Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity

Abstract

The use of cell-cell communication or "quorum sensing (QS)" elements from Gram-negative Proteobacteria has enabled synthetic biologists to begin engineering systems composed of multiple interacting organisms. However, additional tools are necessary if we are to progress toward synthetic microbial consortia that exhibit more complex, dynamic behaviors. EsaR from Pantoea stewartii subsp. stewartii is a QS regulator that binds to DNA as an apoprotein and releases the DNA when it binds to its cognate signal molecule, 3-oxohexanoyl-homoserine lactone (3OC6HSL). In the absence of 3OC6HSL, EsaR binds to DNA and can act as either an activator or a repressor of transcription. Gene expression from P(esaR), which is repressed by wild-type EsaR, requires 100- to 1000-fold higher concentrations of signal than commonly used QS activators, such as LuxR and LasR. Here we have identified EsaR variants with increased sensitivity to 3OC6HSL using directed evolution and a dual ON/OFF screening strategy. Although we targeted EsaR-dependent derepression of P(esaR), our EsaR variants also showed increased 3OC6HSL sensitivity at a second promoter, P(esaS), which is activated by EsaR in the absence of 3OC6HSL. Here, the increase in AHL sensitivity led to gene expression being turned off at lower concentrations of 3OC6HSL. Overall, we have increased the signal sensitivity of EsaR more than 70-fold and generated a set of EsaR variants that recognize 3OC6HSL concentrations ranging over 4 orders of magnitude. QS-dependent transcriptional regulators that bind to DNA and are active in the absence of a QS signal represent a new set of tools for engineering cell-cell communication-dependent gene expression.

Figure 1

Illustration of AHL-dependent activation and repression of gene expression by LuxR and EsaR. (a) LuxR activates expression at P_luxI. (b) EsaR represses transcription at P_esaR. (c) EsaR activates transcription at P_esaS. For a–c, the left and right panels compare gene expression in the absence and presence of 3OC6HSL (red circles), respectively.

Figure 2

Luminescence assay of 3OC6HSL-dependent derepression of P_esaR by evolved EsaR variants. (a) Dose response of wild type and firstgeneration EsaR variants to 3OC6HSL. (b) Dose response of recombined variants and wild-type EsaR. Promoter activity in the absence of any regulator, shown in pink, indicates that complete derepression of P_esaR was observed with the recombined EsaR variants but not with wild type or the first-generation variants. Luminescence data were normalized by OD₆₀₀ to account for subtle differences in cell density following resuspension of cells in bioassay media. Error bars show standard deviations from three independent biological replicates.

Figure 3

Gene activation assays show differences in promoter specificity and 3OC6HSL sensitivity by EsaR, first-generation EsaR variants, and LuxR. (a) Dose response of wild-type LuxR, wild-type EsaR, and EsaR variants at the P_esaS promoter. (b) Dose response of wild-type LuxR and the EsaR variants that exhibited the lowest (EsaR-V220A) and highest (EsaR-I70V) luminescence levels at the P_luxI promoter. Luminescence data were normalized by OD₆₀₀ to account for subtle differences in cell density following resuspension of cells in bioassay media. Error bars show standard deviations from three independent biological replicates.

Figure 4

EsaR homology model highlighting mutations affecting AHL sensitivity. The double-stranded DNA to which EsaR binds is shown in dark gray. The EsaR dimer consists of two peptides shown in red and blue. The anti-parallel β-sheets form a pocket with two strands of α-helices for 3OC6HSL binding. The molecule of 3OC6HSL is shown in space-filling green. Three residues, I70V, D91G, and V220A, are shown in space-filling molecule-specific colors.