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 Sensor Mining for aromatics-sensing transcriptional regulators is "well-studied", it can be expected that some mutants of XylS with novel aromatics-sensing characteristics have been identified in previous studies. As expected, according to the information we collected, there are three groups of XylS mutants. The first group, featured by thr45, includes a XylS mutant that exhibits broader aromatics-sensing profile than wild-type XylS, as well as higher level of transcription activation; this mutant are able to sense the ordinarily non-inducer 4-ethylbenzoate. The second group, featured by leu88 mutation, exhibits constitutive expression from the Pm promoter in the absence of inducers. The third group of mutants, including gly152, his41, val288, phe155, or arg256 mutations, recognizes new inducers, but generally with low induction fold.

See Table 1 for the comprehensive summary of XylS mutants and accompanied novel aromatics-sensing characteristics^[4], which provides a rich repertoire for us to customize the aromatics-sensing characteristics of XylS protein.

Peking iGEM team has adopted thr45 XylS mutant (for the convenient of reading, referred to as “XylS" below) to build a biosensor due to its broader aromatics-sensing profile with high induction ratio. As discussed in Biosensor Introduction, we constructed a Pm/XylS biosensor circuit 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 in the presence of inducers. In this circuit, the strength of Pc promoter and RBS sequence preceding XylS and sfGFP were manipulated, respectively, to fine-tune the performance. Through ON/OFF test following Test Protocol 1, we found the biosensor circuit adopting a weak constitutive promoter, J23114, has the highest induction ratio when exposed to the inducers (Figure 4).

XylS biosensor treated with no inducer showed negligible basal expression of sfGFP. 20 compounds showed apparent activation effects with high induction ratios (higher than 20; Figure. 5). They are 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, and 3-ClTOL (Click Here for the full names of aromatic compounds).

We examined the induction ratios of all aromatics inducers for more details. Induction experiments using different sets of inducer concentrations (10 µM, 30µM, 100µM, 300µM and 1000µM) were performed following Test Protocol 1. The results showed that, along with the increasing of inducer concentration, the induction ratio rises, presenting Hill-function-like curves Figure 6.

After checking the aromatics-sensing profile of XylS biosensor, we found that salicylic acid and its derivatives are also inducers of XylS which has not been reported in previous studies. The dose-response curves are illustrated in Figure 7.

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.

Figure 4.Using a library of constitutive promoters (Pc) to fine-tune the induction ratio of XylS biosensor. Horizontal axis stands for different XylS biosensor circuits with different Pc promoters. These Pc promoters are of different strength to control the expression of XylS. The relative expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 are 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatic compounds, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, shown with different color intensities, were tested following Test Protocol 1. Vertical axis represents the ON-OFF induction ratio. The Pm/XylS biosensor circuit adopting a weak Pc promoter J23114 performed best throughout the four inducers.

Figure 5. (a) The induction ratios of all 78 typical aromatic compounds in the ON/OFF test following Test Protocol 1. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives. (b) The aromatics-sensing profile of XylS biosensor is summarized from (a), highlighted in red in the aromatics spectrum. The structure formula of typical inducers are listed around the central spectrum, near their 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.

Groups	Mutations	Aromatics-sensing Profiles
Broader aromatics-sensing profiles with high induction ratio	Arg45→Thr	SaA; 4-EtBzO; BzO; 2-MeBzO; 4-MeBzO
Constitutive Pm promoter activation	Trp88→Leu	Not mentioned, this mutant causes constitutive expression of Pm promoter
Broader aromatics-sensing profiles with lower induction ratios	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