Reagents

All chemicals were purchased from Sigma-Aldrich unless otherwise specified; plasmids are listed in Supplementary Table 4; and oligonucleotide primers in Supplementary Table 5.

Preparation of dye stock solutions

Aqueous solutions were prepared for all dyes except for Victoria Blue R, which was dissolved in 20% ethanol. Concentrations for all dyes were 10 mM, except for CV, for which we prepared a 1 mM stock solution.

Phylogenetic analysis and design of a consensus operator

The intergenic regions of eilR and eilA homologs in gamma-proteobacteria were extracted using pre-computed gene trees available in MicrobesOnline50. To improve specificity of motif reconstruction, we filtered out intergenic regions with more than 90% of sequence similarity using Jalview51, which resulted in a set of non-redundant intergenic regions from the following bacteria: Enterobacter lignolyticus; Citrobacter koseri; Citrobacter rodentium; Salmonella enterica paratyphi; Salmonella enterica arizonae; Klebsiella pneumoniae 342; Klebsiella pneumoniae NTUH-K2044; Enterobacter sp. 638; Pantoea ananatis LMG 20103; Acinetobacter sp. ADP1; Acinetobacter baumannii. Intergenic regions from these organisms were used to identify putative EilR binding site motifs by MEME52. The MEME algorithm was applied with default parameters, restricting the motifs types to palindromes only and searching any number of site repetition on the same strand. The motif with the lowest E-value was considered as a putative eil-operator.

Plasmid construction for E. coli assays

All plasmids are listed in Supplementary Table 4. To construct the P EilO1t sensor strain, the eilR gene was PCR amplified from a fosmid containing E. lignolyticus genomic DNA that confers ionic liquid tolerance27. The eilR gene was then cloned after the weak constitutive promoter aP FAB254 on pFAB5088 (provided by Vivek Mutalik), containing genes encoding kanamycin resistance and a monomeric red fluorescence protein (RFP) as reporter12. The resulting plasmid, pFAB_eilR was then used as template to generate the library of randomized −10 and −35 regions upstream of rfp. Primers were designed in a way to fit a truncated consensus eil-operator into a 17-bp spacer region between the −35 and −10 sites (see Supplementary Fig. 1). To create the randomized promoter library, pFAB_eilR was PCR amplified with the primers, eilO-pFAB_random_for and eilO-pFAB_random_rev (Supplementary Table 5), digested with DpnI (Thermo-Fisher), phosphorylated (using 100 ng PCR product) with polynucleotide kinase (Thermo-Fisher) and self-ligated with T4 DNA ligase (Thermo-Fisher) at 16 °C for ca. 14 h. One microliter purified ligation product was transformed into chemically competent E. coli DH10B containing plasmid pBbS5c-eilA (SC101 ori, CmR) enabling [C 2 C 1 im]Cl-tolerance via the IPTG-inducible eilA gene. Transformed cells were plated on 200 × 200 mm LB agar plates supplemented with kanamycin (50 mg L−1) and chloramphenicol (12.5 mg L−1) and incubated at 37 °C overnight. One hundred and thirty six colonies were transferred separately into 96-deep-well microtiter plates and grown to stationary phase in EZ-Rich media containing 0.2% glucose and 10 µM IPTG either without or with 300 mM [C 2 C 1 im]Cl. To identify variants that respond to [C 2 C 1 im]Cl, RFP fluorescence of cells was measured in a Tecan F200pro plate reader. Promoter P EilO1t , located on the resulting plasmid pFABeilO1t, is the variant with the highest dynamic range.

After removal of the BglII-site upstream of P EilO1t , the region spanning from eilR to the transcriptional start downstream of P EilO1t was transferred from pFAB_eilR between the AatII and the EcoRI sites of a BglBrick plasmid backbone53 (p15A ori, KanR) containing the rfp gene and its RBS by isothermal DNA assembly, following the manufacturer’s instructions (New England Biolabs), which resulted in plasmid pTR_EilO1t.

