Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix-turn-helix motifs.
The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix-turn-helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.
Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland.|Institute of Molecular Medicine, Trinity Centre, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland.|Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, University of Dublin, Dublin 2, Ireland.
Vivienne
V
Mahon
Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, University of Dublin, Dublin 2, Ireland.
Cyril J
CJ
Smyth
Institute of Molecular Medicine, Trinity Centre, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland.|Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, University of Dublin, Dublin 2, Ireland.|Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, St James's Hospital, Dublin 8, Ireland.
Stephen G J
SGJ
Smith
D000595:Amino Acid Sequence; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D019077:Helix-Turn-Helix Motifs; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C108303:Rns protein, bacteria; D015534:Trans-Activators