title: Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum.
abstract: Flavobacterium psychrophilum is a very significant fish pathogen that secretes two biochemically characterized extracellular proteolytic enzymes, Fpp1 and Fpp2. The genes encoding these enzymes are organized as an fpp2-fpp1 tandem in the genome of strain F. psychrophilum THC02/90. Analysis of the corresponding encoded proteins showed that they belong to two different protease families. For gene function analysis, new genetic tools were developed in F. psychrophilum by constructing stable isogenic fpp1 and fpp2 mutants 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 the fpp2 gene. Phenotypic characterization of the fpp2 mutant 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. LD(50) experiments 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 of F. psychrophilum, open new perspectives for the study of gene function in this bacterium.
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Pérez-Pascual
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Gómez
authors.affiliation: Karolinska Institutet, Institutionen för Laboratoriemedicin, Karolinska Universitetssjukhuset, 14186 Stockholm, Sweden.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Álvarez
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Jessica
authors.initials: J
authors.lastname: Méndez
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Reimundo
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Navais
authors.affiliation: Unité de Virologie et Immunologie Moléculaires, Equipe Infection et Immunité des Poissons, INRA-Domaine de Vilvert, 78352 Jouy en Josas Cédex, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Duchaud
authors.affiliation: Área de Microbiología, Departamento de Biología Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Guijarro
mesh_terms: D000818:Animals; D002364:Caseins / Q000378:metabolism; D005393:Fish Diseases / Q000382:microbiology* / Q000401:mortality; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D007928:Lethal Dose 50; D009154:Mutation; D017686:Oncorhynchus mykiss; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002364:Caseins; D037521:Virulence Factors; D010447:Peptide Hydrolases