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

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.

Complete Genome Sequence of Flavobacterium psychrophilum Strain OSU THCO2-90, Used for Functional Genetic Analysis

“…Its sequence type is ST9 ( 3 ), and it is moderately virulent in a rainbow trout experimental infection model using injection ( 12 ) or bath challenge (unpublished). Importantly, it is so far the only F. psychrophilum strain that can be successfully genetically manipulated ( 13 , 14 ). Indeed, this strain has proved to be a valuable tool for functional genomics and has already contributed to the characterization of several genes involved in pathogenicity ( 12 , 15 – 17 ).…”

More Than Gliding: Involvement of GldD and GldG in the Virulence of Flavobacterium psychrophilum

“…Furthermore, the mechanisms of pathogenicity of this microorganism are still poorly understood (Álvarez et al, 2006 , 2008 ; Pérez-Pascual et al, 2011 , 2015 ; Nakayama et al, 2015 ). Several improvements have been reported during the last decades in bacterial physiology (Álvarez et al, 2004 ; Pérez-Pascual et al, 2009 ), molecular diagnosis (Cepeda and Santos, 2000 ; del Cerro et al, 2002 ; Fujiwara-Nagata and Eguchi, 2009 ; Strepparava et al, 2014 ), molecular epidemiology (Nicolas et al, 2008 ; Siekoula-Nguedia et al, 2012 ; Fujiwara-Nagata et al, 2013 ; Avendaño-Herrera et al, 2014 ; Nilsen et al, 2014 ; Van Vliet et al, 2016 ; Ngo et al, 2017 ), genome analysis (Duchaud et al, 2007 ; Wiens et al, 2014 ; Wu et al, 2015 ; Rochat et al, 2017a , b ), and development of genetic tools (Álvarez et al, 2006 ; Pérez-Pascual et al, 2011 ; Gómez et al, 2012 , 2015 ), opening the way for functional genomics studies.…”

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum

“…A gene deletion procedure was previously developed for F. psychrophilum strain OSU THCO2-90 (43). This double recombination procedure was similar to the one described here, but it relied on an introduced restriction enzyme site for counterselection against strains carrying the integrated plasmid.…”

“…Genetic manipulations remain challenging, however, at least in part because of the low frequencies of gene transfer into F. psychrophilum (42). This is especially challenging for the gene deletion approach described above, which requires multiple rounds of gene transfer (43). Another method to construct gene deletions was recently developed for F. columnare (44) and modified for use in many other Bacteroidetes (45).…”