5-fluoroorotic acid

Transformations were performed with a standard lithium acetate method. To construct Z₃EV and Z₄EV strains, the Gal4dbd of GEV was deleted by PCR-mediated disruption with URA3. Zif268 and Z₄ DNA-binding domains with homology to the ER and ACT1 promoter were then transformed into cells and selected via 5-Fluoroorotic Acid (5-FOA) counter selection of URA3. Single colonies were isolated and sequenced to verify the presence of the proper DNA-binding domain (Supplementary Figure S1A).
The reporter plasmid was created from the base plasmid pRS416 (gift from Megan McClean), a CEN plasmid containing the URA3 selectable marker. The GAL1 promoter region was amplified from genomic DNA. Overlap-extension PCR was used to add the restriction enzyme sites for XbaI and NotI, respectively, on either side of the region of the three continuous Gal4p-binding sites 5′-CGG-N₁₁-CCG-3′ (22 ). This promoter fragment was then cloned into pRS416 in front of green fluorescent protein (GFP) using the restriction enzymes NheI and XmaI. Before zinc-finger-binding sites were added the XbaI site in GFP was re-coded using a silent mutation to remove this restriction site. Finally, the three canonical Gal4p-binding sites were removed via digestion with XbaI and NotI and triplets of dimeric Z₃EV and Z₄EV-binding sites were cloned into respective plasmids (Supplementary Data and Supplementary Figure S1B).
To construct the inducible GCN4 allele, KanMX-Z₄EVpr was amplified from pMN10 with the primers 5′-caatttgtctgctcaagaaaataaattaaatacaaataaaCGCACTTAACTTCGCATCTG-3′ and 5′-tggatttaaagcaaataaacttggctgatattcggacatTATAGTTTTTTCTCCTTGACG-3′ and transformed into Z₄EV-containing parent strain. The uppercase portions of the sequences share homology with the KanMX-Z₄EVpr cassette on the plasmid pMN10.

Strains and plasmids used in this study are listed in Table 1, whereas primers are listed in Table 2. The genes encoding cdtA and cdtB were deleted from C difficile R20291ΔpyrE using allelic-exchange technology [16 (link)]. To achieve this, left and right homology arms corresponding to the regions annealing immediately upstream and downstream of cdtA/B were amplified by polymerase chain reaction (PCR) using cdtAB LAF/RAR and cdtAB RAF/RAR primer sets, respectively. The homology arms were then spliced together by overlap-extension (SOEing) PCR by means of their overlapping 20-base pair (bp) homologous regions before cloning the ensuing product into pMTL-YN4 using flanking SbfI-AscI restriction sites, thus generating the knockout cassette (KOC) pMTL-YN4-cdtAB KOC. The plasmid was then conjugated into C difficile R20291ΔpyrE exactly as previously described, and transconjugants were selected on the basis of thiamphenicol resistance [17 (link)]. Thereafter, single crossover integrants (SCOs) were identified by 2 parallel PCR screens using cdtAB diag F/YN4 primers for left arm recombinants and YN4 F/cdtAB diag R primers for right arm recombinants, respectively (data not shown). To select for double crossover recombinants, SCO integrants were harvested, diluted 1 × 10⁻³, and cultured onto C difficile minimal medium (CDMM) [18 (link)] containing 500 µg/mL 5-fluoroorotic acid and 1 µg/mL uracil, to force plasmid loss through the counter-selection marker pyrE and to select for double crossover mutants before confirming plasmid loss on the basis of thiamphenicol sensitivity. The intended deletions were confirmed by PCR analysis using cdtAB diag F/R primers. The deletion mutant generated an approximately 4-kbp product, while its wild-type (WT) counterpart generated a 4.6-kbp product (Figure 1A). Finally, the pyrE allele was restored to WT using pMTL-YN2 exactly as described previously [17 (link)].
Strains differentially producing CDTa or CDTb were generated by the integration of either cdtA or cdtB at the pyrE locus, under the control of cdtA promoter P_cdtA. First, cdtA coupled with its native promoter was amplified by PCR using P_cdtA F and cdtA R primers, the product of which was cloned into pMTL-YN2C by means of flanking NotI-BamHI restriction sites, thus generating the complementation cassette pMTL-YN2C-P_cdtA-cdtA. In a similar fashion, pMTL-YN2C-P_cdtA-cdtB was generated by amplifying P_cdtA using P_cdtA F/P_cdtA LAR primers, and cdtB was generated using cdtB RAF/cdtB RAR primers, before SOEing the products together and cloning them into pMTL-YN2C by means of flanking NotI-SalI restriction sites. The CDTb-encoding construct could only be generated with a single-nucleotide polymorphism in the promoter region of P_cdtA using an A-G substitution at position-124 relative to the start codon. The resultant plasmids were applied in parallel, to individually integrate the respective CDT constructs at the pyrE locus of R20291ΔpyrEΔcdtAB concomitant with the repair of pyrE, after successful conjugation and selection for uracil prototrophs on CDMM lacking uracil. The PCR analysis using primer pyrE WT F, coupled with either cdtA R or cdtB RAR, demonstrated effective knock-in at the pyrE locus (Figure 1B), thus generating strains R20291ΔcdtAB*P_cdtA-cdtA and R20291ΔcdtAB*P_cdtA-cdtB.