1887

Abstract

The pathogenesis of diarrhoeal disease due to human enterotoxigenic 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 and by a flexible seven amino acid sequence from another 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.

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2010-09-01
2019-10-17
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Expression and purification of His-tagged Rns and mutant derivatives, and Western immunoblot analysis of induced cultures of KS1000/pRare harbouring the indicated mutant Rns proteins. Model of Rns N-terminus (amino acids 15–72) based on TTHA0104 from using PHYRE. Model of Rns C-terminus (amino acids 155–264) based on ToxT using Swiss-Model. are available as a single PDF(486 KB). are available to download here ( Fig. S3, 38 KB; Fig. S5, 70 KB) as PDB files. Please right-click and save the files. These can then be viewed by using Jmol, an open-source Java viewer for chemical structures in 3D.

Expression and purification of His-tagged Rns and mutant derivatives, and Western immunoblot analysis of induced cultures of KS1000/pRare harbouring the indicated mutant Rns proteins. Model of Rns N-terminus (amino acids 15–72) based on TTHA0104 from using PHYRE. Model of Rns C-terminus (amino acids 155–264) based on ToxT using Swiss-Model. are available as a single PDF(486 KB). are available to download here ( Fig. S3, 38 KB; Fig. S5, 70 KB) as PDB files. Please right-click and save the files. These can then be viewed by using Jmol, an open-source Java viewer for chemical structures in 3D.

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