Part:BBa_K4757110

Pm promoter

Pm promoter controlled by the XylS transcription factor

Contribution by iGEM Evry Paris-Saclay 2024

The XylS transcriptional regulator

The Role of XylS Protein in the Regulation of Catabolic Operons

XylS is a transcription factor (TF) identified from the TOL plasmid pWW0 of Pseudomonas putida (P. putida) [Worsey and Williams, 1975] belonging to the AraC/XylS superfamily which includes other notable members such as Ada, AraC, MarA, MelR, RhaR, RhaS, Rob, or SoxS [Gallegos et al., 1997].

XylS plays a key role in regulating the degradation of aromatic hydrocarbons by P. putida (Figure 1) by activating the transcription of genes in the 'meta' operon (XylXYZLTEGFJQKIH operon on the TOL plasmid), involved in the breakdown of benzoate and alkylbenzoates into simpler intermediates, enabling the bacterium to use these compounds as carbon and energy sources, especially in environments contaminated with recalcitrant organic pollutants [Ramos et al., 1986; Ramos et al., 1987; Gallegos et al., 1997; Kim et al., 2016]. This regulatory process is closely linked to the ‘upper’ catabolic pathways (XylUWCMABN operon on the TOL plasmid), which represent the initial steps in the degradation of complex aromatic compounds. In these initial pathways, hydrocarbons such as toluene and xylenes are partially oxidized into intermediate products like carboxylic acids, including benzoate and alkylbenzoates.

These intermediate compounds act as crucial effectors by binding to the N-terminal domain of XylS and inducing a conformational change that activates the C-terminal domain. This activation enables XylS to bind to the Pm promoter of the 'meta' operon and stimulate the transcription of XylXYZLTEGFJQKIH genes responsible for converting these benzoic acids into catechols, facilitating their further breakdown into non-toxic compounds that the bacterium can use. This process is essential for enabling Pseudomonas to metabolize complex aromatic compounds that would otherwise persist in the environment and contribute to pollution.

Thus, effectors like benzoate are not merely by-products of hydrocarbon catabolism but play a pivotal role in the activation of XylS. They ensure precise coordination between the ‘upper’ and ‘meta’ pathways, guaranteeing that hydrocarbon degradation is continuous and efficient. Through this regulation, Pseudomonas can exploit the energy resources of aromatic compounds while reducing their environmental impact [Kaldalu et al., 2000; Cowles et al., 2000].

Figure 1. Schematic representation of the metabolic pathways encoded by the 'meta' and the ‘upper’ operons from the TOL plasmid pWW0 of P. putida involved in the degradation of aromatic compounds into non-toxic simpler intermediates that the bacterium can utilize as sources of carbon and energy (adapted from Kim et al., 2016).

XylS is capable of recognising a broad range of ligands with a certain degree of promiscuity (Figure 2). This variability fine-tunes the activity levels according to the specific ligand present in the context. We have compiled this list based on the data found in the literature. These inducers may be categorized in benzoic acid derivatives (methyl-, fluoro-, chloro-, iodo-, amino-, hydroxybenzoates) which are the most studied because of their ability to modulate XylS activity, phenols and their derivatives, aromatic aldehydes, multifunctional aromatic acids such as phthalic and terephthalic acid, as well as other various aromatic compounds, which do not strictly fit into these categories, were included to broaden the range of induction possibilities. To complement this analysis, we also performed our own experimental tests to evaluate the effect of old and new inducers on XylS activity.

Figure 2. Overview of the XylS effectors / ligands / inducers reported in the literature and their induction ratios. The induction ratios were presented as such by Ramos et al., 1986 or were calculated by us based on the numerical data reported by Zhou et al., 1990 and Michan et al., 1992, or estimated by us based on the graphical data of Xue et al., 2014, Ogawa et al., 2019 and Li et al., 2022.

Structural Analysis and Transcription Mechanism of XylS

XylS is a cytoplasmic monomeric protein composed of 321 residues in its sequence, of 36 kDa [Domínguez-Cuevas et al., 2008]. Currently, like most of the AraC family transcription factors, no experimental 3D structure of XylS has been resolved yet, due to its low solubility and the unsuccessful purification of its active form [Gawin et al., 2017]. Only a predicted model structure from AlphaFold 1 (AF-P07859-F1-v4) is available. As AlphaFold released their latest and more accurate/efficient model version in 2024, AF3 (https://alphafoldserver.com), we predicted a new structure model of XylS by using it and further refined this prediction with SwissDock (https://www.swissdock.ch/). The thus obtained AF-P07859-F3 (with a predicted template modeling (pTM) score of 0,65) structure is presented in Figure 3 with m-toluic acid, one of XylS natural ligands, in the effectors predicted binding pocket.

