Influence of the luxR Regulatory Gene Dosage and Expression Level on the Sensitivity of the Whole-Cell Biosensor to Acyl-Homoserine Lactone.

Sergey Bazhenov,Vladimir Chistyakov,Ekaterina Scheglova,Svetlana Khrulnova,Ilya Manukhov,Uliana Novoyatlova,Olga Melkina,Vadim Fomin

doi:10.3390/bios11060166

Abstract

Aliivibrio fischeri LuxR and Aliivibrio logei LuxR1 and LuxR2 regulatory proteins are quorum sensing transcriptional (QS) activators, inducing promoters of luxICDABEG genes in the presence of an autoinducer (3-oxo-hexanoyl-l-homoserine lactone). In the Aliivibrio cells, luxR genes are regulated by HNS, CRP, LitR, etc. Here we investigated the role of the luxR expression level in LuxI/R QS system functionality and improved the whole-cell biosensor for autoinducer detection. Escherichia coli-based bacterial lux-biosensors were used, in which Photorhabdus luminescens luxCDABE genes were controlled by LuxR-dependent promoters and luxR, luxR1, or luxR2 regulatory genes. We varied either the dosage of the regulatory gene in the cells using additional plasmids, or the level of the regulatory gene expression using the lactose operon promoter. It was shown that an increase in expression level, as well as dosage of the regulatory gene in biosensor cells, leads to an increase in sensitivity (the threshold concentration of AI is reduced by one order of magnitude) and to a two to threefold reduction in response time. The best parameters were obtained for a biosensor with an increased dosage of luxR A. fischeri (sensitivity to 3-oxo-hexanoyl-l-homoserine lactone reached 30–100 pM).

Highlights

Quorum Sensing (QS) is a genetic mechanism enabling bacteria to determine population density through the exchange of specific signaling molecules, called autoinducers (AI)
We investigated the sensitivity of LuxR-based biosensors to AI depending on the regulatory gene dosage and the level of its transcription involving the following three regulatory genes: luxR1 and luxR2 A. logei, and luxR A. fischeri
An increase in the dosage of the A. logei luxR1 gene did not lead to a significant difference in the properties of the corresponding biosensors, which may be associated with the regulation of luxR1 itself

Summary

Introduction

Quorum Sensing (QS) is a genetic mechanism enabling bacteria to determine population density through the exchange of specific signaling molecules, called autoinducers (AI). LuxI produces AI, predominantly a 3-oxohexanoyl-l-homoserine lactone (3OC6-HSL), which passively penetrates the cell membrane and serves for signal transmission between cells [2]. LuxR is a regulatory protein, an AI-sensitive transcription activator. At a sufficient concentration of AI in the medium, the LuxR-AI complex is formed and, by binding to the lux-box in the promoter region, it induces the transcription of luxICDABEG genes [3]. In the closely related psychrophilic bacteria Aliivibrio logei and Aliivibrio salmonicida the LuxI/LuxR QS system differs from that in A. fischeri: the regulatory gene is represented by two homologues: luxR1 and luxR2 [4,5,6]

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Conclusion