Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant
Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study wa...
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| Veröffentlicht in: | Archives of microbiology 2008-04, Vol.189 (4), p.397-405 |
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| creator | Rouws, Luc F. M Simões-Araújo, Jean L Hemerly, Adriana S Baldani, José I |
| description | Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5[trade mark sign]Tnp Transposome[trade mark sign] (Epicentre). Up to 1 x 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool. |
| doi_str_mv | 10.1007/s00203-007-0330-x |
| format | Article |
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Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.</description><identifier>ISSN: 0302-8933</identifier><identifier>EISSN: 1432-072X</identifier><identifier>DOI: 10.1007/s00203-007-0330-x</identifier><identifier>PMID: 18060666</identifier><identifier>CODEN: AMICCW</identifier><language>eng</language><publisher>Berlin/Heidelberg: Berlin/Heidelberg : Springer-Verlag</publisher><subject>Acetobacteraceae - genetics ; Acetobacteraceae - physiology ; Bacteria ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacteriology ; Biochemistry ; Biofilms ; Biofilms - growth & development ; Biological and medical sciences ; Biomedical and Life Sciences ; Biotechnology ; Cell Biology ; Chromosomes, Bacterial - genetics ; DNA Transposable Elements ; Ecology ; Electroporation ; Flagella - genetics ; Flagella - metabolism ; Fundamental and applied biological sciences. Psychology ; Genes ; Genomes ; Gluconacetobacter diazotrophicus ; Life Sciences ; Microbial Ecology ; Microbiology ; Miscellaneous ; Molecular Sequence Data ; Motility ; Mutagenesis ; Mutagenesis, Insertional ; Mutants ; Nitrogen ; Nitrogen fixation ; Original Paper ; Phenotype ; Plant growth ; Polymerase Chain Reaction ; Sugarcane</subject><ispartof>Archives of microbiology, 2008-04, Vol.189 (4), p.397-405</ispartof><rights>Springer-Verlag 2007</rights><rights>2008 INIST-CNRS</rights><rights>Springer-Verlag 2008</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c454t-47105ef43ffedfb10ca0b9bcdc36e503dfdfd175d720339afe1b18ab935e37493</citedby><cites>FETCH-LOGICAL-c454t-47105ef43ffedfb10ca0b9bcdc36e503dfdfd175d720339afe1b18ab935e37493</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00203-007-0330-x$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00203-007-0330-x$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27922,27923,41486,42555,51317</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20213898$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18060666$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Rouws, Luc F. M</creatorcontrib><creatorcontrib>Simões-Araújo, Jean L</creatorcontrib><creatorcontrib>Hemerly, Adriana S</creatorcontrib><creatorcontrib>Baldani, José I</creatorcontrib><title>Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant</title><title>Archives of microbiology</title><addtitle>Arch Microbiol</addtitle><addtitle>Arch Microbiol</addtitle><description>Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5[trade mark sign]Tnp Transposome[trade mark sign] (Epicentre). Up to 1 x 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.</description><subject>Acetobacteraceae - genetics</subject><subject>Acetobacteraceae - physiology</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacteriology</subject><subject>Biochemistry</subject><subject>Biofilms</subject><subject>Biofilms - growth & development</subject><subject>Biological and medical sciences</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnology</subject><subject>Cell Biology</subject><subject>Chromosomes, Bacterial - genetics</subject><subject>DNA Transposable Elements</subject><subject>Ecology</subject><subject>Electroporation</subject><subject>Flagella - genetics</subject><subject>Flagella - metabolism</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genes</subject><subject>Genomes</subject><subject>Gluconacetobacter diazotrophicus</subject><subject>Life Sciences</subject><subject>Microbial Ecology</subject><subject>Microbiology</subject><subject>Miscellaneous</subject><subject>Molecular Sequence Data</subject><subject>Motility</subject><subject>Mutagenesis</subject><subject>Mutagenesis, Insertional</subject><subject>Mutants</subject><subject>Nitrogen</subject><subject>Nitrogen fixation</subject><subject>Original Paper</subject><subject>Phenotype</subject><subject>Plant growth</subject><subject>Polymerase Chain