XylR

Mechanism

Previous Engineering Efforts

Build Our Own Sensor!

XylR is an intensively studied regulatory protein mined from Pseudomonas putida^[1]. It responds strongly to toluene, xylene and 4-chlro-toluene, while weakly to 3-methyl benzyl alcohol^[1].

XylR activates the Pu promoter and expresses TOL "upper pathway" (XylMABC) when exposed to m-xylene. It also activates the Ps1 promoter, thus to produce another transcriptional activator, called XylS, to turn on the expression of the downstream pathway (XylXYZLTEGFJGKIH, the meta-cleavage operon)^[1][2](Fig. 1, Fig. 2). Notably, the entire regulatory network is also controlled by several global regulatory elements, such as IHF. This provides an explanation for the genetic-context-dependent performance of XylR-Pu pair; namely, when expressed in different bacterial species, the regulatory performance of XylR/Pu pair often fails^[3]. Therefore, fine-tuning is often necessary.

The XylR protein consists of FOUR domains (Fig. 3): Domain A is the sensor domain, which will conduct a conformational change upon effector binding. Previous studies showed that Domain A inhibits the DNA binding affinity of Domain C before the conformational change^[4][5]. Domain B is a linker domain; mutations in this domain will disrupt the functional coupling and spatial interactions between Domain A and C^[6].

Domain C is the effector-binding domain with ATPase activity that is crucial for XylR dimerization. A subdomain in domain C is assumed to account for the dimerization. Domain D is the DNA binding domain featured by helix-turn-helix motif whose DNA binding is sequence-specific.

XylR is capable of forming tetramer when bound with ATP ^[7]. Without ATP binding, the XylR dimers bind to two sequence-specific DNA sites. As a typical σ⁵⁴-dependent transcriptional activator, with the binding of ATP, two dimers of XylR tend to further cooperatively tetramerize, thus to bend the promoter region DNA with the help of integrated host factor (IHF). As a result, the transcription will be launched by the interaction between the XylR tetramer and RNA Polymerase (RNAP). ATP hydrolysis provides energy for this process ^[8].

Since one of the criteria of our Sensor Mining for aromatics-sensing transcriptional regulators is "well-studied", it can be expected that some mutants of XylR with novel aromatics-sensing characteristics have been identified. Therefore, we set out to collect the information of XylR. As expected, random mutagenesis on XylR Domain B has been performed in previous studies^[9]. One XylR mutant, referred to as XylR28 in ref. [9], carries 4 point mutations in Domain A and Domain B. These point mutations endow XylR with a remarkably improved response to 2.4-DNT and TNT and a reduced response to its natural inducer, m-xylene, indicating the directed evolution may provide possibility to engineer XylR to respond to compounds that it doesn't naturally sense^[6].

As discussed above, the XylR/Pu pair usually needs fine-tuning. Promoter engineering is considered to be an answer. A XylR-conrolled Pu promoter shows a high basal level. But the cognate promoter of XylR homolog, DmpR, has a fairly low basal level. We found that XylR could activate the Po promoter of DmpR. A hybrid promoter has been accordingly designed using the binding site of XylR from the Pu promoter. This design has shown that the basal level of the hybrid promoter is low and XylR binding affinity is high^[12].

Our team obtained XylR from the TOL plasmid of Pseudomonas putida mt-2 through Polymerase Chain Reaction (PCR) (the strain is from Prof. de Lorenzo’s generous material assistance). We also obtained from Prof. de Lorenzo XylR5 protein coding sequence (on pCON924), which performs well in the original paper^[11][13]. We standardized these regulators' coding sequences, and attached a variety of Pc promoters (iGEM part, J23100 Pc series) in front of these coding sequences to create a library in order to obtain best performance. We first determined to test the wild type XylR for higher engineering potential.

As is shown above, the performance of XylR needed improvement, and we proposed a possible explanation to this .The ON/OFF test was performed on the 96-well plate with sealing film preventing the aromatics' vaporization. But Unfortunately, the main inducers of XylR reported by previous papers are typically hydrophobic aromatic compounds and they can permeate the film and vaporize noticeably faster than some non-inducers. In this case, the experiment result was affected by the inducer's position on the 96-well plate.Regarding this problem, we did meticulous inducing experiment in centrifuge tubes to prevent its vaporization and so we acquired new result,

Figure 1. The regulatory network of TOL pathway, including the xyl gene cluster, XylS and XylR. XylR is the master regulator that regulates Pu promoter (controls "upper pathway", XylMABC) and Ps2 promoter (controls the expression of XylS, thus to indirectly activate the expression of "downstream pathway“, XylXYZLTEGFJGKIH). The xylene or its derivatives are supposed to be the typical inducers of XylR.

