Use of a riboswitch-controlled conditional hypomorphic mutation to uncover a role for the essential csrA gene in bacterial autoaggregation.
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.
Jin
Ye
Y
Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pok Fu Lam, Hong Kong, China.
Watt
Rory M
RM
Danchin
Antoine
A
Huang
Jian-dong
JD
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
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
J Med Microbiol. 1993 Sep;39(3):183-90
8366516
Nat Biotechnol. 2004 Jul;22(7):841-7
15208640
Mol Microbiol. 2005 Jun;56(6):1648-63
15916613
Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83
10811905
Mol Microbiol. 2001 Apr;40(1):245-56
11298291
J Bacteriol. 2007 Aug;189(15):5472-81
17526692
Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14283-8
17709748
J Biol Chem. 1997 Jul 11;272(28):17502-10
9211896
Nat Biotechnol. 2000 Jul;18(7):740-5
10888841
RNA. 2000 May;6(5):659-67
10836787
J Bacteriol. 2004 May;186(9):2673-81
15090508
Science. 1998 Jun 26;280(5372):2114-8
9641917
Mol Microbiol. 2008 Oct;70(1):236-57
18713317
Proc Natl Acad Sci U S A. 2004 May 11;101(19):7287-92
15123812
J Mol Biol. 1995 Oct 20;253(2):277-90
7563089
Nucleic Acids Res. 2004 Mar 05;32(4):1610-4
15004248
Mol Microbiol. 2000 Jul;37(2):424-32
10931336
Gene. 2006 Sep 1;379:109-15
16750601
J Bacteriol. 2003 Aug;185(15):4450-60
12867454
FEMS Microbiol Lett. 1997 Apr 1;149(1):115-20
9103983
BMC Genomics. 2009 Apr 20;10:165
19379489
Curr Opin Genet Dev. 1999 Dec;9(6):709-14
10607608
J Mol Biol. 1999 May 21;288(5):911-40
10329189
J Bacteriol. 2003 Oct;185(19):5673-84
13129938
J Bacteriol. 2004 Dec;186(23):8058-65
15547278
Infect Immun. 2005 Apr;73(4):1954-63
15784535
J Bacteriol. 1995 Jul;177(14):4121-30
7608087
J Bacteriol. 2009 Mar;191(5):1722-4
19103924