Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome
The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed...
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| Veröffentlicht in: | Applied and Environmental Microbiology 2008-11, Vol.74 (21), p.6756-6765 |
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| container_title | Applied and Environmental Microbiology |
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| creator | Humann, Jodi L Schroeder, Brenda K Mortimer, Michael W House, Brent L Yurgel, Svetlana N Maloney, Scott C Ward, Kristel L Fallquist, Heather M Ziemkiewicz, Hope T Kahn, Michael L |
| description | The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed. |
| doi_str_mv | 10.1128/AEM.01468-08 |
| format | Article |
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In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>EISSN: 1098-6596</identifier><identifier>DOI: 10.1128/AEM.01468-08</identifier><identifier>PMID: 18791020</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>Washington, DC: American Society for Microbiology</publisher><subject>Artificial Gene Fusion ; Bacteria ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biological and medical sciences ; Carbon sources ; Cloning ; DNA, Bacterial ; Fundamental and applied biological sciences. Psychology ; Gene expression ; Gene Expression Profiling ; Genes, Reporter ; Genetic Vectors ; Genomics ; Glucuronidase - genetics ; Glucuronidase - metabolism ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; Kinases ; Methods ; Microbiology ; Molecular Sequence Data ; Plasmids ; Proteins ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Recombination, Genetic ; Sequence Analysis, DNA ; Sinorhizobium meliloti ; Sinorhizobium meliloti - genetics ; Sinorhizobium meliloti - metabolism ; Staining and Labeling - methods ; Sugar</subject><ispartof>Applied and Environmental Microbiology, 2008-11, Vol.74 (21), p.6756-6765</ispartof><rights>2009 INIST-CNRS</rights><rights>Copyright American Society for Microbiology Nov 2008</rights><rights>Copyright © 2008, American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c521t-40331ed967f4c463d10c15364bab7d4ffefd8610559782d95ad1d7a3fc06187f3</citedby><cites>FETCH-LOGICAL-c521t-40331ed967f4c463d10c15364bab7d4ffefd8610559782d95ad1d7a3fc06187f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576705/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2576705/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,3175,3176,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=20830321$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/18791020$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Humann, Jodi L</creatorcontrib><creatorcontrib>Schroeder, Brenda K</creatorcontrib><creatorcontrib>Mortimer, Michael W</creatorcontrib><creatorcontrib>House, Brent L</creatorcontrib><creatorcontrib>Yurgel, Svetlana N</creatorcontrib><creatorcontrib>Maloney, Scott C</creatorcontrib><creatorcontrib>Ward, Kristel L</creatorcontrib><creatorcontrib>Fallquist, Heather M</creatorcontrib><creatorcontrib>Ziemkiewicz, Hope T</creatorcontrib><creatorcontrib>Kahn, Michael L</creatorcontrib><title>Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome</title><title>Applied and Environmental Microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.</description><subject>Artificial Gene Fusion</subject><subject>Bacteria</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biological and medical sciences</subject><subject>Carbon sources</subject><subject>Cloning</subject><subject>DNA, Bacterial</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene expression</subject><subject>Gene Expression Profiling</subject><subject>Genes, Reporter</subject><subject>Genetic Vectors</subject><subject>Genomics</subject><subject>Glucuronidase - genetics</subject><subject>Glucuronidase - metabolism</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>Kinases</subject><subject>Methods</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Recombination, Genetic</subject><subject>Sequence Analysis, DNA</subject><subject>Sinorhizobium meliloti</subject><subject>Sinorhizobium meliloti - genetics</subject><subject>Sinorhizobium meliloti - metabolism</subject><subject>Staining and Labeling - methods</subject><subject>Sugar</subject><issn>0099-2240</issn><issn>1098-5336</issn><issn>1098-6596</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0cFv0zAUBvAIgVgZ3DiDhQQnMp7t2E4uSFPVDsTQJLqeLcexW0-JXewEGH89Lq3G4MLJsvzTJ7_3FcVzDGcYk_rd-eLzGeCK1yXUD4oZhqYuGaX8YTEDaJqSkApOiicp3QBABbx-XJzgWjQYCMyKcR58GuOkRxc8Ur5Dix-7aFLaX4NFq2mjIvrkvEoGXUflk45ut8eqRxfGG7Sc9jah9hatk_MbNG4NWjkf4tb9DK2bBjSY3vVhdOjqy9KEwTwtHlnVJ_PseJ4W6-Xiev6hvLy6-Dg_vyw1I3gsK6AUm67hwla64rTDoDGjvGpVK7rKWmO7mmNgrBE16RqmOtwJRa0Gnie09LR4f8jdTe1gOm38GFUvd9ENKt7KoJz8-8W7rdyEb5IwwQWwHPDmGBDD18mkUQ4uadP3ypswJckbgau6Iv-FBDMQlOEMX_0Db8IU8zKzgTwHYaTJ6O0B6RhSisbefRmD3Jcuc-nyd-kS6sxf3B_zDz62nMHrI1BJq97mGrVLd47kDKDk3ue2brP97qKRKg1SmUGKKo8guWA8o5cHZFWQahNz0HpFAFPAeWtNzvoFPhjKJg</recordid><startdate>20081101</startdate><enddate>20081101</enddate><creator>Humann, Jodi