Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum

• Flavobacterium psychrophilumis a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as anfpp2–fpp1tandem in the genome of strainF. psychrophilumTHC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed inF. psychrophilumby constructing stable isogenicfpp1andfpp2mutants via single-crossover homologous recombination. RT-PCR analysis of wild-type and mutant strains suggested that both genes are transcribed as a single mRNA from the promoter located upstream of thefpp2gene. Phenotypic characterization of thefpp2mutant showed lack of caseinolytic activity and higher colony spreading compared with the wild-type strain. Both characteristics were recovered in the complemented strain. One objective of this work was to assess the contribution to virulence of these proteolytic enzymes. LD50experiments using the wild-type strain and mutants showed no significant differences in virulence in a rainbow trout challenge model, suggesting instead a possible nutritional role. The gene disruption procedure developed in this work, together with the knowledge of the complete genome sequence ofF. psychrophilum, open new perspectives for the study of gene function in this bacterium.

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