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. Regulatory circuits controlling expression from the TOL plasmid pWW0[7]. Squares: XylS; circles: XylR; open symbols: transcriptional regulator forms unable to stimulate transcription; closed symbols: forms able to stimulate transcription. The regulatory loops are explained in the text.

Fig 2 structure of Pm promoter[3] The orange arrows indicate the two XylS binding sites (proximal and distal), each composed of conserved A1/A2 and B1/B2 boxes. The -10 and -35 hexamers are in blue. A right-angled arrow indicates the transcription initiation site.

Fig 3 Mechanism of XylS binding to Pm promoter Step 1: Free DNA. The 10/ 35 RNAP binding site and the two XylS binding sites (D: distal; and P: proximal) are depicted. The bending angle is 35°, centered in the XylS proximal binding site. Step 2: A first XylS monomer binds Pm at the proximal site, shifts the bent center to DNA sequence between the two sites, and increases the bending angle to 50°. Step 3: This change favors binding of a second monomer at the distal site, further increasing the DNA curvature to an overall value of 98° (here schematized as a right angle). Contacts are established with RNAP through the α-CTD and probably with the σ-subunit, which as shown in Step 4 favors open complex formation and transcription initiation.

Fig 4. Induction ratio of Pc promoter library. X-axis stands for distinct expression system with different Pc promoter. The expression intensity of promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185 respectively. Four kinds of inducers, namely BzO, 2-MeBzO, 3-MeBzO and 4-MeBzO shown with different color squares were tested following test protocol 1 via flow cytometry. Y-axis represents induction ratio reflected via fluorescence intensity. The Pm/XylS expression system which adopted Pc promoter J23114 clearly performed best of all of the four inducers.

Fig 5. The detective range of Pm/J23114-XylS expression system. (a) The induction ratio column in the On-Off test following protocol 1. XylS could respond to 24 out of 78 aromatics with the induction ratio over 20, mainly benzoate, salicylic and their derivatives. (b) The detection range of sensor strain XylS is profiled in red at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its chemical formula.

Fig 6. Dose-response curve of Pm/J23114-XylS expression system in response to benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratio reflected via fluorescence intensity.

Fig 7. Dose-response curve of Pm/J23114-XylS expression system in response to salicylate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratio reflected via fluorescence intensity.

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.

Group	Amino acid substitution	Inducers
Broader detective range with high induction ratio	Arg45 Thr	SaA; 4-EtBzO; BzO; 2-MeBzO; 4-MeBzO
Constitutive expression of Pm promoter	Trp88 Leu	Not mentioned, this mutant causes constitutive expression of Pm promoter
Broader detective range with low induction ratio as well as reduced response to inducers for wild type XylS	Arg152 Gly	BzO; 2-MeBzO; 2-,3-ClBzO; 2-,3- and 4-MeOBzO
	Arg41 His	BzO; 3-MeBzO; 3-ClBzO; 3-BrBzO; 3-MeOBzO
	Asp288 Val	BzO; 3-MeBzO; 4-MeOBzO; 4-BrBzO; 2,4-MeBzO; 2,5-MeBzO; 3-ClBzO
	Leu155 Phe	2-MeBzO; 3-MeBzO; 4-MeBzO; 2,5-MeBzO, 4-ClBzO
	Pro256 Arg	BzO; 2-MeBzO; 3-MeBzO; 3,5-MeBzO; 3,5-ClBzO