To construct the P JEx -promoter suite, phage promoters P A1 , P H207 , P DE20 , and P L 29 with truncated eilO operators in their 17-bp spacer regions were ordered as gBlocks (IDT), with the flanking regions containing at least 40-bp identity with ends of the PCR-amplified modified version of pFAB_eilR. gBlocks were cloned into the linearized vector backbone by isothermal DNA assembly. The resulting plasmids were PCR-amplified with primers that each contained half an operator at the transcriptional start (Supplementary Table 5). PCR products were self-ligated to obtain promoters with two eilO operators. P JEx1 was generated by taking the same approach, using pTR_EilO1t as template plasmid. All assemblies were transformed into E. coli DH10B, and the promoter region and rfp sequence-verified.

To engineer sacB-plasmids, the sacB gene was PCR-amplified from pKW1 (sacB counterselection suicide plasmid, gift from Kelly Wetmore) to replace the rfp gene on pTR_sJExA1-rfp, pTR_aJExA1-rfp, pTR_sJExD-rfp, and pTR_aJExD-rfp via Golden Gate cloning, while the RBS on these plasmids was maintained. The resulting plasmids pTR_sJExA1-sacB, pTR_aJExA1-sacB, pTR_sJExD-sacB, and pTR_aJExD-sacB were transformed into E. coli DH10B and sequence-verified.

To generate lacZ-plasmids, the lacZ gene was PCR-amplified from E. coli MG1655 genomic DNA to replace the rfp gene on pTR_aJExD-rfp and pBbA7k-rfp via Golden Gate cloning, while the RBS on these plasmids was maintained. Plasmids were transformed into E. coli DH10B and sequence-verified.

E. coli fluorescence measurements

Cells were induced for RFP expression as indicated and measured after growing at 37 °C to stationary phase, unless otherwise described. Microplate measurements were performed in a BioTek Synergy 4 reader for absorbance at 600 nm and fluorescence (575 nm excitation, 620 nm emission).

Flow cytometry

Single-cell fluorescence and population homogeneity were measured in stationary phase E. coli cultures expressing RFP after a 1:200 dilution in PBS buffer. An LSRII Fortessa (BD, CA, USA) instrument, equipped with a yellow–green laser (561 nm excitation) was used to detect mRFP fluorescence during dynamic range measurements shown in Fig. 3a, b. For each sample, 50,000 events were measured with the following settings: FSC-H (forward scatter): 473 V, SSC-H (side scatter): 279 V, PE-Texas Red-H: 450 V (mRFP detection). A Guava easyCyte (Millipore) flow cytometer was used for generating the histogram in Fig. 3c. For each sample, 5000 events were counted by forward and side scatter acquisition, and the cellular accumulation of RFP was measured by fluorescence intensity. Data acquisition was performed using InCyte software version 2.2 (Millipore).

EilR-regulated promoters in other bacteria

Maps of plasmids constructed for assays in P. putida, S. meliloti and C. crescentus are shown in Supplementary Fig. 12. The broad-host-range vector pJC543 was assembled using In-Fusion HD Cloning Kit (Clontech), by inserting tetR from pZS4Int111 into pZE21-MCS1 at the BglII site, and the RK2-based origins of replication and conjugative transfer (oriV-oriT-trfA) from pCM13054 at the SpeI site. Specifically, tetR was amplified using primers DVA00311 (5′-ACGATCCTCATCCTGTCTCTTGATCACGATCGTTAAGACCCACTTTCACATTTAAGTTG) and DVA00312 (5′-AAGGATCTGATGGCGCAGGGGATCAAGATCTATGTCTAGATTAGATAAAAGTAAAGTGA), while oriV-oriT-trfA was amplified using primers DVA00309 (5′-CTCACGTTAAGGGATTTTGGTCATGAACTAGTCTAGCGTTTGCAATGCACCAGG) and DVA00310 (5′-GGGCGTTTTTTATTGGTGAGAATCCAAGCAGCTAGCCTGCCATTTTTGGGGTGAGGCCG).

