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

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 the...

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Autores principales: Gómez, Esther, Álvarez, Beatriz, Duchaud, Eric, Guijarro, José A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333118/
https://www.ncbi.nlm.nih.gov/pubmed/25692569
http://dx.doi.org/10.1371/journal.pone.0117969
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author Gómez, Esther
Álvarez, Beatriz
Duchaud, Eric
Guijarro, José A.
author_facet Gómez, Esther
Álvarez, Beatriz
Duchaud, Eric
Guijarro, José A.
author_sort Gómez, Esther
collection PubMed
description 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 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.
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spelling pubmed-43331182015-02-24 Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum Gómez, Esther Álvarez, Beatriz Duchaud, Eric Guijarro, José A. PLoS One Research Article 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 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. Public Library of Science 2015-02-18 /pmc/articles/PMC4333118/ /pubmed/25692569 http://dx.doi.org/10.1371/journal.pone.0117969 Text en © 2015 Gómez et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Gómez, Esther
Álvarez, Beatriz
Duchaud, Eric
Guijarro, José A.
Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title_full Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title_fullStr Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title_full_unstemmed Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title_short Development of a Markerless Deletion System for the Fish-Pathogenic Bacterium Flavobacterium psychrophilum
title_sort development of a markerless deletion system for the fish-pathogenic bacterium flavobacterium psychrophilum
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333118/
https://www.ncbi.nlm.nih.gov/pubmed/25692569
http://dx.doi.org/10.1371/journal.pone.0117969
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