Supplementary Material

Supplementary Table 1. Bacterial strains and plasmids

Strains and plasmids / Genotype or phenotype / Reference
Escherichia coli strains
DH5α / F−Φ80dlacZΔM15Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK− mK+) supE44λ−thi-1 gyrA relA1 / Hanahan (2)
S17-1 / 294 (recA pro res mod+) Tpr Smr (pRP4-2-Tc::Mu-Km::Tn7) / Simon et al. (7)
BL21(DE3) / F−ompT hsdSB (rB− mB−) gal dcm met (DE3) / Novagen
K-12 / Wild type / DSM 498
Allochromatium vinosum strains
Rif50 / Rifr, spontaneous rifampicin-resistant mutant of A. vinosum DSM 180T / Lübbe et al. (5)
ΔdsrR / Rifr, ΔdsrR (264 bp deletion of dsrR) / This work
ΔdsrR+dsrR / Rifr, Kmr, complementation of ΔdsrR with plasmid pBBRdsrPT-dsrR / This work
Rif50+pTS / Rifr, Kmr, plasmid pTS integrated into Rif50 genome / This work
ΔdsrR+pTS / Rifr, Kmr, plasmid pTS integrated into ΔdsrR genome / This work
Rif50+pTL / Rifr, Kmr, plasmid pTL integrated into Rif50 genome / This work
ΔdsrR+pTL / Rifr, Kmr, plasmid pTL integrated into ΔdsrR genome / This work
Plasmids
pET-15b / Apr, His-Tag (N-terminal) / Novagen
pREX / Apr, NdeI–XhoI fragment of PCR-amplified dsrR in NdeI–XhoI of pET-15b / This work
pTISCA / Kmr, NdeI-BlpI fragment of PCR-amplified iscA in NdeI-BlpI of pET-28b / Ding Clark (1)
pK18mobsacB / Kmr, lacZ', sacB, Mob+ / Schäfer et al. (6)
pK18mobsacBΔdsrR / Kmr, XbaI fragment of PCR-amplified genome region around dsrR with 264 bp deletion of dsrR / This work
pBBR1MCS-2 / Kmr, lacZ', Mob+ / Kovach et al. (4)
pBBRdsrPT1 / Kmr, XbaI–HindIII fragment of PCR-amplified and fused dsrA promoter and dsr terminator in XbaI–HindIII of pBBR1MCS-2 / This work
pBBRdsrPT-dsrR / Kmr, NheI-XmaJI fragment of PCR-amplified dsrR in NheI-XmaJI of pBBRdsrPT1 / This work
pKdsrProm / Kmr, EcoRI-PstI fragment of PCR-amplified dsrA promoter without rbs of dsrA in EcoRI-PstI of pK18mobsacB / This work
pTS / Kmr, PstI-HindIII fragment of PCR-amplified lacZ including rbs in PstI-HindIII of pKdsrProm / This work
pPHU235 / Tcr, broad-host-range lacZ fusion vector / Hübner et al. (3)
pK235 / Kmr, SalI-EcoRI fragment of promoter less lacZ of pPHU235 ligated in HindIII-EcoRI pK18mobsacB / B. Franz, personal communication
pTL / Kmr, PstI-HindIII fragment of PCR-amplified dsrA promoter region including the first 12 bp of dsrA in PstI-HindIII of pK235 / This work


Supplementary Table 2. PCR primers. Restriction sites are marked bold, the T7 promoter is underlined.

