Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis
Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exem...
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| Veröffentlicht in: | Applied and Environmental Microbiology 2005-02, Vol.71 (2), p.728-733 |
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| creator | Kamionka, Annette Bertram, Ralph Hillen, Wolfgang |
| description | Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 [micro]M anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria. |
| doi_str_mv | 10.1128/AEM.71.2.728-733.2005 |
| format | Article |
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We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 [micro]M anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. 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We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 [micro]M anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.</description><subject>Bacillus subtilis - drug effects</subject><subject>Bacillus subtilis - genetics</subject><subject>Bacillus subtilis - metabolism</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>Gene Expression Regulation, Bacterial</subject><subject>Gene Silencing</subject><subject>Genetics and Molecular Biology</subject><subject>Molecular Sequence Data</subject><subject>Operator Regions, Genetic</subject><subject>Promoter Regions, Genetic</subject><subject>Repressor Proteins - metabolism</subject><subject>Tetracycline - pharmacology</subject><subject>Tetracyclines - pharmacology</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkU1v1DAQhi0EokvhJwA5cUvq748Dh7IspWorDrRny3GcXYNjb-0E1H-Pq10VOFkaP-_M6BkA3iLYIYTl2fnmphOow53AshWEdBhC9gysEFSyZYTw52AFoVItxhSegFel_IAQUsjlS3CCGFdIYbICm1s3Z2MfbPDRtZ_d3sXBxblZpzj42adoQnPhomuuYrI_0zI3PjafjPUhLKUpSz_74Mtr8GI0obg3x_cU3H3Z3K6_ttffLi7X59etpUzMrcR4IEjQwQz9KKmSTGKOIBkEM7xnPTEMUmHUgBUbCafO1AJno7NydExIcgo-Hvrul35yg62bZhP0PvvJ5AedjNf__0S_09v0SzPKJaY1_-GYz-l-cWXWky_WhWCiS0vRXFCEIIMVZAfQ5lRKduPTDAT1o39d_WuBNNbVv67-9aP_mnv374J_U0fhFWgOwM5vd799dtqUSRs3PTWryPsDMpqkzTb7ou--Y4hIPadUkHPyB2Z1log</recordid><startdate>20050201</startdate><enddate>20050201</enddate><creator>Kamionka, Annette</creator><creator>Bertram, Ralph</creator><creator>Hillen, Wolfgang</creator><general>American Society for Microbiology</general><scope>FBQ</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>5PM</scope></search><sort><creationdate>20050201</creationdate><title>Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis</title><author>Kamionka, Annette ; Bertram, Ralph ; Hillen, Wolfgang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c457t-822d3174dadbf84985826103d75a6b5b3a5047a9d295f364eaa5065fec8fe5783</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Bacillus subtilis - drug effects</topic><topic>Bacillus subtilis - genetics</topic><topic>Bacillus subtilis - metabolism</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Base Sequence</topic><topic>Gene Expression Regulation, Bacterial</topic><topic>Gene Silencing</topic><topic>Genetics and Molecular Biology</topic><topic>Molecular Sequence Data</topic><topic>Operator Regions, Genetic</topic><topic>Promoter Regions, Genetic</topic><topic>Repressor Proteins - metabolism</topic><topic>Tetracycline - pharmacology</topic><topic>Tetracyclines - pharmacology</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kamionka, Annette</creatorcontrib><creatorcontrib>Bertram, Ralph</creatorcontrib><creatorcontrib>Hillen, Wolfgang</creatorcontrib><collection>AGRIS</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>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>Kamionka, Annette</au><au>Bertram, Ralph</au><au>Hillen, Wolfgang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis</atitle><jtitle>Applied and Environmental Microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2005-02-01</date><risdate>2005</risdate><volume>71</volume><issue>2</issue><spage>728</spage><epage>733</epage><pages>728-733</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><abstract>Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. 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| ispartof | Applied and Environmental Microbiology, 2005-02, Vol.71 (2), p.728-733 |
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| source | American Society for Microbiology; MEDLINE; PubMed Central; Alma/SFX Local Collection |
| subjects | Bacillus subtilis - drug effects Bacillus subtilis - genetics Bacillus subtilis - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism Base Sequence Gene Expression Regulation, Bacterial Gene Silencing Genetics and Molecular Biology Molecular Sequence Data Operator Regions, Genetic Promoter Regions, Genetic Repressor Proteins - metabolism Tetracycline - pharmacology Tetracyclines - pharmacology |
| title | Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis |
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