XylS

Mechanism

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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, and an N-terminal domain required for effector binding and protein dimerization^[5]. XylS detect benzoate and its’ derivatives, mainly methyl and chlorine substitution at 2-, 3- carbon^[4].

On TOL plasmid, the expression of XylS is initiated at two promoters: the strong promoter Ps1 that is σ54-dependent is positively regulated by XylR in the presence of toluene; Ps2, a σ70-dependent promoter, constitutive but weaker^[7].

The cognate promoter regulated by XylS, Pm, is σ54-dependent in Pseudomonas putida; meanwhile in E.coli, it is σ70-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, producing intermediate metabolites as carbon sources (Figure 1).

XylS recognizes two 15-bp repeats (TGCA-N6-GGNTA) in Pm promoter, each featured by box A1/A2 (TGCA) and box B1/B2 (GGNTA), respectively. The arrangement of the two repeats is deposited as shown in Figure 2; the proximal XylS binding site overlaps the -35 box by 2 bp (the sequence for the binding of RNA polymerase)^[3].

The process of Pm activation includes XylS-dependent DNA bending, XylS dimerization^[3] and RNA polymerase (RNAP) recruitment. Binding of effector molecule (e.g., benzoate anion) to XylS N-terminal domain causes the conformational change at the C-terminal domain, which enables XylS protein to bind to upstream activated sequence of Pm promoter. This allows the formation of ready-to-use XylS dimer at Pm promoter. See Figure 3 for detailed explanation on the mechanism of transcription initiation by XylS.

Since one of the criteria of our bioinformatic mining for aromatics-sensing transcriptional regulators is "well-studied", it can be expected that various mutants of XylS have been identified in previous studies. As expected, according to the information we collected, there are three groups of XylS mutants. 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. Mu ant 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 2)

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 a constitutive promoter (Pc), 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.(figure 4, 5)

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 (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.

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. The dose-response curve is illustrated on the left.

Figure 1. Regulatory circuits controlled by XylS and XylR on the TOL plasmid pWW0^[7]. Squares, XylS; circles, XylR; open symbol,s.transcriptional regulator without aromatic effector binding; closed symbols, effector-bound transcription factors that is in active form. See the main text for the detailed explanation for the regulatory loops^[3].

Figure 2 Sequence features of Pm promoter^[3] The orange arrows indicate the two XylS binding sites (proximal and distal), each consisting of conserved A1/A2 and B1/B2 boxes. The -10 and -35 hexamers are in blue. A right-angled arrow indicates the transcription start site (+1).

Figure 3 Mechanism of transcription activation by XylS at Pm promoter. Step 1: Free DNA. The -10/-35 consensus boxes of σ70-dependent promoter and the two XylS binding sites (D: distal; and P: proximal) are highlighted. The bending angle is supposed to be 35°, centered at XylS proximal binding site. Step 2: A first XylS monomer binds to Pm at the proximal site, shifts the bent center to the DNA sequence between the two XylS binding sites, and increases the bending angle to 50°. Step 3: This conformational change facilitates the binding of a second XylS monomer to the distal site, further increasing the DNA curvature to an overall value of 98°. Step 4: Contacts with RNAP, also probably with the σ-subunit, are established through the α-CTD of RNA polymerase, which dramatically facilitates the open complex formation and transcription initiation.

Fig 4.Induction ratio of XylS biosensor library that utilizes different Pc promoters to control the expression of XylS. Horizontal axis stands for different XylS biosensor with Pc promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatics, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, shown with different color intensities, were tested following Test Protocol 1 (a hyper link is needed here). Vertical axis represents the ON-OFF induction ratio. The Pm/XylS expression system which adopted Pc promoter J23114 clearly performed best of all of the four inducers.

Fig 5. (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.

Table 1 Comprehensive summary of XylS mutants and accompanied aromatics-sensing characteristics.