Figure 2. The TOL degradation pathway. The supposed inducer of XylR, the toluene and its derivatives are highlighted in blue.

Figure 3. Schematic organization of XylR protein domains. From N-terminal to C-terminal: the sensor domain A, the linker domain B, the dimerization domain C and the DNA binding domain D.

Figure 4. The mechanism of σ⁵⁴-dependent transcription activation by XylR. Step1, RNAP recruitment by σ⁵⁴; XylR has formed dimers when binding to DNA. Step2, formation of XylR tetramer, coupled with ATP hydrolysis. Step3, RNAP ready to initiate transcription. Step4, transcription start with σ⁵⁴ released. See the main text for more detailed explanation of transcription activation at the Pu promoter.

Figure 5 The induction ratios of all typical aromatic compounds in the ON/OFF test following Test Protocol 1. XylR biosensor could respond to several aromatics with the induction ratio is not sufficiently high.This result was not consisted with the paper's report.^[5].

Figure 6. XylR's main inducers' ON/OFF test. We used the protocols in the papers to test XylR biosensor, and this experiment was processed in Centrifuge tubes to ensure the vaporization was't influence our results. As is shown, the main inducers had an induction effect.

Reference:
[1] Abril, M. A., Michan, C., Timmis, K. N., & Ramos, J. (1989). Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.Journal of bacteriology, 171(12), 6782-6790.
[2] Gerischer, U. (2002). Specific and global regulation of genes associated with the degradation of aromatic compounds in bacteria. Journal of molecular microbiology and biotechnology, 4(2), 111- 122.
[3] Valls, M., Silva‐Rocha, R., Cases, I., Muñoz, A., & de Lorenzo, V. (2011). Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting. Molecular microbiology, 82(3), 591-601.
[4] Devos, D., Garmendia, J., Lorenzo, V. D., & Valencia, A. (2002). Deciphering the action of aromatic effectors on the prokaryotic enhancer‐binding protein XylR: a structural model of its N‐ terminal domain. Environmental microbiology, 4(1), 29-41.
[5] Salto, R., Delgado, A., Michán, C., Marqués, S., & Ramos, J. L. (1998). Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators. Journal of bacteriology,180(3), 600-604.
[6] Garmendia, J., & De Lorenzo, V. (2000). The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida. Molecular microbiology, 38(2), 401-410.
[7] Tropel, D., & Van Der Meer, J. R. (2004). Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, 68(3), 474-500.
[8] Pérez-Martín, J., & de Lorenzo, V. (1996). ATP Binding to the σ< sup> 54-Dependent Activator XylRTriggers a Protein Multimerization Cycle Catalyzed by UAS DNA. Cell, 86(2), 331-339.
[9] de las Heras, A., Chavarría, M., & de Lorenzo, V. (2011). Association of dnt genes of Burkholderia sp. DNT with the substrate‐blind regulator DntR draws the evolutionary itinerary of 2, 4‐ dinitrotoluene biodegradation. Molecular microbiology, 82(2), 287-299.
[10] de las Heras, A., & de Lorenzo, V. (2011). Cooperative amino acid changes shift the response of the σ54‐dependent regulator XylR from natural m‐xylene towards xenobiotic 2, 4‐ dinitrotoluene. Molecular microbiology, 79(5), 1248-1259.
[11] Garmendia, J., De Las Heras, A., Galvão, T. C., & De Lorenzo, V. (2008). Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes. Microbial Biotechnology, 1(3), 236-246.
[12] Kim, M. N., Park, H. H., Lim, W. K., & Shin, H. J. (2005). Construction and comparison of< i> Escherichia coli whole-cell biosensors capable of detecting aromatic compounds. Journal of microbiological methods, 60(2), 235-245.
[13] Garmendia, J., Devos, D., Valencia, A., & De Lorenzo, V. (2001). À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer‐binding protein XylR to non‐natural effectors. Molecular microbiology, 42(1), 47-59.