L</creator><creator>Schroeder, Brenda K</creator><creator>Mortimer, Michael W</creator><creator>House, Brent L</creator><creator>Yurgel, Svetlana N</creator><creator>Maloney, Scott C</creator><creator>Ward, Kristel L</creator><creator>Fallquist, Heather M</creator><creator>Ziemkiewicz, Hope T</creator><creator>Kahn, Michael L</creator><general>American Society for Microbiology</general><general>American Society for Microbiology (ASM)</general><scope>FBQ</scope><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>7QL</scope><scope>7QO</scope><scope>7SN</scope><scope>7SS</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20081101</creationdate><title>Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome</title><author>Humann, Jodi L ; Schroeder, Brenda K ; Mortimer, Michael W ; House, Brent L ; Yurgel, Svetlana N ; Maloney, Scott C ; Ward, Kristel L ; Fallquist, Heather M ; Ziemkiewicz, Hope T ; Kahn, Michael L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c521t-40331ed967f4c463d10c15364bab7d4ffefd8610559782d95ad1d7a3fc06187f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Artificial Gene Fusion</topic><topic>Bacteria</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Biological and medical sciences</topic><topic>Carbon sources</topic><topic>Cloning</topic><topic>DNA, Bacterial</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene expression</topic><topic>Gene Expression Profiling</topic><topic>Genes, Reporter</topic><topic>Genetic Vectors</topic><topic>Genomics</topic><topic>Glucuronidase - genetics</topic><topic>Glucuronidase - metabolism</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>Kinases</topic><topic>Methods</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Recombination, Genetic</topic><topic>Sequence Analysis, DNA</topic><topic>Sinorhizobium meliloti</topic><topic>Sinorhizobium meliloti - genetics</topic><topic>Sinorhizobium meliloti - metabolism</topic><topic>Staining and Labeling - methods</topic><topic>Sugar</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Humann, Jodi L</creatorcontrib><creatorcontrib>Schroeder, Brenda K</creatorcontrib><creatorcontrib>Mortimer, Michael W</creatorcontrib><creatorcontrib>House, Brent L</creatorcontrib><creatorcontrib>Yurgel, Svetlana N</creatorcontrib><creatorcontrib>Maloney, Scott C</creatorcontrib><creatorcontrib>Ward, Kristel L</creatorcontrib><creatorcontrib>Fallquist, Heather M</creatorcontrib><creatorcontrib>Ziemkiewicz, Hope T</creatorcontrib><creatorcontrib>Kahn, Michael L</creatorcontrib><collection>AGRIS</collection><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>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied and Environmental Microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Humann, Jodi L</au><au>Schroeder, Brenda K</au><au>Mortimer, Michael W</au><au>House, Brent L</au><au>Yurgel, Svetlana N</au><au>Maloney, Scott C</au><au>Ward, Kristel L</au><au>Fallquist, Heather M</au><au>Ziemkiewicz, Hope T</au><au>Kahn, Michael L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome</atitle><jtitle>Applied and Environmental Microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2008-11-01</date><risdate>2008</risdate><volume>74</volume><issue>21</issue><spage>6756</spage><epage>6765</epage><pages>6756-6765</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><eissn>1098-6596</eissn><coden>AEMIDF</coden><abstract>The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.</abstract><cop>Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>18791020</pmid><doi>10.1128/AEM.01468-08</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Artificial Gene Fusion Bacteria Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological and medical sciences Carbon sources Cloning DNA, Bacterial Fundamental and applied biological sciences. Psychology Gene expression Gene Expression Profiling Genes, Reporter Genetic Vectors Genomics Glucuronidase - genetics Glucuronidase - metabolism Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Kinases Methods Microbiology Molecular Sequence Data Plasmids Proteins Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Recombination, Genetic Sequence Analysis, DNA Sinorhizobium meliloti Sinorhizobium meliloti - genetics Sinorhizobium meliloti - metabolism Staining and Labeling - methods Sugar |
| title | Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome |
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