Primers / Sequence 5’ à 3’ / Reference
dsrR for cloning in pET-15b
DsrRNdef1 / AATGGAGTCATATGATGTTCAAGCTGACA / This work
DsrRXhor1 / CATGATCCGCTCGAGCGATCAGCCGC / This work
Deletion of dsrR
RXbaf1 / GCGGATCTTCTAGAGATCGAGGGCAATGTC / This work
rrev1 / ATAGGTCGGATCCCTTGTCAGCTTGAACATC / This work
Rfor1 / GATGTTCAAGCTGACAAGGGATCCGACCTAT / This work
rXbar1 / CGATCTGGTCTAGAATCGCTGTCAGCACG / This work
Complementation
PromDsrHindf1 / TCACTGCCAAGCTTGAAGACGGAATCGGCG / This work
PromDsrNher1 / ATCGACGCCCTAGGCTATGCGCTAGCTCTCCTATC / This work
TermDsrXmaJf2 / GATAGGAGAGCTAGCGCATAGCCTAGGGCGTCGAT / This work
TermDsrXbar1 / AGATCTGTCTAGAATCGTGCAACGCTCAGC / This work
DsrRNhef1 / TCAATAGGGCTAGCATGATGTTCAAGCTGAC / This work
DsrRXmaJr1 / ATGATCCGCCTAGGCGATCAGCCGCCG / This work
Transcriptional and translational gene fusion
GfdsrPromf2 / CTGCGAATTCTGAAGACGGAATC / This work
GfTKdsrProm2 / CTCCTCTGCAGATAACCAGGAACG / This work
lacZrbsf1 / ACAATCTGCAGCAGGAAACAGCTATG / This work
lacZrbsr1 / GCGAAAGCTTGCAGACATGGCC / This work
GfdsrPromf1 / CTGCCTGCAGTGAAGACGG / This work
TLGfdsrPromr1 / GTCTCAAGCTTGTCGATAGCC / This work
RT-PCR
RNAdsrAf1 / CGTTCAAGAAGCTGGAGACC / This work
RNAdsrAr1 / ATCCATGCGGATGTAGGAAC / This work
RNAdsrEf1 / CCGGGTCTTCTTCTATCACG / This work
RNAdsrEr1 / GAATTTGGGATGGATGTTGG / This work
RNAdsrCf1 / GGAGGGCTATCTCTCCAACC / This work
RNAdsrCr1 / CTTCTCCTTGCCGAGCTTCT / This work
RNAdsrLf1 / GAACACGTCTGGGACAACCT / This work
RNAdsrLr1 / ATACTCCTGCCAGTCCATGC / This work
RNAdsrRf1 / CCGATGGAAGCATCGATTAC / This work
RNAdsrRr1 / CCTGGGATTGAGGAAGATGA / This work
RNAdsrSf2 / GGTATCCGGTCTATCTCAGC / This work
RNAdsrSr2 / TTGCCGGCGTCCTCCATC / This work
StandarddsrAf1 / TAATACGACTCACTATAGGGCCGCCAGTACTACATCGACA / This work
StandarddsrAr1 / CAGTGTGAAGACGTCGAGGA / This work
StandarddsrEf1 / TAATACGACTCACTATAGGGCATGATCGAACGACAATCCA / This work
StandarddsrEr1 / GAGCTGGTAGACACCGTCGT / This work
StandarddsrCf1 / TAATACGACTCACTATAGGGGAATCCTGCCTGAAGTTTGC / This work
StandarddsrCr1 / CGGCGAAATAGAACAGGAAG / This work
StandarddsrLf1 / TAATACGACTCACTATAGGGCGATCCGAACGAAGAAGAAG / This work
StandarddsrLr1 / GTCTTGAGCCTGACCTCGAC / This work
StandarddsrRf1 / TAATACGACTCACTATAGGGAGTTTCATCATTCGGCCATC / This work
StandarddsrRr1 / GGTATTGGGATTGAGCTGGA / This work
StandarddsrSf1 / TAATACGACTCACTATAGGGTCCAGCTCAATCCCAATACC / This work
StandarddsrSr1 / GGATCAGAGGACGGGAAGTC / This work


Supplementary Table 3. Structural statistics for the ensemble of 20 A. vinosum DsrR structures

Distance restraints
Total / 953
Intraresidue / 181
Sequential / 181
Medium range (1 < |i-j| < 5) / 177
Long range / 414
Hydrogen bonds (2 restraints per H-bond) / 2*21
Dihedral angle restraints
Total / 74
Φ / 36
Ψ / 38
Χ1 / 0
Total restraints / 1069
Restraints per restrained residue (99 residues) / 10.8
Long range NOE restraints restrained residue (99 residues) / 4.2
Average restraint violations per structure
Distance restraints (all > 0.0 Å) / 32.1±4.1
Maximum violation (Å) / 0.15
Dihedral restraints (all > 0.00) / 2.7±1.3
Maximum violation (°) / 0.55°
Average pair wise RMSD to average structures (Å):
Residues 1-92 (92 residues)
Backbone atoms (N,Cα,C’) / 0.94
All heavy atoms / 1.33
Residues 1-16,24-32,38-47,51-56,59-80,86-92 (70 PSVS ordered residues)
Backbone atoms (N,C,C’) / 0.67
All heavy atoms / 1.10
Ramachandran (PROCHECK)
Residues 1-92 inclusive (79 non-glycine and non-proline residues)
Most favored regions (%) / 89.3
Additional allowed regions (%) / 10.0
Generously allowed regions (%) / 0.3
Disallowed regions (%) / 0.4
Ordered residues (PSVS),residues 1-16,24-32,38-47,51-56,59-80,86-92, inclusive (65 non-glycine and non-proline residues)
Most favored regions (%) / 90.2
Additional allowed regions (%) / 9.7
Generously allowed regions (%) / 0.1
Disallowed regions (%) / 0.0


Reference List

1. Ding, H. and R. J. Clark. 2004. Characterization of iron binding in IscA, an ancient iron-sulphur cluster assembly protein. Biochem. J. 379:433-440.

2. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.

3. Hübner, P., J. C. Willison, P. Vignais, and T. A. Bickle. 1991. Expression of regulatory nif genes in Rhodobacter cpasulatus. J. Bacteriol. 173:2993-2999.

4. Kovach, M. E., P. H. Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. II. Roop, and K. M. Peterson. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175-176.

5. Lübbe, Y. J., H.-S. Youn, R. Timkovich, and C. Dahl. 2006. Siro(haem)amide in Allochromatium vinosum and relevance of DsrL and DsrN, a homolog of cobyrinic acid a,c diamide synthase for sulphur oxidation. FEMS Microbiol. Lett. 261:194-202.

6. Schäfer, A., A. Tauch, W. Jäger, J. Kalinowski, G. Thierbach, and A. Pühler. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69-73.

7. Simon, R., U. Priefer, and A. Pühler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784-791.

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