Synthetic Homoserine Lactone Sensors for Gram-Positive Bacillus subtilis using LuxR-type Regulators

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Abstract A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that gram-negative and gram-positive bacteria generally communicate via homoserine lactones (HSL) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (–35, –16, –10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors (≥ 20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p -coumaroyl-HSL, 3-oxohexanoyl-HSL, n -butyryl-HSL, and n -(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.

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