XylR

Overview

Build Our Own Sensor!

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]. 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[ ][ ]. 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[ ]. (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.

Several experiments all conformed that NahR tightly binds to DNA in vivo in the presence or absence of salicylate. Either the amount or the affinity of NahR binding to DNA will be affected by salicylate in engineered E. coli and its native host Pseudomonas putida [7]. This fact, along with the evidence from methylation protection experiments, suggested a conformational change in the NahR•DNA complex which results in transcriptional activation (Fig. 4)[8].

Wide Type of NahR responds to its authentic inducer salicylate with the induction ratio over 20 [7]. In the attempt of building different whole-cell biosensors, NahR has been artificially evolved or somehow reshaped by mutagenesis to respond to new signals such as substituted salicylates and substituted benzoates [6,9]. New inducers obtained from mutagenesis are summarized in Table 1.

We ligated BBa_J61051 which contains the constitutively expressed NahR and sal promoter with the reporter gene sfGFP (Fig. 5) via standard assembly. The plasmid verified by Beijing Genomics Institute was transformed into E. coli (TOP10, TransGen Biotech). Single clone of bacteria was picked and grown in rich LB medium added chloromycetin (170 μg/ml) overnight and stored at -80℃ in 20% glycerol, waiting for induction test.

On-off test were first carried out for sensor strain NahR following test protocol 1 (hyperlink is needed). NahR strain with no inducer showed low basal expression of sfGFP and 18 compounds showed apparent activation effect with the induction ratios over 20 (Fig. 6). They are listed as follows: SaA, 2-ABzO, 3-MeSaA, 4-MeSaA, 4-ClSaA, 5-ClSaA, AsPR, 2,4,6-TClPhl, 3-IBzO, 2-MeBzO, 3-MeBzO, 4- FBzO, 3-ClBzO, 3-MeOBzO, 3-HSaA, 4-HSaA, 5-ClSaD, 4-ClBzO(For the full name, CHICK HERE). Besides salicylate derivatives, our sensor strain specially responded to 2,4,6-TClPhl (a kind of polychlorinated phenol (short for PCP)), which is of significant hazard to water environment and human health.

One step further, NahR strain was subjected to induction experiments with concentration of inducer ranging from 0.03 μM to 1 or 3 mM. Dose-response curves of inducers listed above are obtained according to test protocol 1 (Fig. 7). Hundreds fold of induction can be reached at micro-molar concentration for SaA and its derivatives. Substituted benzoate also functions to activate NahR but with slightly lower induction ratio.

In summary, NahR strain works as a highly-sensitive and robust biosensor for salicylates, benzoate derivatives and water-hazard 2,4,6-TClPhl.

Fig. 1. Degradation pathway of naphthalene in Pseudomonas putida and the gene cluster encoding this function. (a) Gene cluster on the NAH7 plasmid that degrades naphthalene: Naphthalene is transformed into salicylate under the enzymes encoded by the upper operon; salicylate is further degraded to enter TCA cycle via the gene products of the lower operon. Both of the operons are regulated by the transcription factor NahR in response to salicylate, the metabolic intermediate in the pathway. (b) Metabolism of naphthalene encoded by the NAH7 plasmid: Naphthalene is degraded by a series of 13 enzymatic reactions, each catalyzed by a specific nah gene product represented by a capital letter. A through M: A, Naphthalene dioxygenase; B, cis-dihydroxy-naphthalene dioxygenase; D, 2-hydroxychromene-2-carboxylate isomerase; E, 2-hydroxybenzalpyruvate aldolase; F, salicylaldehyde dehydrogenase; G, salicylate hydroxylase; H, catechol 2,3-dioxygenase; I, 2- hydroxymuconate semialdehyde dehydrogenase; J, 2-hydroxymuconate tautomerase; K, 4-oxalcrotonate decarboxylase; L, 2-oxo-4-pentenoate hydratase; M, 2-oxo-4-hydroxypentanoate aldolase.

Fig. 2. Location of the functional domains of NahR transcriptional factor. Domain marked by green near the N terminal accounts for DNA binding, which contains a typical helix-turn-helix motif; red domain functions to bind inducer, while the orange domain is putatively involved in multimerization of NahR in the activation.

Fig. 3. Schematic diagram of the consensus structure of the nahR-regulated promoter nah and sal. Alignment of sal and nah promoter is shown and the consensus forward sequences are marked in color. NahR binding sequence and RNAP binding sequence are shown in green and yellow respectively.

Fig. 4. Schematic diagram of the activation of sal (or nah) promoter via NahR in presence of inducer salicylate: 1. The DNA structure of sal promoter: A,B,C and D represent the binding sites for the putative tetramer of NahR; the yellow arrow shows the direction of sal promoter. 2. RNAP and σ70 bind to the sal promoter by recognizing -35 and -10 region; 3. Transcription factor NahR tightly binds to sal promoter and forms a tetramer no matter whether there is salicylate or not; 4. When salicylate is present, NahR•DNA complex undergoes a conformational change. After the hydrolysis of ATP, DNA is opened and transcription is activated.

Fig. 5. Schematic diagram of the plasmid built for sensor strain NahR. iGEM part BBa_J61051 was ligate with reporter sfGFP in the backbone pSB1C3. Promoters are in orange, RBS in light green, CDS in dark blue and terminators in red.

Fig. 6. Response of sensor strain NahR to various aromatics. (For the full name of the compounds, CLICK HERE(hyperlink is needed here)). (a) The induction ratio column in the On-Off test. NahR could respond to 18 out of 78 aromatics with the induction ratio over 20. (b) The detection range of sensor strain NahR is profiled in green at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its chemical formula.

Fig. 7. Dose response curves of inducers of NahR. (a) Salicylate and its homologs and derivatives; (b) Benzoate, its derivatives and special inducers like 5-ClSaD and 2,4,6-TClPhl. For the full name of the compounds, CLICK HERE(hyperlink is needed here)).

Reference:
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[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.
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