Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity.
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.
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA.
Jasmine
J
Shong
Yao-Ming
YM
Huang
Christopher
C
Bystroff
Cynthia H
CH
Collins
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D019020:Directed Molecular Evolution; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020636:Pantoea; D053038:Quorum Sensing; D011995:Recombination, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; C095459:EsaR protein, Pantoea stewartii; D014157:Transcription Factors