XylR

Overview

Mechanism

Previous Engineering Effort

Our Work

XylR is an intensively studied regulatory protein originated from Pseudomonas putida^[1], with numerous researches about it published prior to our project. XylR mainly responds to toluene, xylene, 4-chlro-toluene, while responds weakly to 3-methyl benzyl alcohol^[1]. These responses to aromatic compounds bring about the necessity to study its functioning mechanism.

XylR activates the Pu promoter and opens TOL upper pathway when exposed to m-xylene. XylR also activates another transcriptional factor called XylS, which controls the downstream pathway^[1][2].(Fig. 1,2)

This process is also controlled by several global regulatory elements, and the difference of global regulatory elements may provide an explanation to different performance in different genetic context^[3].

The XylR protein consists of FOUR domains:
A Domain is the sensor domain, which will change its secondary structure upon binding of proper ligands. Previous researches showed that A domain inhibits C domain’s DNA binding activity before the conformational change^[4][5].
B Domain is a linker; mutations of this domain change the interaction between domain A and C through changing the spatial position of two domains^[6].(Fig. 3)
C Domain is the effector domain that has ATPase activity. A subdomain in domain C is assumed to have the ability of dimerization. The ATPase activity is crucial for its dimerization.
D Domain is DNA binding domain with helix-turn-helix motif, which is capable of binding specific DNA sequence.

XylR is capable of forming tetramer with ATP’s binding^[7]. This procedure is significant because the XylR may only bind to DNA after forming a dimer. A σ54-dependent transcriptional factor typically has two binding site, and the binding of XylR is cooperative, which means binding of the first protein will facilitate the binding of the other transcriptional factor(TF) as well as its dimerization with the first TF. After dimerization, two dimers of one promoter may form a tetramer to bend the promoter region DNA with the help of integrated host factor (IHF). After DNA binding, transcription will be launched by the interaction between the XylR tetramer and RNA Polymerase (RNAP). ATP hydrolysis will provide energy for this process. After ATP has been hydrolyzed to ADP, XylR tetramer will break apart from RNA polymerase, waiting for other RNAP and ATP’s interaction to start transcription again^[8].

A random mutation of XylR B domain has been selected by previous work^[9]. This result suggested that mutations in several specific sites may cause XylR to respond to 2.4-DNT. The XylR28 referred in this paper which carries 4 point mutations in A and B domains, has a remarkably improved performance responding to 2.4-DNT and TNT, while reduced response to its natural inducer m-xylene, indicating directed evolution may engender possibility for XylR to respond to compounds that it doesn't naturally sense^[6].

Apart from the point mutation, another protein engineering method is called 'shuffling', which means the hybrid of homologous proteins or protein domains. Proteins of the same family may have similar functions and structures, and the specific active sites for recognizing natural inducers may distribute in different subdomains. If the proteins of the same family exchange some subdomain, the result, hybrid protein, may have the capacity of recognizing inducers including a proportion of both the original inducers of the two original transcriptional factors, or furthermore, new inducers with new detection range. Previous studies showed that the shuffling product between XylR and its homolog DmpR has a higher induction ratio for the original protein's natural inducers and starts to response to several new inducers, for example, 2,4-DNT^[10][11].

To extend detection range and improve performance, promoter engineering is considered to be a more reliable alternative to protein engineering. A XylR-conrolled Pu promoter has a high basal level because the ATP binding doesn't coupled to this transcription activation. But its homolog, DmpR, has a fairly low basal level. For the similarity of XylR and DmpR, XylR can activate the Po promoter for DmpR. Based on this crosstalk phenomenon, a promoter engineering is possible. They designed a hybrid promoter of Po promoter’s basal structure and XylR’s binding site from Pu promoter. This design ensured that the hybrid promoter’s basal level 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.

Fig. 1. TOL pathway, the xyl gene cluster and regulatory mechanism, XylR controls Pu promoter and Ps2 promoter. Ps1 is a weak constitutive promoter, which controls the level of XylS.When xylene or its homologous compounds exist, the upper pathway and another promoter called XylS is activated.

Fig. 2. TOL degradation pathway, XylR’s inducers, Toluene’s homologous compounds, is shown in blue.

Fig. 3. Protein domains of XylR: From N terminal to C terminal are the functional domain A, linker B, dimer domain C and DNA binding domain D as discussed below.

Fig. 4. σ54-dependent TF mechanism for XylR activation.
Step1: RNAP recruit facilitated by σ54, XylR have formed dimers when binding to DNA.
Step2: Formation of XylR tetramer, this process is coupled with ATP hydrolysis.
Step3: RNAP ready to transcription
Step4: Transcription start, with σ54’s divorce from its position.

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.