Figure 3. Predicted three-dimensional structure of XylS (UniProt P07859) binding to m-toluic acid. Predicted model of XylS from AlphaFold3 (AF3) binding to m-toluic acid ligand (blue) upon docking with SwissDock (A) and the corresponding topology diagram representation (B). The DNA-binding domain (HTH) is highlighted in cyan and the Effector-Binding Domain (EBD) is shown in turquoise.

Predicting structure by homologous modeling is a preferable strategy option but no 3D structure of homologous protein with more than 50-80 % ID is available yet. For now it is difficult to find a protein template that shares more than 35 % ID with XylS in order to resolve more accurately and precisely.

XylS, like many members of the AraC superfamily, is an asymmetrical monomer protein containing two domains connected by a linker. The 200-amino-acid N-terminal domain, the Effector-Binding Domain (EBD), is responsible for the effector recognition, protein dimerization and interaction with the RNA polymerase (RNAP) complex [Ruíz et al., 2001]. This domain is characterized by a β-barrel core of eleven β strands (strand β1-β11) forming a “jelly roll” fold similar to the well characterized structure of an homologous transcriptional factor ToXT (PDB: 3GBG) [Lowden et al., 2010] that contain the binding pocket (Figure 3). The C-terminal domain is characterized by two Helix-Turn-Helix (HTH) motifs, composed of seven alpha-helix (helix α6-α12) commonly found in DNA Binding Domain (DBD) in most TFs (PROSITE: PS01124) (Figure 3) and likely interacts with the Pm promoter.

The monomeric XylS is inactive. Upon ligand binding, it dimerizes leading to its active form that could bind DNA (Figure 4) and activate the RNAP complex and transcription initiation (Figures 4 and 5) [Ruíz et al., 2001 ; Domínguez-Cuevas et al., 2010]. More precisely, binding of the inducer causes conformational changes that prevent repression by the DNA-binding domain (DBD) and favor dimerization of XylS monomers. This dimerization is stabilized by hydrophobic interactions in the antiparallel coiled-coil regions of the effector-binding domain (EBD), which are crucial for the interaction between the two domains (DBD and EBD) via the linker [Ruiz et al., 2003]. Mutations (L193A, L194A, I205A) next to the linker prevent from coiled-coil interactions and highlight their essential role for dimerization [Domínguez et al., 2008].

Figure 4. Schematic representation of the activation of XylS upon binding m-toluic acid (adapted from Ogawa et al., 2019).

The activation of the Pm promoter occurs in several steps (Figures 5 and 6):

STEP 1: Upon inducer binding, a first XylS monomer is activated and binds to proximal regions of the Pm promoter located into a bending angle of 35°. Each HTH motif binds to two boxes, consensus binding sites, “TGCA” (A box) and “GGATA” (B box) separated by six nucleotides.

STEP 2: After inducer binding, the activated XylS monomer form binds to the P site, inducing a 50° DNA bending angle that brings the P and D binding sites closer.

STEP 3: The binding of a second XylS monomer to the distal binding site (D), facilitates the dimerization of the two XylS and induce further conformational changes of the DNA topology by increasing the DNA bending angle to 98°.

STEP 4: XylS then recruits and interact with RNA polymerase (RNAP) through direct contact with carboxyl-terminal domain of the RNAP alpha subunit (α-CTD) (probably thanks D137 and H153 residues) and potentially with the σ32/38 factor. These interactions favor the assembly of RNAP complexes and promote the initiation transcription [Ruíz et al., 2001; Domínguez-Cuevas et al., 2005, 2008, 2010; Gawin et al., 2017].

Figure 5. Schematic sequence of Pm promoter and XylS binding sites corresponding to A and B boxes (D: distal; P: proximal; colored in blue) and the ‐10/‐35 consensus boxes of σ32/38 RNAP subunit (colored in green) (adapted from Domínguez-Cuevas et al., 2010).

Figure 6. Schematic model of the sequential Pm activation mechanism:

1: Free DNA showing XylS Pm binding sites (D: distal; P: proximal; blue boxes) and the ‐10/‐35 consensus boxes (green boxes) of σ32/38 RNAP subunit. Proximal binding site is located into the bending angle of 35°.

2: After inducer binding, the activated XylS monomer form binds to the P site, inducing a 50° DNA bending angle that brings the P and D binding sites closer.

3: The binding of a second XylS monomer to the D binding site favorized dimerization, further increasing the bending angle to 98° and facilitating RNAP recruitment through interaction with σ and the two α-CTD subunits that finally favorized RNAP complex formation and transcription initiation (Adapted from Domínguez-Cuevas et al., 2005, 2008, 2010). The targeted gene transcription level will depend on two factors: Pm sensitivity depending on intracellular XylS expression level (activated and dimerized at high XylS concentration without inducer) and the activity of XylS depending on the ligand (nature, concentration).

