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Article: Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation
Title | Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation | ||||||
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Authors | |||||||
Issue Date | 2009 | ||||||
Publisher | American Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/ | ||||||
Citation | Journal Of Biological Chemistry, 2009, v. 284 n. 42, p. 28738-28745 How to Cite? | ||||||
Abstract | Essential genes encode biological functions critical for cell survival. Correspondingly, their null mutants are often difficult to obtain, which impedes subsequent genetic and functional analysis. Here, we describe the development and utility of a theophylline-responsive riboswitch that enables target gene expression to be specifically "tuned" from low to high levels, which may be used to generate conditional hypomorphic mutants. Low levels of gene activity in the absence of the ligand (theophylline) permit cell survival, enabling gene activities to be investigated. Normal gene expression levels and wild-type phenotypes can be restored by the addition of the ligand. We demonstrate the utility of this approach with csrA, an essential gene in Escherichia coli that encodes the global regulatory protein CsrA. We placed the theophylline-responsive riboswitch immediately upstream of the csrA ribosome binding site, with the resulting mutant named switch-csrA. Hypomorphism of switchc-srA and its specific responsiveness to theophylline were verified by phenotypic examination and translation analysis. The utility of switch-csrA revealed a previously unidentified function for CsrA, namely its role as a repressor of cellular autoaggregation. Specifically, switch-csrA in the non-ligand-bound form produced low levels of CsrA, and its cells autoaggregated. Theophylline binding induced conformational changes in the riboswitch and permitted efficient csrA translation; consequently, autoaggregation did not occur. Our results indicate that CsrA modulates autoaggregation via the polysaccharide adhesin poly-β-1,6-N-acetyl-D-glucosamine. In summary, the use of ligand-responsive riboswitches to construct conditional hypomorphic mutants represents a novel approach for investigating the activities of essential genes, which effectively complements traditional genetic approaches. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. | ||||||
Persistent Identifier | http://hdl.handle.net/10722/124507 | ||||||
ISSN | 2020 Impact Factor: 5.157 2023 SCImago Journal Rankings: 1.766 | ||||||
PubMed Central ID | |||||||
ISI Accession Number ID |
Funding Information: This work was supported by the University of Hong Kong Committee on Research and Conference Grants Seed Funding Program for Basic Research and by Research Grants Council of Hong Kong Grant HKU 7485/06M (to J. D. H.). | ||||||
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Grants |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Jin, Y | en_HK |
dc.contributor.author | Watt, RM | en_HK |
dc.contributor.author | Danchin, A | en_HK |
dc.contributor.author | Huang, JD | en_HK |
dc.date.accessioned | 2010-10-31T10:38:20Z | - |
dc.date.available | 2010-10-31T10:38:20Z | - |
dc.date.issued | 2009 | en_HK |
dc.identifier.citation | Journal Of Biological Chemistry, 2009, v. 284 n. 42, p. 28738-28745 | en_HK |
dc.identifier.issn | 0021-9258 | en_HK |
dc.identifier.uri | http://hdl.handle.net/10722/124507 | - |
dc.description.abstract | Essential genes encode biological functions critical for cell survival. Correspondingly, their null mutants are often difficult to obtain, which impedes subsequent genetic and functional analysis. Here, we describe the development and utility of a theophylline-responsive riboswitch that enables target gene expression to be specifically "tuned" from low to high levels, which may be used to generate conditional hypomorphic mutants. Low levels of gene activity in the absence of the ligand (theophylline) permit cell survival, enabling gene activities to be investigated. Normal gene expression levels and wild-type phenotypes can be restored by the addition of the ligand. We demonstrate the utility of this approach with csrA, an essential gene in Escherichia coli that encodes the global regulatory protein CsrA. We placed the theophylline-responsive riboswitch immediately upstream of the csrA ribosome binding site, with the resulting mutant named switch-csrA. Hypomorphism of switchc-srA and its specific responsiveness to theophylline were verified by phenotypic examination and translation analysis. The utility of switch-csrA revealed a previously unidentified function for CsrA, namely its role as a repressor of cellular autoaggregation. Specifically, switch-csrA in the non-ligand-bound form produced low levels of CsrA, and its cells autoaggregated. Theophylline binding induced conformational changes in the riboswitch and permitted efficient csrA translation; consequently, autoaggregation did not occur. Our results indicate that CsrA modulates autoaggregation via the polysaccharide adhesin poly-β-1,6-N-acetyl-D-glucosamine. In summary, the use of ligand-responsive riboswitches to construct conditional hypomorphic mutants represents a novel approach for investigating the activities of essential genes, which effectively complements traditional genetic approaches. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. | en_HK |
dc.language | eng | en_HK |
dc.publisher | American Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/ | en_HK |
dc.relation.ispartof | Journal of Biological Chemistry | en_HK |
dc.rights | Journal of Biological Chemistry. Copyright © American Society for Biochemistry and Molecular Biology, Inc. | - |
dc.rights | This research was originally published in [Journal Name]. Author(s). Title. Journal Name. Year. Vol:pp-pp. © the American Society for Biochemistry and Molecular Biology | - |
dc.subject.mesh | Escherichia coli Proteins - genetics - metabolism | - |
dc.subject.mesh | Gene Expression Regulation, Bacterial | - |
dc.subject.mesh | Mutation | - |
dc.subject.mesh | RNA, Untranslated - genetics - metabolism | - |
dc.subject.mesh | RNA-Binding Proteins - genetics - metabolism | - |
dc.title | Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation | en_HK |
dc.type | Article | en_HK |
dc.identifier.openurl | http://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0021-9258&volume=284&issue=42&spage=28738&epage=28745&date=2009&atitle=Use+of+a+riboswitch-controlled+conditional+hypomorphic+mutation+to+uncover+a+role+for+the+essential+csrA+gene+in+bacterial+autoaggregation | en_HK |
dc.identifier.email | Watt, RM:rmwatt@hku.hk | en_HK |
dc.identifier.email | Huang, JD:jdhuang@hkucc.hku.hk | en_HK |
dc.identifier.authority | Watt, RM=rp00043 | en_HK |
dc.identifier.authority | Huang, JD=rp00451 | en_HK |
dc.description.nature | link_to_OA_fulltext | - |
dc.identifier.doi | 10.1074/jbc.M109.028076 | en_HK |
dc.identifier.pmid | 19706608 | - |
dc.identifier.pmcid | PMC2781419 | - |
dc.identifier.scopus | eid_2-s2.0-70350366781 | en_HK |
dc.identifier.hkuros | 176363 | en_HK |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-70350366781&selection=ref&src=s&origin=recordpage | en_HK |
dc.identifier.volume | 284 | en_HK |
dc.identifier.issue | 42 | en_HK |
dc.identifier.spage | 28738 | en_HK |
dc.identifier.epage | 28745 | en_HK |
dc.identifier.eissn | 1083-351X | - |
dc.identifier.isi | WOS:000270678900030 | - |
dc.publisher.place | United States | en_HK |
dc.relation.project | Towards a visualized proteome of <I>Escherichia coli</I> | - |
dc.identifier.scopusauthorid | Jin, Y=29467538100 | en_HK |
dc.identifier.scopusauthorid | Watt, RM=7102907536 | en_HK |
dc.identifier.scopusauthorid | Danchin, A=7103235597 | en_HK |
dc.identifier.scopusauthorid | Huang, JD=8108660600 | en_HK |
dc.identifier.issnl | 0021-9258 | - |