Reaction</subject><subject>Sugarcane</subject><issn>0302-8933</issn><issn>1432-072X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp9kUFv1DAQhS0EotvCD-ACFlK5BcaeJI6PVQUFqRIHWsQtmjj2bqpsvNiO1PbO_8bbrKDigHzwaPy9NyM_xl4JeC8A1IcIIAGLXBaACMXtE7YSJcoClPzxlK0AQRaNRjxixzHeAAjZNM1zdiQaqKGu6xX79Z3Goac0-Il7x4lfTRVPgaa48zH3tnOitZ1sHCKPdzHZLXc-8ItxNn4iY5PvyCQbeD_QvU_B7zaDmSNPm-Dn9YabDYUHYLh_NMSN2XQcKTz4T-kFe-ZojPbl4T5h158-Xp1_Li6_Xnw5P7ssTFmVqSiVgMq6Ep2zvesEGIJOd6Y3WNsKsHf5CFX1Kn8LanJWdKKhTmNlUZUaT9i7xXcX_M_ZxtRuh2j2m0zWz7EVupKVRMzg23_AGz-HKe-WGY2lrlWdIbFAJvgYg3XtLgxbCnetgHYfULsE1O7LfUDtbda8PhjP3db2fxWHRDJwegAoGhpdjsIM8Q8nQQpsdJM5uXAxP01rGx5t-J_pbxaRI9_SOmTj628SBAI0qlJa4W9s4LWa</recordid><startdate>20080401</startdate><enddate>20080401</enddate><creator>Rouws, Luc F. 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Psychology</topic><topic>Genes</topic><topic>Genomes</topic><topic>Gluconacetobacter diazotrophicus</topic><topic>Life Sciences</topic><topic>Microbial Ecology</topic><topic>Microbiology</topic><topic>Miscellaneous</topic><topic>Molecular Sequence Data</topic><topic>Motility</topic><topic>Mutagenesis</topic><topic>Mutagenesis, Insertional</topic><topic>Mutants</topic><topic>Nitrogen</topic><topic>Nitrogen fixation</topic><topic>Original Paper</topic><topic>Phenotype</topic><topic>Plant growth</topic><topic>Polymerase Chain Reaction</topic><topic>Sugarcane</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rouws, Luc F. 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M</au><au>Simões-Araújo, Jean L</au><au>Hemerly, Adriana S</au><au>Baldani, José I</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant</atitle><jtitle>Archives of microbiology</jtitle><stitle>Arch Microbiol</stitle><addtitle>Arch Microbiol</addtitle><date>2008-04-01</date><risdate>2008</risdate><volume>189</volume><issue>4</issue><spage>397</spage><epage>405</epage><pages>397-405</pages><issn>0302-8933</issn><eissn>1432-072X</eissn><coden>AMICCW</coden><abstract>Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium, which was originally isolated from the interior of sugarcane plants. The genome of strain PAL5 of G. diazotrophicus has been completely sequenced and a next step is the functional characterization of its genes. The aim of this study was to establish an efficient mutagenesis method, using the commercial Tn5 transposon EZ::Tn5[trade mark sign]Tnp Transposome[trade mark sign] (Epicentre). Up to 1 x 10⁶ mutants per microgram of transposome were generated in a single electroporation experiment. Insertion-site flanking sequences were amplified by inverse PCR and sequenced for 31 mutants. For ten of these mutants, both insertion flanks could be identified, confirming the 9 bp duplication that is typical for Tn5 transposition. Insertions occurred in a random fashion and were genetically stable for at least 50 generations. One mutant had an insertion in a homolog of the flagellar gene flgA, and was therefore predicted to be affected in flagella-dependent traits and used to validate the applied mutagenesis methodology. This mutant lacked flagella and was non-motile on soft agar. Interestingly, it was also strongly affected in the ability to form biofilm on glass wool.</abstract><cop>Berlin/Heidelberg</cop><pub>Berlin/Heidelberg : Springer-Verlag</pub><pmid>18060666</pmid><doi>10.1007/s00203-007-0330-x</doi><tpages>9</tpages></addata></record> |
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| ispartof | Archives of microbiology, 2008-04, Vol.189 (4), p.397-405 |
| issn | 0302-8933 1432-072X |
| language | eng |
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| subjects | Acetobacteraceae - genetics Acetobacteraceae - physiology Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacteriology Biochemistry Biofilms Biofilms - growth & development Biological and medical sciences Biomedical and Life Sciences Biotechnology Cell Biology Chromosomes, Bacterial - genetics DNA Transposable Elements Ecology Electroporation Flagella - genetics Flagella - metabolism Fundamental and applied biological sciences. Psychology Genes Genomes Gluconacetobacter diazotrophicus Life Sciences Microbial Ecology Microbiology Miscellaneous Molecular Sequence Data Motility Mutagenesis Mutagenesis, Insertional Mutants Nitrogen Nitrogen fixation Original Paper Phenotype Plant growth Polymerase Chain Reaction Sugarcane |
| title | Validation of a Tn5 transposon mutagenesis system for Gluconacetobacter diazotrophicus through characterization of a flagellar mutant |
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