Dimerization of the quorum sensing regulator RhlR: development of a method using EGFP fluorescence anisotropy.
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.
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Laboratoire d'Ingéniérie des Systèmes Macromoléculaires, UPR9027, IBSM/CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Ledgham
Fouzia
F
Prima
Valérie
V
Lazdunski
Andrée
A
Foglino
Maryline
M
Sturgis
James N
JN
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; C074896:rhiR protein, Rhizobium leguminosarum