The two PCR products were combined with the two fragments of pZE21-MCS1 resulting from digestion with BglII and SpeI. The assembled pJC543 plasmid, which contains the P LtetO-1 promoter and encodes its cognate regulator TetR, can be conjugated into a wide range of species by selecting for resistance to kanamycin or neomycin.

Next, the EcoRI-AvrII fragment containing rfp (iGEM part BBa_E1010) and the dbl transcriptional terminators (part BBa_B0015) from pBbB2k-RFP53 was inserted into pJC543, using the same restriction sites downstream of P LtetO-1 , thus replacing the rrnB T1 transcriptional terminator and generating pJC548. Expression of RFP from this plasmid can be induced with anhydrotetracycline.

The first series of plasmids (pJC566—pJC573) containing EilR-regulated promoters (P JEx1 , P JExA1 , P JExA2 , P JExL , P JExD , P JExH1 , and P JExH2 , respectively) was constructed by inserting each promoter and the divergently transcribed eilR into pJC548 to replace P LtetO-1 and tetR, using In-Fusion HD. Promoters and eilR were amplified from the pTR_aJEx plasmid suite using primers Kan-R-ig-F (5′-GCGAAACGATCCTCATCCTG) and RFP-R (5′-GTCTTCGCTACTCGCCATATG), and the PCR products were each combined with the desired PvuI-EcoRI fragment from pJC548. This series of plasmids could be introduced into P. putida KT2440 and S. meliloti Rm1021 but not C. crescentus NA1000. Repeated attempts led to recovery of plasmids with transposon insertions between nptII and oriV, suggesting possible interference of plasmid replication due to transcription read-through from eilR and nptII.

The EilR-regulated expression plasmids were refined by inverting the region containing the origins of replication and transfer and inserting the V. fischerii luxG terminator (iGEM part BBa_B0011) downstream of trfA, to generate pJC575—pJC582 (containing P JEx1 , P JExA1 , P JExA2 , P JExL , P JExD , P JExH1 , and P JExH2 , respectively). For example, to construct pJC575, the oriV-trfA region was amplified from pJC566 with primers DVA00608 (5′-CACGTTAAGGGATTTTGGTCATGAACTAGTCTGCCATTTTTGGGGTGAGGCCG) and DVA00609 (5′-CGGGCGTTTTTTATTGGTGAGAATCCAAGCAAAATAATAAAAAAGCCGGATTAATAAT CTGGCTTTTTATATTCTCTGCTAGCGTTTGCAATGCACCAGG), and the resulting PCR product was combined with the appropriate NheI-SpeI fragment from pJC566. For comparison, a TetR-regulated expression plasmid pJC583 was constructed by ligating the EcoRI-SacI fragment containing rfp, colE1, oriV, oriT, and trfA from pJC575 and the EcoRI-SacI fragment containing nptII, tetR, and P LtetO-1 from pJC548. A plasmid without rfp, pJC586, which served as the vector control, was constructed by ligating the SphI-AvrII fragment containing tetR, P LtetO-1 , and rrnB T1 from pJC543 with the AvrII-SphI fragment containing colE1, oriV, oriT, and trfA from pJC575.

Plasmid pJC681 was constructed by amplifying SMc02369 from pJC476 with primers pleC −20F BamHI (5′-GCGGGATCCAGGACGACAAATTGGATAAG) and SMc02369+17R BamHI (5′-TTAGGATCCAGCGTAAGCGGGCGTGGTCA), digested with BamHI, and inserted into pJC581, digested with BglII and BamHI to replace rfp. Sequences of all fragments amplified by PCR were verified in the resultant plasmids. DNA parts used for a subset of the plasmid construction were designed using DeviceEditor and j5 software tools55 and assembled via isothermal DNA assembly.

