Directed evolution of the autoinducer selectivity of <i>Vibrio fischeri</i> LuxR

Abstract

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory. We found an orthogonal pair of LuxR mutants specific either to 3-oxo-hexanoyl homoserine lactone or to 3-oxo-octanoyl homoserine lactone. Interestingly, the majority of the specificity changes did not arise from modulating the recognition event but rather from changing the efficiency of the transition from the inactive form to the active form upon signal binding. This finding explains how quorum sensing systems can rapidly diverge in nature and in the laboratory and how signal orthogonality and mutual inhibition frequently occur among closely related diverging systems.

Characterization of Diverse Homoserine Lactone Synthases inEscherichia coli

“…In conclusion, our work adds to the rich, growing body of knowledge to inform design principles and best practices for the use of HSL-modulated quorum sensing networks in engineered circuits [18,19] . A series of recent publications has greatly expanded the number of HSL Receiver-type devices available to genetic engineers [18,30,44,45] . Our work to build and characterize a library of diverse senders complements these efforts to enable researchers to build more complex networks with minimal crosstalk, and provides an approach to identify functionally orthogonal sender-receiver pairs for parallel computation in multiple contexts.…”

“…We considered the length of the acyl chain, the chain saturation, and the number of different HSLs produced by a single synthase. From our previously published list of reported synthase proteins and their HSL profiles [30] , we selected the following sythases: RpaI, BraI, RhlI, BjaI, EsaI, LuxI, AubI, LasI, and CerI (Table 1). This set represents HSLs with chain lengths from 3 to 18 carbons and modifications including phenol, phenyl, carbonyl, and methyl groups.…”

Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors

“…Further, when multiple sensors are combined into one cell, they can interfere with each other's response functions (Figure 1b). Some small molecules bind non-cognate regulators and this can lead to off-target activation (cross reactivity) or competitive inhibition with the cognate small molecule (antagonism) [22][23][24][25][26][27] . Finally, each sensor requires cellular resources (e.g., ribosomes) to function and the activation of one sensor can influence another indirectly due to resource competition 28 .…”

Marionette:E. colicontaining 12 highly-optimized small molecule sensors

“…Sensors can be improved using biophysical models, rational engineering and directed evolution 5, 23, 26, 30-36 . Improving performance requires that screens or selections be performed in the presence and absence of inducer 37 .…”

“…Such dual selections have been accomplished by sorting cells based low and high fluorescence, utilizing proteins that can be both toxic and selective ( e.g. , TetA or HSV-TK), or by deploying separate positive and negative selections 26, 35, 37-44 . This has been applied to improving the response functions and eliminating cross-reactivity between pairs of regulators 23, 26, 35, 45 .…”