Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum
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 co...
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| description | 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. |
| doi_str_mv | 10.1099/mic.0.046938-0 |
| format | Article |
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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.</description><identifier>ISSN: 1350-0872</identifier><identifier>EISSN: 1465-2080</identifier><identifier>DOI: 10.1099/mic.0.046938-0</identifier><identifier>PMID: 21292745</identifier><language>eng</language><publisher>Reading: Society for General Microbiology</publisher><subject>Animals ; Bacteriology ; Biological and medical sciences ; Caseins - metabolism ; Fish Diseases - microbiology ; Fish Diseases - mortality ; Flavobacterium - enzymology ; Flavobacterium - genetics ; Flavobacterium - isolation & purification ; Flavobacterium psychrophilum ; Fundamental and applied biological sciences. Psychology ; Gene Expression Profiling ; Gene Knockout Techniques ; Genetic Complementation Test ; Lethal Dose 50 ; Life Sciences ; Microbiology ; Miscellaneous ; Mutation ; Oncorhynchus mykiss ; Peptide Hydrolases - genetics ; Peptide Hydrolases - metabolism ; Promoter Regions, Genetic ; Recombination, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic ; Virulence ; Virulence Factors - genetics ; Virulence Factors - metabolism</subject><ispartof>MICROBIOLOGY-SGM, 2011-04, Vol.157 (Pt 4), p.1196-1204</ispartof><rights>2015 INIST-CNRS</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c468t-4a10cafbf5a3d296c7dbd429a9d4d027f4b2ae2dd310e068d1a28e629faf75643</citedby><cites>FETCH-LOGICAL-c468t-4a10cafbf5a3d296c7dbd429a9d4d027f4b2ae2dd310e068d1a28e629faf75643</cites><orcidid>0000-0003-3353-4714</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24047132$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21292745$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.inrae.fr/hal-02645914$$DView record in HAL$$Hfree_for_read</backlink><backlink>$$Uhttp://kipublications.ki.se/Default.aspx?queryparsed=id:122485690$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>PEREZ-PASCUAL, David</creatorcontrib><creatorcontrib>GOMEZ, Esther</creatorcontrib><creatorcontrib>ALVAREZ, Beatriz</creatorcontrib><creatorcontrib>MENDEZ, Jessica</creatorcontrib><creatorcontrib>REIMUNDO, Pilar</creatorcontrib><creatorcontrib>NAVAIS, Roberto</creatorcontrib><creatorcontrib>DUCHAUD, Eric</creatorcontrib><creatorcontrib>GUIJARRO, Jose A</creatorcontrib><title>Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum</title><title>MICROBIOLOGY-SGM</title><addtitle>Microbiology</addtitle><description>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.</description><subject>Animals</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Caseins - metabolism</subject><subject>Fish Diseases - microbiology</subject><subject>Fish Diseases - mortality</subject><subject>Flavobacterium - enzymology</subject><subject>Flavobacterium - genetics</subject><subject>Flavobacterium - isolation & purification</subject><subject>Flavobacterium psychrophilum</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Profiling</subject><subject>Gene Knockout Techniques</subject><subject>Genetic Complementation Test</subject><subject>Lethal Dose 50</subject><subject>Life Sciences</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Mutation</subject><subject>Oncorhynchus mykiss</subject><subject>Peptide Hydrolases - genetics</subject><subject>Peptide Hydrolases - metabolism</subject><subject>Promoter Regions, Genetic</subject><subject>Recombination, Genetic</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Transcription, Genetic</subject><subject>Virulence</subject><subject>Virulence Factors - genetics</subject><subject>Virulence Factors - metabolism</subject><issn>1350-0872</issn><issn>1465-2080</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0r2O1DAQAOAIgbjjoKVEbhCiyDJ2nL_ytOI4pJVooLYm9vjWkMTBThbCQ_DMeNnlEBWNPRp_M7LsybLnHDYc2vbN4PQGNiCrtmhyeJBdclmVuYAGHqa4KCGHphYX2ZMYPwOkQ-CPswvBRStqWV5mP7d-mDDg7A7EcMR-jS6mwLBhmVPWj4ysJT1H5i2z0yTytHA2J0IDu6ORIqNRe-PGOzYFP5Pv19lpRt_ngJr6fukxJPJjHeh3k5seD75DPVNwy8CmuOp98NPe9cvwNHtksY_07LxfZZ9u3n7c3ua7D-_eb693uZZVM-cSOWi0nS2xMKKtdG06I0WLrZEGRG1lJ5CEMQUHgqoxHEVDlWgt2rqsZHGV5ae-8RtNS6em4AYMq_Lo1Dn1JUWkSlkVskj-9cnvsf8H317v1DEHopJly-WBJ_vqZNNrfF0ozmpw8fgQOJJfompByqaVTfVf2TRl-qdCQpKbk9TBxxjI3l-CgzoOQqrUCtRpENSx4MW59dINZO75n59P4OUZYNTY24CjdvGvkyBrXojiF62kv1U</recordid><startdate>20110401</startdate><enddate>20110401</enddate><creator>PEREZ-PASCUAL, David</creator><creator>GOMEZ, Esther</creator><creator>ALVAREZ, Beatriz</creator><creator>MENDEZ, Jessica</creator><creator>REIMUNDO, Pilar</creator><creator>NAVAIS, Roberto</creator><creator>DUCHAUD, Eric</creator><creator>GUIJARRO, Jose A</creator><general>Society for General Microbiology</general><general>Microbiology Society</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7QL</scope><scope>C1K</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope><scope>1XC</scope><scope>ADTPV</scope><scope>AOWAS</scope><orcidid>https://orcid.org/0000-0003-3353-4714</orcidid></search><sort><creationdate>20110401</creationdate><title>Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum</title><author>PEREZ-PASCUAL, David ; GOMEZ, Esther ; ALVAREZ, Beatriz ; MENDEZ, Jessica ; REIMUNDO, Pilar ; NAVAIS, Roberto ; DUCHAUD, Eric ; GUIJARRO, Jose A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c468t-4a10cafbf5a3d296c7dbd429a9d4d027f4b2ae2dd310e068d1a28e629faf75643</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Caseins - metabolism</topic><topic>Fish Diseases - microbiology</topic><topic>Fish Diseases - mortality</topic><topic>Flavobacterium - enzymology</topic><topic>Flavobacterium - genetics</topic><topic>Flavobacterium - isolation & purification</topic><topic>Flavobacterium psychrophilum</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Profiling</topic><topic>Gene Knockout Techniques</topic><topic>Genetic Complementation Test</topic><topic>Lethal Dose 50</topic><topic>Life Sciences</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Mutation</topic><topic>Oncorhynchus mykiss</topic><topic>Peptide Hydrolases - genetics</topic><topic>Peptide Hydrolases - metabolism</topic><topic>Promoter Regions, Genetic</topic><topic>Recombination, Genetic</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Transcription, Genetic</topic><topic>Virulence</topic><topic>Virulence Factors - genetics</topic><topic>Virulence Factors - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>PEREZ-PASCUAL, David</creatorcontrib><creatorcontrib>GOMEZ, Esther</creatorcontrib><creatorcontrib>ALVAREZ, Beatriz</creatorcontrib><creatorcontrib>MENDEZ, Jessica</creatorcontrib><creatorcontrib>REIMUNDO, Pilar</creatorcontrib><creatorcontrib>NAVAIS, Roberto</creatorcontrib><creatorcontrib>DUCHAUD, Eric</creatorcontrib><creatorcontrib>GUIJARRO, Jose A</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Hyper Article en Ligne (HAL)</collection><collection>SwePub</collection><collection>SwePub Articles</collection><jtitle>MICROBIOLOGY-SGM</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>PEREZ-PASCUAL, David</au><au>GOMEZ, Esther</au><au>ALVAREZ, Beatriz</au><au>MENDEZ, Jessica</au><au>REIMUNDO, Pilar</au><au>NAVAIS, Roberto</au><au>DUCHAUD, Eric</au><au>GUIJARRO, Jose A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum</atitle><jtitle>MICROBIOLOGY-SGM</jtitle><addtitle>Microbiology</addtitle><date>2011-04-01</date><risdate>2011</risdate><volume>157</volume><issue>Pt 4</issue><spage>1196</spage><epage>1204</epage><pages>1196-1204</pages><issn>1350-0872</issn><eissn>1465-2080</eissn><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.</abstract><cop>Reading</cop><pub>Society for General Microbiology</pub><pmid>21292745</pmid><doi>10.1099/mic.0.046938-0</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0003-3353-4714</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Bacteriology Biological and medical sciences Caseins - metabolism Fish Diseases - microbiology Fish Diseases - mortality Flavobacterium - enzymology Flavobacterium - genetics Flavobacterium - isolation & purification Flavobacterium psychrophilum Fundamental and applied biological sciences. Psychology Gene Expression Profiling Gene Knockout Techniques Genetic Complementation Test Lethal Dose 50 Life Sciences Microbiology Miscellaneous Mutation Oncorhynchus mykiss Peptide Hydrolases - genetics Peptide Hydrolases - metabolism Promoter Regions, Genetic Recombination, Genetic Reverse Transcriptase Polymerase Chain Reaction Transcription, Genetic Virulence Virulence Factors - genetics Virulence Factors - metabolism |
| title | Comparative analysis and mutation effects of fpp2-fpp1 tandem genes encoding proteolytic extracellular enzymes of Flavobacterium psychrophilum |
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