Growth and induction of non-E. coli strains

Plasmids were maintained in E. coli DH10B (Invitrogen), which was cultured using lysogeny broth (LB) supplemented with 30 (in liquid medium) or 50 (in solid medium) μg ml−1 kanamycin. C. crescentus NA100056 and S. meliloti Rm102157 were grown in peptone-yeast extract (PYE) medium and P. putida KT244058 in LB medium, with antibiotics when appropriate: chloramphenicol (12.5 μg ml−1 for KT2440), kanamycin (5 (liquid) or 25 (solid) μg ml−1 for NA1000; 25 μg ml−1 for Rm1021; 50 μg ml−1 for KT2440), nalidixic acid (20 μg ml−1 for NA1000 and Rm1021), and neomycin (50 μg ml−1 for Rm1021).

Mobilization of plasmids from E. coli DH10B to S. meliloti or C. crescentus was accomplished by triparental mating, with the help of strain MT616, carrying pRK60059 (conferring resistance to chloramphenicol); nalidixic acid was used to select against E. coli donor and helper strains. Similarly, E. coli strain HB101/pRK207360 (conferring resistance to spectinomycin) facilitated conjugation into P. putida; chloramphenicol was used to select against the donor and helper strains.

RFP expression was monitored with 160 μL cultures grown in 96-well plates (Corning Falcon 353072) at 30 °C for up to 24 h. For P. putida KT2440-derived strains, overnight cultures were diluted to an optical density at 600 nm of 0.015, and 80 μL aliquots of the diluted cultures were dispensed into each well containing 80 μL of LB medium containing kanamycin, with varying concentrations of anhydrotetracycline or crystal violet. Absorbance at 600 nm and fluorescence (575 nm excitation, 620 nm emission) were measured every 20 min in a Tecan Infinite F200 PRO plate reader. For C. crescentus NA1000, overnight cultures were diluted to an initial optical density at 600 nm of 0.1 in PYE medium containing kanamycin for distribution into wells, and absorbance at 590 nm and fluorescence (535 nm excitation, 620 nm emission) were measured in a Tecan Infinite F200 plate reader. For S. meliloti Rm1021, overnight cultures were diluted to an initial optical density at 600 nm of 0.2 in PYE medium containing kanamycin, and absorbance at 600 nm and fluorescence (575 nm excitation, 620 nm emission) were measured in a BioTek Synergy 4 plate reader.

Spotting assays

E. coli DH10B containing pTR_sJExA1-sacB, pTR_aJExA1-sacB, pTR_sJExD-sacB, pTR_aJExD-sacB or the non-sacB control plasmid pBbA0k were grown overnight in LB/Kan (50 µg/mL) and diluted to an OD 600nm = 1 for 10-fold serial dilutions. Three microliter were spotted on LB/Kan (50 µg/mL) supplemented with 8% sucrose and/or 1 µM CV, and colonies from OD 600nm 10−2 to 10−5 dilutions were photographed after 20 h growth at 37 °C.

The S. meliloti pleC gene under the control of P JExH1 (pJC681) was introduced into strain JOE360839 [ΔpleC::Ω/pJC476 (P tau -pleC)] by triparental mating, selecting for neomycin resistance in the presence of 100 mM taurine and 1 µM CV, to replace the complementing plasmid pJC476, resulting in strain JOE5635. Stationary-phase cultures of JOE5635, JOE3608 (ΔpleC/P tau -pleC) and JOE3593 [Rm1021 (pleC+)/P tau -pleC) were washed and resuspended in PYE to an optical density at 600 nm (A600) of 0.1, and serially diluted 10-fold in water. Five microliter of each dilution (from 10−2 to 10−6) was spotted onto PYE plates, without or with 100 mM taurine or 1 µM CV, and incubated at 30 °C for 4 days prior to imaging.

