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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

TitleUse of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation
Authors
Issue Date2009
PublisherAmerican 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?
AbstractEssential 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 Identifierhttp://hdl.handle.net/10722/124507
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
University of Hong Kong Committee on Research and Conference
Research Grants Council of Hong KongHKU 7485/06M
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.).

References
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DC FieldValueLanguage
dc.contributor.authorJin, Yen_HK
dc.contributor.authorWatt, RMen_HK
dc.contributor.authorDanchin, Aen_HK
dc.contributor.authorHuang, JDen_HK
dc.date.accessioned2010-10-31T10:38:20Z-
dc.date.available2010-10-31T10:38:20Z-
dc.date.issued2009en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2009, v. 284 n. 42, p. 28738-28745en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/124507-
dc.description.abstractEssential 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.languageengen_HK
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_HK
dc.relation.ispartofJournal of Biological Chemistryen_HK
dc.rightsJournal of Biological Chemistry. Copyright © American Society for Biochemistry and Molecular Biology, Inc.-
dc.rightsThis 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.meshEscherichia coli Proteins - genetics - metabolism-
dc.subject.meshGene Expression Regulation, Bacterial-
dc.subject.meshMutation-
dc.subject.meshRNA, Untranslated - genetics - metabolism-
dc.subject.meshRNA-Binding Proteins - genetics - metabolism-
dc.titleUse of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregationen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://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+autoaggregationen_HK
dc.identifier.emailWatt, RM:rmwatt@hku.hken_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1074/jbc.M109.028076en_HK
dc.identifier.pmid19706608-
dc.identifier.pmcidPMC2781419-
dc.identifier.scopuseid_2-s2.0-70350366781en_HK
dc.identifier.hkuros176363en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-70350366781&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume284en_HK
dc.identifier.issue42en_HK
dc.identifier.spage28738en_HK
dc.identifier.epage28745en_HK
dc.identifier.eissn1083-351X-
dc.identifier.isiWOS:000270678900030-
dc.publisher.placeUnited Statesen_HK
dc.relation.projectTowards a visualized proteome of <I>Escherichia coli</I>-
dc.identifier.scopusauthoridJin, Y=29467538100en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.scopusauthoridDanchin, A=7103235597en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK

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