XylS

Overview

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XylS is an archetype transcriptional activator of AraC/XylS family, mined from the TOL plasmid pWW0 of the bacterium Pseudomonas putida. It is composed of a C-terminal domain (CTD) involved in DNA binding containing two helix-turn-helix motifs and an N-terminal domain required for effector binding and protein dimerization[5].
XylS detect benzoate and its’ derivatives, mainly methyl and chlorine substitutes at 2-, 3- carbon[4] .

On TOL plasmid, the expression of XylS is initiated from two promoters. The strong promoter Ps1 which depends on factor is positively regulated by XylR at the presence of toluene. Ps2, which depends on factor, is constitutive but weak[7].
The cognate promoter regulated by XylS, Pm, is 70-dependent in E.coli, while in Pseudomonas putida, it is 32/38-dependent[6]. It acts as the master regulator to control the ON/OFF expression of meta-operon on TOL plasmid pWW0[2]. In this meta-operon, XylXYZLTEGFJQKIH genes encode enzymes for the degradation of benzoate and its derivatives, generating intermediate products in TCA cycle. (Fig 1)

XylS recognizes two 15-bp direct repeats (TGCA-N6-GGNTA) on Pm promoter, each consisting of two half-sites: box A1/A2 (TGCA) and box B1/B2 (GGNTA). The arrangement of the two repeats is deposited like this so that the proximal XylS binding site overlaps the RNA polymerase -35 binding box by 2 bp[3]. (Fig 2)

The process of Pm activation includes XylS-dependent DNA bending, XylS dimerization[3] and RNAP recruitment. Binding of effector molecule (e.g., benzoate anion) to XylS N-terminal domain causes the conformational change of C-terminal domain, which is released from inhibition[3], thus enabling XylS protein to bind Pm promoter on upstream activated sequence. The binding of XylS to DNA and accompanied DNA bending is a sequential process: A first XylS monomer binds to Pm at the proximal site with a bending angle to 50°. This DNA bending facilitates the binding of a second monomer at the distal site, increasing the DNA curvature up to 98°. This allows the formation of ready-to-use XylS dimer at Pm promoter. It is widely speculated that the XylS dimer recruits RNAP through contact with  subunit and -CTD (C-terminal domain of  subunit), thus to allow following open complex formation at -10 region and transcription is initiated[1].(Fig 3.)

Various mutants of XylS are recognized, and they are divided into three groups. The first group, consisting of thr45, includes XylS mutant that exhibits broader detective range than XylS wild type as well as mediating high level reporter expression. This mutant recognizes the ordinary non-effectors salycilate and 4-ethylbenzoate. The second group, consisting of mutant leu88, exhibits constitutive expression from Pm promoter in the absence of inducers. The third group of mutants, including gly152, his41, val288, phe155, arg256, recognize new inducers, but differ from group one in that their induction ratio with new inducers are generally low, and the response to wild type inducers in some cases is reduced, even absent . For example, mutant gly152 is activated by new inducers 2-, 3- and 4-methoxybenzoate, but not by 2-fluorobenzoate and only weakly by methylbenzoates that are activators for wild type XylS. Mutant his41 is strongly activated by 5mM 3-methoxybenzoate, but it doesn't recognize the usual inducers 2- and 4-fluoro-3-iodo-, 3,4-dimethyl- and 3,4-dichlorobenzoate[4]. (Table 1)

2013 Peking iGEM team adopted thr45 XylS mutant and obtained the coding sequence via synthesis. We constructed Pm/XylS expression system using sfGFP as reporter gene, XylS is constitutively expressed under the control of Pc promoter, and the expression of sfGFP is positively regulated by XylS at the presence of inducers. In this system, the strength of Pc promoter and RBS in front of sfGFP coding sequence is manipulated. In order to obtain better response performance, we contructed Pc library containing Pc of different strength, thus controlling the amount of XylS protein.
From On/Off test via ELIZA following test protocol 1 we found the Pm/XylS expression system adopting Pc promoter J23114 has the highest induction ratio when exposed to natural inducers (Fig 4.).

XylS strain with no inducer showed low basal expression of sfGFP, and 20compounds showed apparent activation effect with the induction ratios over 20 (Fig 5), they are listed as follows: BzO, 2-MeBzO, 3-MeBzO, 4-MeBzO, 2-FBzO, 4-FBzO, 2-ClBzO, 3-ClBzO, 4-ClBzO, 2-BrBzO, 4-BrBzO, 3-IBzO, 3-MeOBzO, SaA, 3-MeOSaA, 4-ClSaA, 5-ClSaA, 3-MeBAD, 3-ClTOL (Fig 5.)(click here for full name).

After construction of well-tuned expression system, Pm/J23114-XylS expression system was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM, 100µM, 300µM and 1000µM according to protocol 1. The results showed along with the raise of inducer concentration, the induction ratio rises (Fig 6.).

We induced Pm/J23114-XylS expression system with 78 types of aromatic compounds, and found that salicylic acid and its’ derivatives are also inducers of XylS which is not reported in previous work (Fig 7.).

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)).

REFERENCES: [1] Domínguez-Cuevas, P., Ramos, J. L., & Marqués, S. (2010). Sequential XylS-CTD binding to the Pm promoter induces DNA bending prior to activation. Journal of bacteriology, 192(11), 2682-2690. [2] Kaldalu, N., Mandel, T., & Ustav, M. (1996). TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence. Molecular microbiology,20(3), 569-579. [3] Domínguez-Cuevas, P., Marín, P., Busby, S., Ramos, J. L., & Marqués, S. (2008). Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding. Journal of bacteriology, 190(9), 3118-3128. [4] Ramos, J. L., Michan, C., Rojo, F., Dwyer, D., & Timmis, K. (1990). Signal-regulator interactions, genetic analysis of the effector binding site of xyls, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. Journal of molecular biology, 211(2), 373-382. [5] Kaldalu, N., Toots, U., de Lorenzo, V., & Ustav, M. (2000). Functional domains of the TOL plasmid transcription factor XylS. Journal of bacteriology, 182(4), 1118-1126. [6] 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. [7] Ramos, J. L., Marqués, S., & Timmis, K. N. (1997). Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annual Reviews in Microbiology, 51(1), 341-373.