SDS-PAGE

For RFP expression, low-copy-number RFP expression plasmids pBbS7k-RFP (LacI/P T7 ) and pTR_sJExD-rfp (EilR/ PJExD ) were transformed into BL21-(DE3), which were then grown in 50 mL TB+Kan (50 µg/mL) to an OD600 of ~1.2. Five ml of each culture was transferred to 25 mL glass culture tubes. One sample of each strain was stored at −20 °C as un-induced sample. The other cultures were induced with the indicated amount of CV or IPTG and grown at 30 °C for 24 h at 200 rpm. Samples were then centrifuged, re-suspended in 50 µL of 1× SDS-PAGE sample buffer (SB) and heated for 6 min in the microwave. Samples (1 µL) and a Novex sharp prestained protein standard were loaded onto 8–16% gels.

For β-galactosidase (LacZ) expression with either T7 or P JEx promoters, BL21-DE3 cells were transformed with the medium-copy-number plasmids pBbA7k-lacZ (LacI/P T7 ) and pTR_aJExD-lacZ (EilR/P JExD ). Cells were grown in 5 mL LB + Kan (50 µg/mL) in glass culture tubes at 37 °C until an OD 600nm of ~1.0. The cultures were then induced with either 500 µM IPTG or 1 µM CV and grown at 37 °C for 4 h. OD7 pellets (~1 ml culture at OD 600nm of 7) were collected and stored at −20 °C. An uninduced culture for each plasmid was also grown and sampled. The pellets were thawed, re-suspended in 700 µL PBS with 3 µg/mL DNase I. The cells were lysed by sonication, and the lysate was centrifuged to separate the soluble from insoluble fractions. The insoluble fraction was resuspended in 700 µL PBS and 7 µg of both the soluble and insoluble samples were analyzed by SDS-PAGE.

β-Galactosidase (LacZ) activity measurement

Activity of β-galactosidase was measured in the soluble fraction of the lysates used for the SDS-PAGE analysis, following an established spectrophotometric assay using ONPG (ortho-nitrophenyl-β-galactoside) as the substrate61. This involved mixing 1 µL of lysate with 276 µL 0.1 M Na-phosphate (pH 7.5) buffer containing 20 µL ONPG (4 mg/mL in 0.1 M Na-phosphate buffer), 3 µL 0.1 M MgCl 2 , and 4.9 M β-mercaptoethanol. Mixtures were incubated for 15 s at room temperature, prior to stopping the reaction with 600 µL 1 M Na 2 CO 3 . Absorbance was read on a spectrophotometer at 420 nM, using H 2 O as blank. Readouts were 0.189 (P T7 /IPTG), 0.586 (P JExD /1 µM CV), and 0.021 (sample without lysate).

Purification of the EilR protein

EilR-His 8 was expressed in E. coli harboring a pET-derived expression plasmid, pLane-eilR, with an IPTG-inducible T5 promoter and a TEV protease-cleavable his8-tag. Cells were grown to stationary phase overnight, and diluted 1:100 in 500 mL Terrific Broth (TB) supplemented with 2 mM MgSO 4 for cultivation in 2-L non-baffled flasks. These cultures were grown at 37 °C shaking at 200 r.p.m. until the OD 600 was ~1.3, then the temperature was lowered to 20 °C, IPTG was added to 0.5 mM, and the cultures continued for 3 days. For crystallography, selenomethionine (Se-Met) labeled protein was produced using the method described by Studier62. At this time the cells were harvested and the pellets stored at −80 °C. Expression levels were estimated using SDS-PAGE. Protein purification was begun by thawing the paste and re-suspending it in 50 mM Tris buffer, pH 8.0, containing 600 mM NaCl, 50 mM Na-glutamate, 50 mM arginine-HCl, 10 mM MgCl 2 , and 0.5 mM dithiothreitol (high salt buffer, HSB). The re-suspended cells were lysed using the Emulsiflex C3 homogenizer. The lysate was clarified by centrifugation at 40,000×g for 40 m at 4 °C. The clarified lysate was loaded onto a 5 mL His-Trap column and fractionated using an AKTA FPLC. The column was washed with HSB to establish an OD 280 baseline prior to applying 100 mL (20 column volume) of a gradient of 2–99% of 1 M imidazole in HSB. To remove both imidazole and the His 8 tag, fractions containing EilR-His 8 were pooled and TEV protease was added at a 1:100 molar ratio. The pooled fraction was dialyzed against 1 L of HSB overnight at 4 °C. Cleavage was monitored by SDS-PAGE analysis of an aliquot. The dialysate was passed through a 1 mL His-Trap column to capture TEV and remaining His-tagged EilR. EilR lacking the His 8 tag was collected in the flowthrough. For crystallographic studies, purified EilR was dialyzed against 1 L of 50 mM Tris buffer, pH 8.0, containing 150 mM NaCl, 50 mM Na-glutamate, 50 mM Arginine-HCl, 10 mM MgCl 2 , and 0.5 mM dithiothreitol, and concentrated to 10.5 mg mL−1.