Experimental characterisation of gene regulation by XylS

DESIGN

Our experimental setup, schematised in Figure 7 follows a classical architecture for whole-cell biosensors. It is composed of two parts:

-> the expression plasmid (EP) carrying the XylS gene under the control of the J23104 promoter, a custom made RBS and the B0011 terminator in the BBa_K5061003 backbone composed of the chloramphenicol resistance gene (CmR) and the CloDF13 origin of replication. As plasmids having a CloDF13 ori are medium copy (20-40 / cell), we choose a strong promoter for the XylS gene: the J23104, one of the strongest of the Anderson library promoters according to the data presented on its page in the Registry. We also designed a specific RBS library for XylS using the De Novo DNA’s Library Calculator v2.0 [Reis et al., 2020; Farasat et al., 2014; Ng et al., 2015] and selected the one predicted to have a Translation Initiation Rate (TIR) of about 25000 (BBa_K5061024).

-> the test plasmid (TP) carrying the sfGFP-LVAtag gene under the control of the Pm promoter regulated by XylS in the BBa_K5061002 backbone composed of the ampicillin resistance gene (AmpR) and the pSC101 origin of replication. The choice of sfGFP fused to the LVA degradation tag (BBa_K2675006) was based on the experience of our PI with this reporter gene from previous iGEM projects. Indeed, the LVA degradation tag (AANDENYALVA), which is an ssrA tag that accelerates protein degradation in Escherichia coli at 37°C [Pédelacq et al., 2006], allows a rapid degradation of the reporter gene and thus finely observe the synthetic gene networks dynamics. The backbone carries the pSC101 origin of replication, which is low copy and thus allows reducing the background expression of leaky promoters.

Figure 7. Experimental setup for in vivo transcriptional whole-cell biosensors for charactérisation of XylS activity in the presence of various natural and synthetic ligands including m-toluic acid, 3-chlorobenzoic acid, salicylic acid, benzoic acid, 3,4-dichlorobenzoic acid, 3,4-dihydroxybenzoic acid, anthranilic acid, 4-aminobenzoic acid, 3-(dimethylamino)benzoic acid, PA and TPA with detection via GFP fluorescence (adapted from Alvarez-Gonzalez and Dixon, 2019).

BUILD

The construction of our EP (BBa_K5061060) and TP (BBa_K5061056) plasmids for XylS characterisation was achieved by Golden Gate in two steps using established molecular biology protocols.

First we have built EP and TP cloning platforms (BBa_K5061033 and BBa_K5061034, respectively) in which Golden Gates adapters with BsaI sites are present between the J23104 promoter and B0011 terminator in the case of EP and upstream the RBS of sfGFP in the case of TP. Those plasmids were assembled by Golden Gate too, using BsmBI as a restriction enzyme, and DNA fragments obtained either by PCR from plasmid templates purchased from Addgene (MP6 and pJC175e) or provided by the hosting lab or synthesized.

Using these ‘universal’ Golden Gate cloning platforms for EP and TP, we readily built the XylS specific EP (BBa_K5061060) and TP (BBa_K5061056) plasmids. For this, XylS was synthesized as a DNA fragment containing the designed RBS sequence with flanking convenient BsaI type IIS restriction sites. The Pm promoter was synthesized also, but as two long oligonucleotides, one for each DNA strand, that were annealed to form the double stranded DNA that was used in the Golden Gate reaction.

As positive control, we have built an sfGFP expressing cassette under the control of the constitutive promoter J23110 (BBa_K5061046).

TEST

To evaluated the XylS capacity to regulate the expression from the Pm promoter in our experimental setup, E. coli DH10B cells transformed with the XylS specific EP (BBa_K5061060) and TP (BBa_K5061056) plasmids were grown overnight at 37°C with shaking at 200 rpm in 96-DeepWell plates containing 1 mL of LB medium supplemented with 50 µg/mL ampicillin and 17.5 µg/mL chloramphenicol. The cells were then diluted 1:40 in a fresh LB medium containing the same antibiotics and incubated for 3-4 hours at 37°C with shaking. Following this incubation, the cells were further diluted 1:20 in LB medium containing the antibiotics and varying concentrations of specific inducers: m-toluic acid, 3-chlorobenzoic acid, terephthalic acid (TPA), phthalic acid (PA), salicylic acid, benzoic acid, 3,4-dichlorobenzoic acid, 3,4-dihydroxybenzoic acid, anthranilic acid, 4-aminobenzoic acid, and 3-(dimethylamino)benzoic acid, all as sodium or potassium salts. Inducer stock solutions were prepared at 5 mM in LB, and dilutions were performed to achieve final concentrations ranging from 0.01 µM to 5 mM. For each test, 10 µL of the 1:40 diluted cultures were added to 190 µL of the media containing the desired concentration of each inducer in an opaque 96-well polystyrene microplate (COSTAR 96, Corning). The microplates were then incubated at 37°C with shaking at 200 rpm. Fluorescence measurements (λ_{excitation</sup> 483 nm, λemission</sup> 530 nm) and optical density at 600 nm (OD600) were taken every 15 minutes for 24 hours using a CLARIOstar (BMGLabtech) plate reader. Fluorescence values were normalized by OD600 to account for variations in cell density.}

