<i>B. subtilis</i> strains and plasmids.

<p>Unless otherwise noted, <i>trxB-lacZ</i> contains a region between -115 and +47 of the <i>trxB</i> promoter.</p

Selected Crystallographic Data and Statistics.

a<p>R_sym = ∑∑|Ihkl-Ihkl(j)|/∑Ihkl, where Ihkl(j) is the observed intensity and Ihkl is the final average value of intensity.</p>b<p>R_work = ∑||F_obs|−|F_calc||/∑|F_obs| and R_free = ∑||F_obs|−|F_calc||/∑|F_obs|; where all reflections belong to a test set of 5% randomly selected data.</p

Interaction of αCTD and Spx variants with the regulatory region of the <i>trxB</i> promoter.

<p>The <i>trxB</i> probe (−56 to −21) was generated by annealing of oligonucleotides followed by labeling of the 3′-end of the template strand using Klenow fragment and [³²P]dATP. Bands corresponding to the <i>trxB</i>/αCTD and <i>trxB</i>/Spx/αCTD complexes are marked with arrows. (A) EMSA analysis of αCTD and Spx binding to the <i>trxB</i> probe in reactions containing Spx variant or mixtures of mutant Spx proteins or mutant with the wild-type Spx (each at 5 µM). Abbreviations: W, wild-type Spx; G, Spx^G52R; R, Spx^R60E. (B) Redox-sensitive interaction was examined in the presence of DTT.</p

Effect of base substitutions of the <i>trxB</i> promoter on <i>trxB</i> expression.

<p>Single base pair substitutions were generated in the <i>trxB</i> promoter (−115 to +47). The mutated promoters fused to <i>lacZ</i> were introduced in <i>spx</i> mutant strains expressing <i>spx^DD</i> from the IPTG-inducible P<i>spank-hy</i> promoter. Expression of <i>trxB-lacZ</i> was determined in at least two independent isolates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone-0008664-g003" target="_blank">Fig. 3</a>. The effect of each base substitution is shown as a percentage of the peak <i>trxB</i> transcribed from the wild-type promoter, which was used as a control in each experiment. The peak expression was generally seen around 1.5 hr after the addition of IPTG.</p

Structure of reduced C10S Spx in complex with αCTD.

<p>(A) Spx and αCTD are shown as teal and green ribbons, respectively, and their secondary structures are labelled. Helix α4, which is observed in oxidized Spx <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a> but has unraveled in the reduced form, is colored magenta. The residues mutated in this study, R60 and K62, are labelled and shown as sticks with carbon atoms colored white and nitrogen atoms either blue or magenta. Residues S10 and C13 are labelled and shown as sticks with carbon and sulphur atoms colored yellow and the γ-oxygen of S10, red. (B) Close up of the region surrounding helix α4 and residues C10/S10 and C13 after the superposition of the oxidized and reduced αCTD-Spx complex structures. Reduced Spx is shown as a magenta ribbon and oxidized Spx as a teal ribbon. The C10-C13 disulfide bond is shown in orange sticks and S10 and C13 from the reduced structure are shown as yellow sticks. In the reduced form residue R92 has moved 2.8 Å away from its position in the ammonium sulphate-containing oxidized form <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a>. The side chain of residue R60 beyond the Cβ atom is disordered in the sulphate-containing crystal form of oxidized Spx, which was used in the superposition visualized here <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone.0008664-Newberry1" target="_blank">[9]</a>.</p

Effect of <i>trxB</i> base substitutions on interaction with αCTD and Spx.

<p>The wild-type (W) and the <i>trxB</i> (G-44A/G-33A) mutant (M) probes were incubated with different concentrations of αCTD and Spx as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#pone-0008664-g007" target="_blank">Figure 7</a>.</p

Effect of the A-34T substitution on <i>trxB</i> transcription activated by the wild-type and mutant Spx.

<p>Strains carrying <i>trxB-lacZ</i> fusions were grown in DS medium. When the OD₆₀₀ was 0.4 to 0.5, each culture was divided into two flasks, and 1 mM IPTG was added to one flask. Samples were taken at time intervals and β-galactosidase activities were measured. (A) Expression of the wild-type <i>trxB-lacZ</i>. Symbols: squares, ORB7276 with Spx^DD; triangles, ORB7282 with Spx^DD-R60E; diamonds, ORB7316 with Spx^DD-C10A; circles, ORB7337 with Spx^DD-G52R. (B) Expression of <i>trxB</i>(A-34T)-<i>lacZ</i>. Symbols: squares, ORB7342 with Spx^DD; triangles, ORB7343 with Spx^DD-R60E; diamonds, ORB7347 with Spx^DD-C10A; circles, ORB7348 with Spx^DD-G52R. Open symbols represent cells cultured without IPTG and closed symbols represent cells cultured with IPTG.</p

In vitro transcription from the wild-type and <i>trxB</i>(A-34T) promoters in the absence and presence of the wild-type and R60E Spx.

<p>Either the wild-type or A-34T <i>trxB</i> template (1 nM) was incubated with 25 nM RNAP together with 25 nM σ^A in the presence of 7.5 nM Spx. The arrow shows the 66-base <i>trxB</i> transcript.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Production of the wild-type and mutant Spx in <i>B. subtilis</i>.

<p><i>B. subtilis</i> cells expressing the wild-type and the mutant Spx^DD (the C-terminal two amino acids are substituted with aspartate residues, which renders the Spx protein insensitive to ClpXP protease) were grown in DS medium in the absence (lanes 1, 3, 5, and 7) and the presence of IPTG (lanes 2, 4, 6, and 8) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008664#s2" target="_blank">Materials and Methods</a>. The lysate was prepared by the protoplast lysis method as described and 15 µg of total protein was resolved by SDS-polyacrylamide gel electrophoresis. Western blot analysis was carried out to detect Spx as shown previously. Lanes: M, molecular weight marker; 1 and 2, ORB6894 (Spx^DD), ORB6895 (Spx^DD-R60E), ORB6896 (Spx^DD-K62E), and ORB6897 (Spx^DD-K66E).</p