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Flavobacterium psychrophilum  is a Gram-negative fish pathogen that causes important economic losses in aquaculture worldwide. Although the genome of this bacterium has been determined, the function and relative importance of genes in relation to virulence remain to be established. To investigate their respective contribution to the bacterial pathogenesis, effective tools for gene inactivation are required. In the present study, a markerless gene deletion system has been successfully developed for the first time in this bacterium. Using this method, the  F. psychrophilum   fcpB  gene, encoding a predicted cysteine protease homologous to  Streptococcus pyogenes  streptopain, was deleted. The developed system involved the construction of a conjugative plasmid that harbors the flanking sequences of the  fcpB  gene and an I- Sce I meganuclease restriction site. Once this plasmid was integrated in the genome by homologous recombination, the merodiploid was resolved by the introduction of a plasmid expressing I- Sce I under the control of the  fpp2 F. psychrophilum  inducible promoter. The resulting deleted  fcpB  mutant presented a decrease in extracellular proteolytic activity compared to the parental strain. However, there were not significant differences between their LD 50  in an intramuscularly challenged rainbow trout infection model. The mutagenesis approach developed in this work represents an improvement over the gene inactivation tools existing hitherto for this “fastidious” bacterium. Unlike transposon mutagenesis and gene disruption, gene markerless deletion has less potential for polar effects and allows the mutation of virtually any non-essential gene or gene clusters.

Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum