC), fluorescence emission studies, inductively coupled plasma mass spectrometry (ICP-MS), and UV-Vis spectroscopy, to confirm the absence of any post-translational modifications and copper binding in isolated FcoP.These findings suggest that without coexpression of FcoH, FcoP is unable to bind copper and that FcoH is essential for introducing modifications that enable FcoP to bind copper.