title: Modeling Analysis of Signal Sensitivity and Specificity by Vibrio fischeri LuxR Variants.
abstract: The LuxR protein of the bacterium Vibrio fischeri belongs to a family of transcriptional activators that underlie pheromone-mediated signaling by responding to acyl-homoserine lactones (-HSLs) or related molecules. V. fischeri produces two acyl-HSLs, N-3-oxo-hexanoyl-HSL (3OC6-HSL) and N-octanoyl-HSL (C8-HSL), each of which interact with LuxR to facilitate its binding to a "lux box" DNA sequence, thereby enabling LuxR to activate transcription of the lux operon responsible for bioluminescence. We have investigated the HSL sensitivity of four different variants of V. fischeri LuxR: two derived from wild-type strains ES114 and MJ1, and two derivatives of LuxRMJ1 generated by directed evolution. For each LuxR variant, we measured the bioluminescence induced by combinations of C8-HSL and 3OC6-HSL. We fit these data to a model in which the two HSLs compete with each other to form multimeric LuxR complexes that directly interact with lux to activate bioluminescence. The model reproduces the observed effects of HSL combinations on the bioluminescence responses directed by LuxR variants, including competition and non-monotonic responses to C8-HSL and 3OC6-HSL. The analysis yields robust estimates for the underlying dissociation constants and cooperativities (Hill coefficients) of the LuxR-HSL complexes and their affinities for the lux box. It also reveals significant differences in the affinities of LuxRMJ1 and LuxRES114 for 3OC6-HSL. Further, LuxRMJ1 and LuxRES114 differed sharply from LuxRs retrieved by directed evolution in the cooperativity of LuxR-HSL complex formation and the affinity of these complexes for lux. These results show how computational modeling of in vivo experimental data can provide insight into the mechanistic consequences of directed evolution.
authors.affiliation: Department of Microbiology, University of Georgia, Athens, GA, United States of America.
authors.forename: Deanna M
authors.initials: DM
authors.lastname: Colton
authors.affiliation: Department of Microbiology, University of Georgia, Athens, GA, United States of America.
authors.forename: Eric V
authors.initials: EV
authors.lastname: Stabb
authors.affiliation: Physics Department, University of Florida, Gainesville, FL, United States of America.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hagen
mesh_terms: D054742:Acyl-Butyrolactones / Q000378:metabolism; D048908:Aliivibrio fischeri / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D015964:Gene Expression Regulation, Bacterial; D008163:Luminescent Measurements; D011485:Protein Binding
chemical_list: D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins