Bacterial strains used in this study are listed in Supplementary Table S1. Strains were preserved in glycerol stocks (80% glycerol, −80°C). Unless noted otherwise, P. fluorescens and E. coli were cultivated at 30°C and 37°C, respectively, in Lysogeny Broth (LB) (10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract) or M9 medium (1.63 g/L NaH₂PO₄, 3.88 g/L K₂HPO₄, 2 g/L (NH₄)₂SO₄, 10 mg/L EDTA, 100 mg/L MgCl₂.6H₂O, 2 mg/L ZnSO₄.7H₂O, 1 mg/L CaCl₂.2H₂O, 5 mg/L FeSO₄.7H₂O, 0.2 mg/L Na₂MoO₄.2H₂O, 0.2 mg/L CuSO₄.5H₂O, 0.4 mg/L CoCl₂.6H₂O, and 1 mg/L MnCl₂.2H₂O) supplemented with 50 mM glucose at 250 rpm. Antibiotics and inducers were added as needed as listed on Supplementary Table S2.

To produce AHL-containing conditioned medium (CM), E. coli pSEVAb64_PCQS, a strain capable of synthesizing AHL, was cultured overnight in 10 mL of LB with the appropriate antibiotic. The cells were washed three times with 20:80 LB:M9 medium supplemented with 50 mM glucose. A 1:100 dilution of the resulting cell suspension was then used to inoculate 10 mL of fresh 20:80 LB:M9 + 50 mM glucose and the appropriate antibiotic. After overnight incubation, the culture was centrifuged for 5 min at 4,700 rpm, and the supernatant, referred to as conditioned medium (CM), was filter-sterilized using a 0.2 μm filter. The CM was supplemented with 50 mM glucose and freshly prepared for each subsequent assay.

Plasmids were assembled using the SEVA 3.1 platform (Damalas et al., 2020). DNA fragments were PCR-amplified using customized or standard primers and NEB Q5 High-Fidelity DNA polymerase, purified via agarose gel electrophoresis (1% w/v), and extracted with the ZymocleanTM Gen DNA Recovery Kit (Zymo Research). DNA was eluted in Milli-Q waster and quantified by NanoDrop spectrophotometry.

PCR products were ligated into SEVAb backbones using SEVAbrick assembly (Damalas et al., 2020). Plasmids were transformed into chemically competent E. coli DH5α or chemically competent P. fluorescens SBW25 cells and selected on LB agar with antibiotics. Colony PCR screening was performed with Phire Hot Start II polymerase, and positive colonies were identified via agarose gel electrophoresis (1% w/v). Subsequently, plasmids from positive colonies were isolated using the GeneJET plasmid Miniperp Kit (Thermo Scientific) from overnight liquid LB cultures. Again, plasmid DNA was eluted in Milli-Q water and quantified by NanoDrop spectrophotometry. Plasmid sequences were verified by Sanger sequencing (MACROGEN Inc. DNA Sequencing Service; Amsterdam, Netherlands). All plasmids constructed and used in this study along with primers and templates are listed and can be found in Supplementary Tables S3-S5.

To study responses to root exudates, five inducible expression systems were tested: pBAD/AraCE, pCym/CymR, pTtg/TtgR, pSal/nahR, and pVan/VanR. Promoters, transcription factors and regulatory elements were PCR-amplified from Addgene plasmids pAJM.677, pAJM.657, pAJM.611, pAJM.771 and pAJM.773 (Meyer et al., 2019), respectively, and assembled into pSB1C3 backbones following the scheme of Figure 1. Customized primers for each amplification can be found in Supplementary Table S4. Expression constructs were optimized by reversing promoter orientations relative to their transcription factors. As indicated, BBa_J23100 promoter and BBa_J34801 RBS were employed to drive the expression of the transcription factors.

All inducible systems in the pSB1C3 plasmids were amplified using primers 448 and 647. The sfgfp gene was amplified with primers 619 and 827 following the protocol by Damalas et al. (2020). The sfgfp insert was assembled downstream of the inducible promoter in pSEVAb23 backbones. The pSEVAb23_EV control plasmid was constructed by amplifying the pSEVAb23 backbone with primers 760 and 476 and assembling it without additional DNA inserts.

The pSEVAb64_PCQS and pSEVAb64_GFP_LuxR plasmids used in quorum sensing experiments to produce conditioned medium (CM) and respond to AHL (Sigma Aldrich #K3255), respectively, were kindly provided by Dr. Anna Doloman. The pSEVAb64_ EV control was constructed similarly to pSEVAb23_EV.

The pSEVAb23_Toehold2.1_V1_GFP plasmid, used to characterize the toehold switch from Green et al. (2014), was previously constructed in house for Asin-Garcia et al. (2024). To assemble the pSEVAb23_LuxR_SwGFP plasmid, the luxR gene and luxpR promoter were amplified from pSEVAb64_GFP_LuxR using NVD_LuxR_FW-RV. The gfp gene downstream of the switch RNA was amplified from pSEVAb23_Toehold2.1_V1_GFP using primers NVD_SwRNA_FW and 827. Both amplicons were inserted into a pSEVAb23 backbone.

To assemble the pSEVAb64_SalTr plasmid, the pSal/nahR system along with a fragment of the trigger RNA was amplified from pSB1C3_Sal using primers 448 and NVD_Trig2.1_pSAL_RV. The amplicon was inserted into a pSEVAb64 backbone containing the remaining part of the trigger RNA using primers NVD_Trig2.1_pSV23_FW and 448.

The constructed plasmids were transformed into chemically competent P. fluorescens SBW25 cells using heat shock. The presence of the correct plasmids was confirmed by colony PCR and Sanger sequencing. Strains harboring the correct plasmids were used for fluorescence plate reader assays.

A single colony of P. fluorescens SBW25 was transferred into LB medium in sterile conditions and incubated overnight at 30°C and 250 rpm. The overnight culture was chilled on ice and centrifuged at 7,000 × g for 2 min at 4°C. The supernatant was gently decanted, taking care not to disturb the pellet. Cells were resuspended in 20 mL ice-cold 0.1 M CaCl₂ by gentle pipetting, followed by a second centrifugation under the same conditions. This wash step was repeated once more for a total of two washes. The final pellet was resuspended in ice-cold 0.1 M CaCl₂ containing 15% (v/v) glycerol; the resuspension volume was calculated as 1/10 of the overnight culture volume. Cell suspensions were thoroughly mixed, then aliquoted (100 μL per tube) into sterile, autoclaved 1.5 mL microcentrifuge tubes. Aliquots were flash-frozen in a dry ice–ethanol bath (optional) and stored at −80 °C until further use.

High-performance liquid chromatography (HPLC) standard formulations of root exudates L-arabinose, cuminic acid, naringenin, salicylic acid, and vanillic acid were obtained from Sigma Aldrich (Lenga, n.d.). The exudates were diluted in their respective solvents listed in Supplementary Table S2 to the concentrations 1 M, 100 mM, 1 M, 1 M and 100 mM, respectively. All solutions were filter-sterilized and stored at −20°C.

To assess their toxicity in the bacterial host, overnight cultures of P. fluorescens SBW25 were prepared in biological triplicates, with technical replicates included. The following root exudate concentrations, dissolved in M9 medium supplemented with 50 mM glucose and 50 mg/mL kanamycin, were tested in a growth assay: 4 mM L-arabinose, 1 mM cuminic acid, 1 mM naringenin, 1 mM salicylic acid and 0.1 mM vanillic acid.

OD₆₀₀ was measured using a BioTek Synergy H1 Microplate Reader (BioTek Instruments, Inc., VT, U.S.) for 24 h at 30°C, with continuous shaking taking OD₆₀₀ readings taken every 5 min, using volumes of 200 µL. The average OD₆₀₀ of technical replicates was used to calculate the relative OD₆₀₀ of each biological replicate. Standard deviations between biological replicates were determined, and the average OD₆₀₀ of biological triplicates was calculated. Statistical differences between samples were determined using a Two-Sample t-Test Assuming Equal Variances. Data analysis and visualization were performed using Microsoft Excel.

The BLAST search engine from NCBI (Altschul et al., 1990) was used to determine whether P. fluorescens SBW25 possesses native proteins, transcription factors or promoters that could interfere with Vibrio fischeri’s LuxI/LuxR quorum system. BLASTp was employed to identify amino acid similarities between luxI and luxR genes and the P. fluorescens SBW25 genome. Additionally, BLASTn was used to search for sequence similarities between the luxpR promoter and the P. fluorescens SBW25 genome. Default BLAST settings were applied, and the result with the highest score was reported.

Multiple aspects of this study were assessed using fluorescence assays. These experiments were conducted in 96-well plates to measure both absorbance and fluorescence in a total volume of 200 µL per well. Optical density (OD₆₀₀) and fluorescence (excitation: 467 nm, emission: 508 nm) were monitored over 24 h using a BioTek Synergy H1 Microplate Reader (BioTek Instruments, Inc., VT, U.S.). Unless stated otherwise, each condition was tested using three biological replicates, with three technical replicates per strain.

Overnight cell precultures were washed three times with M9 medium supplemented with 50 mM glucose and diluted to an initial OD₆₀₀ of 0.3. Inducers were added when necessary. Relative fluorescence values were calculated by normalizing fluorescence readings to OD₆₀₀ values. The average relative fluorescence of technical replicates was used to determine biological replicates, and standard deviations were calculated from the three biological replicates. Statistical significance was assessed using a Two-Sample t-Test Assuming Equal Variances. Data analysis and visualization were performed using Microsoft Excel.

To characterize the response of the root exudate-inducible expression systems, fluorescence assays were performed with P. fluorescens strains carrying pSEVAb23_Ara, pSEVAb23_Cym pSEVAb23_Ttg and pSEVAb23_ Sal. The range of root exudate concentrations analyzed was based on the maximum and minimum induction levels reported by Meyer et al. (2019) and Ryu et al. (2019).

To investigate the AHL-sensing component of the LuxI/LuxR quorum system and assess the strain’s response to varying AHL concentrations, two fluorescence assays were conducted using the P. fluorescens pSEVAb64_GFP_LuxR strain. In the first assay, the following AHL concentrations were tested: 0 nM, 1 nM, 3 nM, 5 nM and 10 nM. The second assay tested an extended range of AHL concentrations: 0 nM, 0.025 nM, 0.05 nM, 0.1 nM, 0.25 nM, 0.5 nM, 1 nM, and 5 nM. In both assays, the P. fluorescens pSEVAb64_EV strain was cultivated under all conditions to account for autofluorescence.

To evaluate the response of the LuxI/LuxR quorum system to bacterially produced AHL, a fluorescence assay was performed using the P. fluorescens pSEVAb64_GFP_LuxR strain. The strain was cultivated under varying CM concentrations: 0% CM, 0.01% CM, 0.1% CM, 1% CM, 10% CM, 25% CM, and 50% CM.

To assess the toehold switch developed by Green et al. (2014), a fluorescence assay was performed using P. fluorescens pSEVAb23_Toehold2.1_V1_GFP. The strain was cultivated and tested under the following conditions over 24 h: no inducers, 1 mM 3-methylbenzoate, 3.75 mM rhamnose, and both inducers combined. Following the redesign of the toehold switch to respond to salicylic acid and AHL, its functionality was assessed instead under the following conditions: no inducers, 150 μM salicylic acid, 5 nM AHL, and both inducers combined.

We selected five root exudates as proxies for root proximity that could induce gene expression in bacteria: L-arabinose, cuminic acid, naringerin, salicylic acid and vanillic acid. To ensure these molecules were safe for detecting root proximity, we verified that they were not toxic to P. fluorescens SBW25. We tested 4 mM L-arabinose, 1 mM cuminic acid 1 mM naringenin, 1 mM salicylic acid and 0.1 mM vanillic acid, which corresponds to the highest concentrations used in subsequent experiments (Supplementary Figure S1).

Next, we characterized the inducible systems corresponding to these root exudates in P. fluorescens SBW25. Each inducible expression system examined in this study consists of a promoter and its corresponding transcription factor. Upon binding its respective root exudate, the transcription factor activates the promoter, initiating transcription of the downstream genes. The arabinose-inducible expression system was the only exception, as it included the arabinose transporter AraE to facilitate sugar transport across the membrane. All inducible expression systems, pBAD/AraCE, pCym/CymR, pSal/nahR, pTtg/TtgR and pVan/VanR, were cloned into pSEVAb23 vectors controlling a sfgfp gene, allowing fluorescence-based measurement of induction.

When introducing pCym/CymR, pTtg/TtgR and pVan/VanR, deletion mutations were observed. Since these transcription factors repress the promoter in the absence of their corresponding inducers, we suspected cross-regulation in which essential genes with similar promoters might have been repressed, leading to cell death. To counteract this, we supplemented the medium with 100 μM cuminic acid and 1 mM of naringenin during cloning, which successfully facilitated the introduction of pCym/cymR and pTtg/TtgR. However, the vanillic acid system could not be introduced, even with inducer concentrations up to 1 mM.

Dynamic range and induction range for each system was calculated and compared in Table 1 to the values obtained by Meyer et al. (2019) and Ryu et al. (2019). All tested systems responded to increasing inducer concentrations, as shown in Figure 2. Notably, pCym/CymR and pSal/nahR exhibited the highest dynamic ranges, with values of 9.57 and 9.13, respectively, as seen in Figures 2B,D. These responses occurred within narrow concentration ranges: 0.01 μM–10 μM for pCym/CymR and 1 μM–150 μM for pSal/nahR. In contrast, the precise inducer concentration ranges for pBAD/AraCE and pTtg/TtgR could not be determined, as maximum induction, indicated by a plateau in relative fluorescence, was not reached (Figures 2A,C). Additionally, these two systems displayed relatively low dynamic ranges of 3.77 and 1.3, respectively.

To respond to fluctuations in cell density, the LuxI/LuxR quorum sensing system from V. fischeri was selected. Before implementing this system in P. fluorescens SBW25, we investigated whether the bacterium naturally possesses a similar system, comprising a luxpR-like binding site and luxI- and luxR-like genes. A BLASTn search for luxpR did not yield any significant similarity, while BLASTp searches for LuxI and LuxR revealed significant similarity only for the latter, identifying a putative LuxR-family regulatory protein. These findings suggest that P. fluorescens SBW25 does not naturally produce acyl-homoserine lactones (AHLs) and lacks a complete LuxI/LuxR system, though it may encode a LuxR-like component, which matches previous work (I. De Bruijn and Raaijmakers, 2009).

To characterize the quorum sensing system, we set out to separately test its response to quorum signals and its ability to produce signals in P. fluorescens SBW25. The response element was tested using the pSEVAb64_GFP_LuxR plasmid in which gfp expression is controlled by the luxR gene which binds to the luxpR promoter under sufficiently high AHL concentrations as shown in Figure 3A. Notably, this plasmid deliberately excludes the gene encoding LuxI to allow for precise control of AHL levels to determine the concentrations triggering induction. In a plate reader fluorescence assay, we tested P. fluorescens carrying pSEVAb64_GFP_LuxR over a range of 0 nM, 0.025 nM, 0.05 nM, 0.1 nM, 0.25 nM, 0.5 nM, 1 nM, and 5 nM AHL. Figure 3B shows that there is an increasing signal response observed as AHL concentrations are increased plateauing around 0.5 nM.