Electrophoretic mobility shift assays (EMSA)

In the assay comparing EilR affinity to various operator versions, the purified EilR molecules and the duplexed oligonucleotides (Supplementary Table 6) were mixed in 30 mM Tris, pH 7.7, 100 mM NaCl, 25 mM arginine, 25 mM glutamine, 5 mM MgCl 2 and left at room temperature for 1.5 h before their run in a 2% agarose gel in Tris-borate-EDTA buffer. A total of 231 pmoles of protein were used for the 2:1 EilR:DNA, and 462 pmoles of protein for the 4:1 EilR-DNA mixtures. The samples were run on a 2% agarose gel in TBE buffer stained with SYBR safe dye (Invitrogen), and imaged with an Alpha Innotech FluorChemQ instrument.

In the EilR-mutant EMSA assay, EilR versions were mixed with the duplexed oligonucleotide 5′-AAAAAGTTGGACACGTGTCCAACTTTCC-3′ (eilOc operator in bold letters) in 50 mM Tris, pH 8.0, 150 mM NaCl, 50 mM arginine, 50 mM glutamine, 10 mM MgCl 2 , and 0.5 mM DTT and left at room temperature for 30 min before running in a 2% agarose gel in Tris-borate-EDTA buffer.

Crystallization of EilR in complex with eilO c and inducers

The final concentration of EilR used for crystallization trials was 10 mg mL−1. Oligonucleotides were synthesized at the 1 μmol scale and purified to remove small-molecule impurities by commercial vendors, such as IDTDNA (Coralville, Iowa). The oligonucleotides were resuspended in 20 mM Tris–HCl pH 7.5 containing 10 mM MgCl 2 . Oligonucleotide pairs were annealed in equimolar ratios by heating at 95 °C for 10 min and gradual cooling to room temperature. The EilR–eilO c complexes were formed by adding the duplexed oligonucleotide AAAAAGTTGGACACGTGTCCAACTTTCC-3′ (eilOc operator in bold letters) to the protein solution in a 2:1 (protein:DNA) molar ratio. The EilR apoenzyme and EilR-eilO complexes were screened using the sparse matrix method63 with a Phoenix Robot (Art Robbins Instruments, Sunnyvale, CA) and the following crystallization screens: Berkeley Screen (Lawrence Berkeley National Laboratory, Berkeley, CA), Crystal Screen, SaltRx, PEG/Ion, Index, and PEGRx (Hampton Research, Aliso Viejo, CA). Crystals of EilR apoenzyme were formed in 0.2 M trisodium citrate and 20% (w/v) polyethylene glycol 3350. Crystals of EilR-MG and EilR-CV complexes were obtained by soaking the crystallized EilR apoenzyme in 1 mM MG or CV solution for 5 h. The EilR-eilO complex was crystallized in 0.1 M Li 2 SO 4 , 0.1 M MgCl 2 , 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5), 20% (w/v) polyethylene glycol 3350 and 10% hexanediol. EilR apoenzyme and EilR-eilO crystals were obtained after 3 days by the sitting-drop vapor-diffusion method with the drops consisting of a mixture of 0.2 μL of protein solution and 0.2 μL of reservoir solution.

X-ray data collection and structure determination