Premium
Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy
Author(s) -
Ventre Isabelle,
Ledgham Fouzia,
Prima Valérie,
Lazdunski Andrée,
Foglino Maryline,
Sturgis James N.
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03422.x
Subject(s) - quorum sensing , biology , regulator , green fluorescent protein , microbiology and biotechnology , pseudomonas aeruginosa , inducer , fusion protein , intracellular , biochemistry , virulence , gene , bacteria , genetics , recombinant dna
Summary Of considerable interest in the biology of pathogenic bacteria are the mechanisms of intercellular signalling that can lead to the formation of persistent infections. In this article, we have examined the intracellular behaviour of a Pseudomonas aeruginosa quorum sensing regulator RhlR believed to be important in this process. We have further examined the modulation of this behaviour in response to various auto‐inducers. For these measurements, we have developed an assay based on the fluorescence anisotropy of EGFP fusion proteins that we use to measure protein–protein interactions in vivo . We show that the transcriptional regulator, RhlR, expressed as an EGFP fusion protein in Escherichia coli , forms a homodimer. This homodimer can be dissociated into monomers by the auto‐inducer N ‐(3‐oxododecanoyl)‐ l ‐homoserine lactone (3O‐C12‐HSL) whereas N ‐(butanoyl)‐ l ‐homoserine lactone (C4‐HSL) has little effect. These observations are of particular interest as RhlR modulation of gene expression depends on the presence of C4‐HSL, whereas 3O‐C12‐HSL modulates the expression of genes regulated by LasR. These observations thus provide a framework for understanding the regulatory network that links the various different QS regulators in P. aeruginosa . Furthermore, the technique we have developed should permit the study of numerous protein/protein or protein/nucleic acid interactions in vivo and so shed light on natural protein function.