Following this characterization of the luxpR/LuxR system with known AHL concentrations, we assessed its responsiveness to AHL produced by bacteria. For this E. coli K12 was transformed with pSEVAb64_PCQS, a plasmid containing the complete LuxI/LuxR system, as it has been verified to enable AHL production. E. coli pSEVAb64_PCQS was cultured overnight in 20:80 LB:M9 supplemented with 50 mM glucose and subsequently filtered to obtain AHL-containing conditioned medium (CM). A fluorescence assay was conducted on P. fluorescens pSEVAb64_GFP_LuxR cultured in varying concentrations of CM (Figure 3C). The strain exhibited no response in samples containing 0%–25% CM. However, in 50% CM, fluorescence levels increased 14-fold compared to the 0% CM sample, indicating strong induction. Notably, this sample also displayed a 90% reduction in growth relative to the others, a trend also observed at higher known concentrations of AHL (Supplementary Figures S2 and S3). These results demonstrate that it is possible to engineer a quorum sensing signal sensor in P. fluorescens SBW25 and that its activation can be made dependent on cell population density.

Next, we investigated whether P. fluorescens is also capable of producing AHL. To test this, pSEVAb64_PCQS (Figure 4A) was transformed into P. fluorescens. Fluorescence levels after overnight incubation were comparable to those observed in E. coli pSEVAb64_PCQS (Figure 4B). Since GFP production depends on LuxR binding to AHL, these observations strongly suggest that AHL production can be successfully engineered in P. fluorescens SBW25.

The toehold switch employed in this study is based on the highest-performing design from Green et al. (2014). This design was later adapted into the pSEVAb23_Toehold2.1_V1_GFP plasmid, illustrated in Figure 5A, and characterized in Pseudomonas putida by Asin-Garcia et al. (2024).

The toehold system functions as a genetic AND gate, where the trigger RNA (trRNA) and switch RNA (swRNA) are transcribed in the presence of rhamnose and 3-MB, respectively (Figures 5A,B). To evaluate its functionality and orthogonality to native cellular machinery, pSEVAb23_Toehold2.1_V1_GFP was introduced into P. fluorescens SBW25. When both inducers were added simultaneously, fluorescence increased by 22-fold compared to the control without inducers, as shown in Figures 5C,D. This response significantly exceeded that of the individual inducers, confirming the correct functionality of the toehold. However, some decrease in cell growth was observed when adding both inducers individually and together. In addition, leakiness was observed when rhamnose and 3-MB were added separately, resulting in 4.9- and 3.6-fold increases in GFP production, respectively.

Following the successful validation of pSEVAb23_Toehold2.1_V1_GFP in P. fluorescens, the toehold system was redesigned to integrate inputs from the salicylic acid-inducible expression system and the quorum sensing system. The new toehold architecture was constructed using two plasmids of similar copy numbers, as illustrated in Figure 6A. The first plasmid, pSEVAb64_SalTrg, was designed to express the trRNA under the control of the pSal/nahR inducible system in the presence of salicylic acid. The second plasmid, pSEVAb23_LuxRSw_GFP, was designed to express the swRNA upstream of the gpf gene, which is driven by the luxpR/LuxR system when AHL concentrations are sufficiently high. The redesigned toehold was expected to produce GFP only in the presence of both root exudate and quorum signals (Figure 6B).

After successfully co-transforming both plasmids into P. fluorescens SBW25, the redesigned toehold was tested using a fluorescence assay. Over time, an increase in GFP production was observed exclusively in the sample exposed to both salicylic acid and AHL, as shown in Figure 6C. Initially, high fluorescence levels were detected in the AHL-only sample. However, as cell density increased and fluorescence values remained constant, the corrected fluorescence signal in this sample decreased. After 24 h, a 322-fold induction was observed in the sample containing both inducers, a significant increase compared to all other conditions (Figure 6D).

Furthermore, when individual inducers were added, no significant GFP levels were detected. Notably, no differences in cell growth were observed between samples (Supplementary Figure S4). These results suggest that that the redesigned toehold enables the biosensor to monitor multiple environmental conditions, inducing high levels of gene expression only under user-defined conditions (i.e., optimal delivery conditions into the roots), without imposing a burden on the cells.