*2.5. ß-Galactosidase-Measurements*

The ß-galactosidase activity of transcriptional fusions was measured by the hydrolysis of O-nitrophenyl- ß-D-galactopyranoside (ONPG) (Serva, Heidelberg, Germany) and expressed as Miller Units, as described in [36].

## *2.6. Di*ff*erential RNA-seq*

RNA isolation, TEX (Terminator EXonuclease) treatment, library construction and sequencing, read mapping, and transcription start site prediction have been described in detail in [37]. The RNA-seq data sets are available at the NCBI Gene Expression Omnibus database under accession number GSE71844.

#### *2.7. RNA Isolation and Quantitative RT-PCR*

Twenty milliliters of *R. sphaeroides* cells were harvested by centrifugation when an OD660 of 0.5 was reached. Total RNA for quantitative RT-PCR was isolated by using the peqGOLDTriFast kit (Peqlab, Erlangen, Germany), as described by the manufacturer. Remaining traces of DNA were removed by the TURBO DNaseI (Invitrogen, Schwerte, Germany). To further confirm the absence of DNA, PCR was performed targeting *gloB* (RSP\_0799) with the primers listed in Table S2. Quantitative RT-PCR was performed in a Bio-Rad CFX96 Real-Time system, as described in our previous study [20]. The reference gene *rpoZ* encoding the ω-subunit of RNA polymerase of *R. sphaeroides* was used to normalize the mRNA expression levels [38] according to the formula given by Pfaffl [39]. Primers are listed in Table S2.

#### *2.8. Analysis of isc-suf Operon Synteny*

The synteny analysis was performed by applying the EDGAR software [40].

Purification of the IscR, Irr, and OxyR proteins and electrophoretic mobility shift assays with dsDNA and protein.

*E. coli* M15 (pREP4/pQE2.4.1oxyR) was used for the overexpression of His-tagged OxyR protein, and the purification of OxyR was performed, as described earlier [41]. Isolation of His-tagged Irr from *E. coli* M15 (pREP4/pQE2.4.1*irr*) is described in [30], and the purification of His-tagged IscR from *E. coli* M15 (pREP4/pQE2.4.1*iscR*) in [31]. Binding of the proteins to the promoter regions of the *isc-suf* operon was determined by an electrophoretic mobility shift assay, as described previously [30,38]. DNA fragments containing the promoter regions of the *isc-suf* operon were produced by PCR. Primers used in the PCR are listed in Table S2. The final volume of the binding reactions was 15–20 μL. The reaction mixtures contained varying amounts of protein, ~3–7 fmol <sup>γ</sup>-32PATP labeled DNA fragment (2000 cpm, Hartmann Analytik, Braunschweig, Germany), salmon sperm DNA (1 μg), and binding buffer, as described elsewhere [30,42]. After the binding reactions were incubated for 30 min at room temperature or 4 ◦C, the samples were loaded onto a 4% (*w*/*v*) polyacrylamide gel in 0.5×TBE buffer (22 mM Tris-HCl, 22 mM boric acid, 0.5 mM EDTA, pH 8.3), and the electrophoresis was performed at 130–180 V for 2–5 h at room temperature or at 4 ◦C. Irr and IscR were isolated and tested under non-reducing conditions. OxyR was purified and tested under reducing and non-reducing conditions with the same outcome.
