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Best New Basic Part (MexL)

Usage and Biology

Pseudomonas aeruginosa is an opportunistic pathogen widely distributed in aquatic environments, posing a serious threat to public health safety. MexL is a TetR-family transcriptional regulator derived from Pseudomonas aeruginosa, whose unique function is to specifically sense the pathogen's characteristic signaling molecule—pyocyanin (PYO)—and thereby regulate gene expression.

The core function of this component (MexL protein coding sequence) is to serve as a PYO-sensing switch. In the absence of PYO, MexL protein acts as a transcriptional activator, specifically binding and activating its target promoter PphzA1, thereby driving high-level expression of downstream reporter genes (such as gus). When PYO is present in the environment, it acts as an effector molecule and binds to MexL, inducing a conformational change that results in the loss of DNA-binding ability, thus relieving the activation of PphzA1 and causing the suppression of downstream reporter gene expression [1].

Therefore, based on the negative-correlation detection mode of "signal decreases in the presence of PYO and increases in the absence of PYO," MexL is an ideal core sensing element for constructing high-specificity, high-sensitivity biosensors for detecting Pseudomonas aeruginosa.

Detailed Molecular Mechanism

In its natural state, the MexL protein can directly bind to the PphzA1 promoter region of key phenazine biosynthesis genes. It activates transcription of these genes by recognizing a conserved palindromic DNA sequence (5'-TGTAATTT-3' and its complementary strand 5'-AAATTACA-3').

1. Activated State (without PYO): In the absence of the effector molecule PYO, MexL exists in its naturally active conformation. The N-terminal DNA-binding domain can efficiently recognize and tightly bind to the specific palindromic sequence within the target promoter PphzA1, thereby recruiting RNA polymerase and strongly activating transcription of downstream genes.

2. Repressed State (with PYO): As an effector molecule, PYO specifically embeds into the hydrophobic pocket of the C-terminal ligand-binding domain of MexL, inducing a significant conformational change (allosteric effect). This change prevents the N-terminal DNA-binding domain from stably associating with DNA, causing MexL to dissociate from the PphzA1 promoter, thus relieving the activation and allowing transcription levels of downstream genes to return to a weak basal level.

This "ligand-induced inactivation" mechanism makes MexL an ideal "genetic switch" that can convert the chemical signal of PYO into a detectable change in gene expression signals (such as a decrease in fluorescence).

Three-Dimensional Structure of the Protein

Sequence and structural analysis indicate that this protein is a bacterial transcriptional regulatory protein belonging to the TetR family. Its three-dimensional structure exhibits typical features of this family (Figure 1): the N-terminus (amino acids 15-59) contains a helix-turn-helix DNA-binding domain responsible for recognizing specific DNA sequences; the C-terminus (amino acids 69-207) forms a ligand-binding domain that creates a hydrophobic pocket for binding the small-molecule ligand PYO. These two domains are conformationally coupled: when PYO binds to the C-terminal domain, it triggers an overall conformational change in the protein, preventing the N-terminal helix-turn-helix domain from effectively binding DNA, thereby losing its transcriptional activation function.

Figure 1: Three-dimensional structure of MexL protein

Design Philosophy

Based on these characteristics, we designed MexL as the core element of a highly specific biosensor for detecting PYO, the signature signaling molecule of Pseudomonas aeruginosa. The working principle is as follows (Figure 2):

we couple MexL protein with the phzA1 gene promoter (PphzA1) it regulates and the gus reporter gene (encoding β-glucuronidase). In the absence of PYO, MexL is in an active state, binds to the PphzA1 promoter, and drives efficient expression of the gus gene. The GUS enzyme catalyzes the hydrolysis of the substrate 4-methylumbelliferyl-β-D-glucuronide (MUG), producing an intense fluorescent signal (4-methylumbelliferone, MU). When PYO is present in the environment, it binds to MexL, causing a conformational change and dissociation from the promoter, resulting in the suppression of gus gene transcription, decreased GUS enzyme expression, and a subsequent reduction in the fluorescent signal. By monitoring changes in fluorescence intensity, we can achieve precise and sensitive detection of Pseudomonas aeruginosa.

Figure 2：Working Principle Diagram of the PYO Biosensor Based on MexL-PphzA1

Functional Characterization and Validation

To confirm the reliability of MexL as a PYO sensor, we performed rigorous multi-level functional validation.

1. Prediction and Validation of Binding Ability (Molecular Docking Simulation)

Principle: Molecular docking simulation is a computational biology method that predicts binding mode, binding energy, and stability by simulating the three-dimensional interactions between a ligand (PYO) and a receptor (MexL protein), providing a theoretical basis for specific binding between the two [2].

Method: We used molecular docking software (AutoDock 4) to dock PYO molecules into the ligand-binding domain (C-terminal hydrophobic pocket) of the MexL crystal structure.

Conclusion: The results in Figure 3 show that PYO can form stable interactions with key amino acid residues of the MexL protein, such as TYR-165 and LYS-160. Generally, a negative binding affinity indicates the possibility of spontaneous binding between ligand and receptor, while a binding energy below -6 kcal/mol usually indicates strong binding ability. The simulation calculated the binding affinity between PYO and MexL protein as -7.10 kcal/mol, indicating high binding affinity between the two.

Figure 3: Interaction between MexL protein and PYO molecule

a. AutoDock4 results of double-PYO docking with MexL protein.

b. Convergence curves of binding energy predictions from two sets of 100 docking simulations.

