Fig. S1. Cysteine auxotrophy of the PAO1ΔcysB mutant. The wild-type PAO1 and the PAO1ΔcysB mutant strains were grown for 24 h at 37°C in succinate M9 minimal medium plates supplemented (lower panels) or not (upper panels) with L-cysteine (40 μg ml−1).

Fig. S2. Expression and purification of the CysB-His6 (A) and MvaT-His6 (B) in E. coli. Protein samples from key steps of purification were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Lanes have been loaded with the following samples: M, molecular weight marker with size (kDa) on the left; 1, uninduced whole-cell extract; 2, IPTG-induced whole-cell extract; 3, insoluble protein fraction from IPTG-induced cells; 4, soluble protein fraction from IPTG-induced cells; 5, column flow-through; 6 and 7, proteins eluted with 10 mM imidazole (first and last washes, respectively); 8, pooled fractions eluted with 250 mM imidazole.

Fig. S3. Sequence of the 376 bp long pvdS promoter fragment used in our analyses. The potential −35 and −10 binding sites for RNA polymerase and the ribosome binding site (RBS) are underlined, while the Fur box is in bold (Cunliffe et al., 1995; Ochsner and Vasil, 1996). The proposed binding sites for CysB are highlighted in yellow. The A/T content of the upper and lower regions is 80% and 70%, respectively, while the whole DNA fragment has an A/T content of 49%.

Fig. S4. Binding of CysB-His6 to the cysP promoter (PcysP). Electrophoretic mobility shift assay was performed using the purified CysB-His6 protein and 32P-labelled PcysP fragment (0.5 nM) as the probe. The probe was obtained by PCR using the primers PcysP_FW and PcysP_RV (Table S1) and labelled following the same protocol used or the PpvdS probe, as outlined in Experimental procedures. CysB-His6 concentrations (nM) are shown below each lane.

Fig. S5. Expression of selected CysB-dependent genes under the experimental conditions used in this study. Activity of the atsB, atsR and cysP promoters in P. aeruginosa strains PAO1 and PAO1ΔcysB (filled and striped histograms, respectively) harbouring either the PatsB::lacZ (green) or the PatsR::lacZ (red) or the PcysP::lacZ (yellow) transcriptional fusion. Strains were cultured for 20 h in TSBD medium at 37°C with vigorous aeration. Values are expressed in Miller units and represent the mean of two independent assays (SD < 10% of the mean value).

Table S1. Primers used in this study.

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