LEARN

In our exploration of the effects of novel and classical inducers of XylS, we decided to test 11 compounds among which 7 are known XylS effectors, 3 were previously reported in the literature as ineffective and one that has not been yet assessed, to the best of our knowledge. The results of our experiments are presented in Figure 8. We can first notice that both the negative and the positive controls behaved as expected (Figure 8A): the fluorescence/OD600 values remained very low when the sfGFP had no promoter upstream and that they readily increased when the constitutive promoter J23110 was present.

Moreover, when sfGFP was preceded by the Pm promoter, in the absence of XylS or in the presence of XylS and in the absence of an inducer, as expected, the sfGFP expression remained at very low levels comparable to those of the negative control. These results demonstrate that the leakiness of Pm promoter is negligible.

Furthermore, when the 3-methylbenzoic acid (m-toluic acid), the most potent XylS inducer (see Figure 2 above), was added in the media, the sfGFP expression quickly and gradually increased to even higher levels then those obtained for the positive control. The fluorescence/OD600 fold changes in the presence of this inducer compared to the values in the absence of the inducer reached 24.4 at 1 mM m-toluic acid, a value which is in the range of those reported in the literature for this effector (see Figure 2 above), thus validating our experimental setup. Benzoic acid was the second most potent XylS effector we tested for which obtained a maximum fluorescence/OD600 fold change of 16.4, a value also comparable to those reported in the literature (see Figure 2 above).

3-Chlorobenzoic acid, 2-hydroxybenzoic acid (salicylic acid), 2-aminobenzoic acid (anthranilic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid) and 3,4-dichlorobenzoic acid are the other know XylS effectors we included in our experiments. All of them are weaker XylS inducers (see Figure 2 above) and, for three of them, our detection system did not prove sensitive enough to detect their action.

In our set of compounds we also included 3 compounds that were reported as ineffective XylS effectors in the literature (see Figure 2 above): 4-aminobenzoic acid, phthalic acid (PA) and terephthalic acid (TPA). As expected, no sfGFP fluorescence was observed in the presence of PA or TPA indicating that their structure negatively affects their interaction with XylS, limiting their effectiveness as inducers for XylS. In contrast, 4-aminobenzoic acid showed a weak, but not inexistante induction capacity.

The XylS effectors list is not a short one. Many compounds were already tested for their ability to induce the Pm promoter. We took advantage of our testing system and added to this list a compound never assessed before, to the best of our knowledge: the 3-(dimethylamino)benzoic acid (CAS 99-64-9). This compound is a known biochemical substrate used in chromogenic assays for peroxidase and peroxidase-coupled reactions [Ngo and Lenhoff, 1980]. XylS demonstrated its broad spectrum capacity to detect a wide range of benzoic acid derivatives, and proved to be compatible also with the 3-(dimethylamino)benzoic acid. Indeed, the sfGFP expression gradually increased with increasing levels of this compound up to 5 mM, the highest concentration we tested. The induction levels were not extremely high, but reached a non negligible level of 3, higher than those we obtained with established XylS effectors like 4-aminobenzoic acid, 2-aminobenzoic acid (anthranilic acid), 3,4-dihydroxybenzoic acid (protocatechuic acid) or 3,4-dichlorobenzoic acid.

We would like to specify that none of the 11 tested compounds had a negative influence on E. coli growth up to the highest concentration tested of 5 mM.

Figure 8. In vivo characterization of sfGFP-LVAtag expression by E. coli DH10B cells carrying the XylS expressing plasmid (BBa_K5061060) together with the reporter plasmid carrying the sfGFP-LVAtag gene under the control of Pm promoter (BBa_K5061056) in the presence of increasing concentrations of different XylS effectors. The negative controls (no XylS and no promoter) were performed with the EP and TP cloning platforms BBa_K5061033 and BBa_K5061034, respectively. As positive control, we used BBa_K5061046 in which sfGFP-LVAtag is expressed from the constitutive promoter J23110. (A, B) Fluorescence/OD600 values obtained in the late exponential growth phase as a function of effectors' concentration. (C) Fluorescence/OD600 fold changes in the presence of the inducer compared to the values in the absence of the inducer. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]