2. Validation of DNA Binding Ability and Specificity between MexL Protein and PphzA1 Promoter (EMSA Experiment)

Principle: Electrophoretic Mobility Shift Assay (EMSA) is an in vitro technique for detecting specific protein-DNA interactions. When a protein binds to a specific DNA sequence, a protein-DNA complex is formed, which migrates more slowly in gel electrophoresis than the free DNA probe [3].

Method: We purified the MexL protein and performed binding reactions in vitro with two different DNA probes:

Specific probe: Contains the putative target sequence of MexL—the PphzA1 promoter region.

Non-specific control probe: Contains part of the coding sequence of the phzA1 gene, used as a control for binding specificity.

The reaction system was set up as follows:

Lane 1: DNA probe containing only the PphzA1 promoter sequence.

Lane 2: DNA probe containing only part of the phzA1 gene sequence.

Lane 3: Mixture of PphzA1 promoter probe and partial phzA1 gene sequence probe.

Lane 4: Reaction system containing PphzA1 promoter probe, partial phzA1 gene sequence probe, and purified MexL protein.

Lane 5: Reaction system containing PphzA1 promoter probe, partial phzA1 gene sequence probe, and bovine serum albumin (BSA).

After the reaction, non-denaturing polyacrylamide gel electrophoresis was performed, and binding was analyzed by observing DNA band migration.

Conclusion: The EMSA results (Figure 4) clearly show that in Lane 4 (containing MexL protein), only the band of the PphzA1 promoter probe showed significant migration retardation, while the position of the phzA1 gene sequence probe band remained unchanged. This demonstrates that the MexL protein can specifically bind to its target PphzA1 promoter but not to the non-specific phzA1 gene sequence.

In Lane 5 (containing BSA), neither DNA probe showed migration retardation, ruling out the possibility of non-specific protein-DNA interactions.

Figure 4: Validation of MexL protein binding ability to PphzA1 promoter

3. Validation of PYO-Induced Transcriptional Repression and Signal Molecule Specificity in Cellular Systems

Principle: By co-transforming the MexL expression unit and the gus reporter gene controlled by the PphzA1 promoter into E. coli and using the fluorescent substrate MUG to detect reporter gene expression, we can simultaneously evaluate MexL's response to PYO in living cells and its specific recognition of PYO.

Method: We constructed an engineered strain containing the MexL expression unit (pAZ2-Pcat-RBS-MexL) and the PphzA1-gus reporter module (pBZ2-PphzA1-gus). The strain was cultured under conditions without signal molecules (DMSO added), with PYO, with its precursor PCA, and with other common bacterial signal molecules (such as PQS). Fluorescent signals produced by MU were detected under UV light at 366 nm.

Conclusion: In the absence of any signal molecules, the strain produced an intense fluorescent signal; only after adding PYO did the fluorescent signal decrease, while the groups with PCA or PQS added showed no significant change in signal (Figure 5). This indicates that MexL can functionally and specifically respond to PYO in living cells and inhibit transcription of downstream reporter genes.

Figure 5: Response of MexL to PYO in living cells

4. Validation of Binding Specificity between MexL Protein and PphzA1

Principle: By comparing reporter gene expression driven by different promoters, we can verify whether MexL specifically recognizes only its natural target, the PphzA1 promoter.

Method: We constructed plasmids containing the MexL expression unit and the gus reporter gene driven by different promoters (such as PphzA1, PmexG, PbrlR), transformed them into engineered bacteria, and detected reporter signals in the absence of PYO.

Conclusion: Intense background fluorescence was observed only in engineered bacteria containing the PphzA1 promoter, while strains controlled by other promoters showed weak signals (Figure 6), demonstrating the high specificity of MexL for activating PphzA1.

Figure 6: Validation of binding specificity between MexL protein and PphzA1

Usage Method

Core concept: This component (MexL coding sequence) needs to be used in conjunction with its target promoter PphzA1 (available as a separate component) to form a complete sensing system.

Expression recommendation: To ensure sufficient and constant concentration of MexL protein in the host cell (such as E. coli) to maintain the basal activation level of PphzA1, we place this component (mexL gene) downstream of a strong constitutive promoter (cat Promoter).

System assembly: Sensor construction method:

1. Sensing module: Constitutive promoter → MexL coding sequence.

2. Reporter module: PphzA1 promoter → reporter gene gus.

Place the two modules on two compatible, different plasmids.

Detection process: Incubate the constructed engineered bacteria with the sample to be tested, add the reporter gene substrate (MUG), and detect the fluorescence signal intensity. A weaker fluorescence signal indicates a higher concentration of PYO in the environment, meaning a greater likelihood of Pseudomonas aeruginosa presence.

Precautions

1. Host Strain Selection: The host strain used in this system is a gus gene deletion mutant of Escherichia coli EC1000 (EC1000 △gus). This strain effectively eliminates background signal interference that may arise from endogenous β-glucuronidase activity, thereby ensuring the specificity and sensitivity of the detection results.

2. Biosafety Level:The MexL coding sequence was amplified by PCR using the Pseudomonas aeruginosa genome as the template. Since the experimental process involves genetic material derived from a pathogenic bacterium, all our experiments are conducted under Biosafety Level 2 (BSL-2) conditions in accordance with relevant biosafety protocols.

References

[1] Yu, Z., Wu, Z., Liu, D. et al. Dual-function regulator MexL as a target to control phenazines production and pathogenesis of Pseudomonas aeruginosa. Nat Commun 16, 2000 (2025).

[2] Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc. 2007;2(8):1849-1861.

[3] Shahidi, F., Dissanayaka, C.S. Phenolic-protein interactions: insight from in-silico analyses – a review. Food Prod Process and Nutr 5, 2 (2023).