Abstract
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-SceI 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-SceI 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 LD50 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.
Highlights
Flavobacterium psychrophilum is the etiological agent of the bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), which affects juvenile rainbow trout (Oncorhynchus mykiss), causing important economic losses in salmonid aquaculture worldwide
The fcpB gene is predicted to encode a 394 amino-acid protein with significant homology with bacterial cysteine endopeptidases such as streptopains and others C10 family peptidases from different bacterial species including Dyadobacter fermentans, Spirosoma linguale, Bacteroides intestinalis, S. pyogenes and Flavobacterium branchiophilum
Two major determinants were important in this procedure: i) large sequences of about 1.5 kb flanking the fcpB gene were used to maximize the probability that the first cross-over recombination event took place, ii) the expression of the I-SceI meganuclease gene under the inducible fpp2 promoter [16] to Development of a Deletion System in Flavobacterium psychrophilum facilitate the second recombination event to obtain the fcpB deletion mutant
Summary
Flavobacterium psychrophilum is the etiological agent of the bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), which affects juvenile rainbow trout (Oncorhynchus mykiss), causing important economic losses in salmonid aquaculture worldwide. 14°C [1] and, as there is no commercial vaccine, its control requires the massive use of antibiotics. It is considered a “fastidious” bacterium because it is difficult to isolate and manipulate [2]. The knowledge regarding its virulence factors is still fairly limited. In this respect, some factors associated with pathogenesis have been described. Two of them (Fpp and Fpp2) were suggested to have a nutritional role [12]
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