title: Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation.
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 switch-csrA 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-beta-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.
authors.affiliation: Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pok Fu Lam, Hong Kong, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Jin
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Antoine
authors.initials: A
authors.lastname: Danchin
authors.forename: Jian-dong
authors.initials: JD
authors.lastname: Huang
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry; D018829:Adhesins, Bacterial / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002470:Cell Survival; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011134:Polysaccharides / Q000737:chemistry; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013806:Theophylline / Q000737:chemistry
chemical_list: D018829:Adhesins, Bacterial; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D008024:Ligands; D011134:Polysaccharides; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D013806:Theophylline; D000117:Acetylglucosamine
references.citation: J Med Microbiol. 1993 Sep;39(3):183-90
references.pmid: 8366516
references.citation: Nat Biotechnol. 2004 Jul;22(7):841-7
references.pmid: 15208640
references.citation: Mol Microbiol. 2005 Jun;56(6):1648-63
references.pmid: 15916613
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
references.pmid: 10811905
references.citation: Mol Microbiol. 2001 Apr;40(1):245-56
references.pmid: 11298291
references.citation: J Bacteriol. 2007 Aug;189(15):5472-81
references.pmid: 17526692
references.citation: Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14283-8
references.pmid: 17709748
references.citation: J Biol Chem. 1997 Jul 11;272(28):17502-10
references.pmid: 9211896
references.citation: Nat Biotechnol. 2000 Jul;18(7):740-5
references.pmid: 10888841
references.citation: RNA. 2000 May;6(5):659-67
references.pmid: 10836787
references.citation: J Bacteriol. 2004 May;186(9):2673-81
references.pmid: 15090508
references.citation: Science. 1998 Jun 26;280(5372):2114-8
references.pmid: 9641917
references.citation: Mol Microbiol. 2008 Oct;70(1):236-57
references.pmid: 18713317
references.citation: Proc Natl Acad Sci U S A. 2004 May 11;101(19):7287-92
references.pmid: 15123812
references.citation: J Mol Biol. 1995 Oct 20;253(2):277-90
references.pmid: 7563089
references.citation: Nucleic Acids Res. 2004 Mar 05;32(4):1610-4
references.pmid: 15004248
references.citation: Mol Microbiol. 2000 Jul;37(2):424-32
references.pmid: 10931336
references.citation: Gene. 2006 Sep 1;379:109-15
references.pmid: 16750601
references.citation: J Bacteriol. 2003 Aug;185(15):4450-60
references.pmid: 12867454
references.citation: FEMS Microbiol Lett. 1997 Apr 1;149(1):115-20
references.pmid: 9103983
references.citation: BMC Genomics. 2009 Apr 20;10:165
references.pmid: 19379489
references.citation: Curr Opin Genet Dev. 1999 Dec;9(6):709-14
references.pmid: 10607608
references.citation: J Mol Biol. 1999 May 21;288(5):911-40
references.pmid: 10329189
references.citation: J Bacteriol. 2003 Oct;185(19):5673-84
references.pmid: 13129938
references.citation: J Bacteriol. 2004 Dec;186(23):8058-65
references.pmid: 15547278
references.citation: Infect Immun. 2005 Apr;73(4):1954-63
references.pmid: 15784535
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: J Bacteriol. 2009 Mar;191(5):1722-4
references.pmid: 19103924