Control of ytmI and ytlI by Spx and CymR. A. Expression of ytlI′-lacZ in the wild type (ORB5691) and spx (ORB4871) and cymR (ORB5733) mutant strains. Cells of each fusion strain were grown in TSS medium containing MgSO₄ (SO₄) or cysteine (Cys) as sole sulfur source. Samples were collected at log phase and assayed for β-galactosidase activity, which was expressed in Miller units. B. Expression of ytmI′-lacZ in wild-type (ORB4794), cymR (ORB5730), and cymR ytlI (ORB5732) strains. Mutation of ytlI (ORB5732) eliminates the derepression of ytmI in the cymR mutant. C. Complementation of cymR with a thrC::cymR construct. ytlI-directed β-galactosidase activity of the cymR mutant strain bearing a ytlI-lacZ fusion (strain ORB5733) and the fusion-bearing partial diploid strain, ORB5809, with a thrC::cymR⁺ construct are shown.

Control of cymR expression by Spx. Expression of a translational cymR-lacZ fusion over time in ORB6080 (cymR-lacZ; diamonds) and ORB6081 (spx::aphA3 cymR-lacZ; squares) grown in TSS medium containing either MgSO₄ (SO₄) (A) or cysteine (Cys) (B) is shown. The x axis represents the time (in hours) of cell growth.

Spx and CymR control of the yrrT operon. A. Organization of the yrrT operon (see Fig. 1 for details of gene functions.). B. Expression of yrrT′-lacZ in wild-type (WT) and cymR mutant cells. Cells were grown to mid-log phase in TSS medium containing either MgSO₄ (SO₄), cysteine (Cys), or methionine (Met) as the sole sulfur source. Samples were collected for assay of β-galactosidase activity. C. Expression of yrrT′-lacZ in wild-type and spx mutant cells. Cultures were grown and samples were processed as for panel B.

Diagram summarizing the proposed role of Spx in the control of organosulfur metabolism. Spx is required for the optimal production of the CymR repressor, which exerts negative control of the organosulfur utilization operons ytmI, yxeI, and ssu, and the methionine utilization pathway that generates cysteine via reactions catalyzed by the products of the yrrT operon. Spx exerts direct positive control of yrrT transcription under conditions that result in elevated Spx protein concentration (oxidative stress) and when cells must produce cysteine from alternative forms of sulfur, such as methionine.

Diagram of the organosulfur metabolic pathways of B. subtilis (18, 40). Cysteine can be produced from sulfate or from methionine/SAM. Production of sulfite is from sulfate and organosulfonates, and the genes believed to function in the process are shown. The SAH shunt-transsulfuration pathways (indicated) function in converting methionine to cysteine via SAM and SAH. The genes whose products function at each step are indicated, as are the effects of Spx activity; an arrowhead pointing up denotes Spx-dependent stimulation of expression, and an arrowhead pointing down indicates Spx-dependent repression (34). α-kb, α-ketobutyrate; O-ahs, O-acetylhomoserine; pyr, pyruvate; AI-2, autoinducer-2 (48).

A. The diagram top right shows the organization of the cymR operon containing the yrvO and trmU genes, based on primer extension analysis of cymR RNA in wild-type and spx mutant cells. Cultures were grown in TSS minimal medium containing either MgSO₄ (S), cysteine (C), or methionine (M) to an OD₆₀₀ of 0.7, and total RNA was isolated. Primer extension reactions and the sequencing reaction were performed using oligonucleotide oSY09. Primer extension products were resolved by polyacrylamide gel electrophoresis and visualized by phosphorimaging. Four lanes on the left are the gel profiles of sequencing reactions A, G, C, and T. Primer extension was also done with an rpsD-specific primer for a control of constitutive expression. B. Diagram of the nucleotide sequence of the cymR promoter region, showing the −10 sequence (TG promoter) and −35 along with the regions of dyad symmetry upstream of the promoter (dotted arrows). Coding sequences of the cymR and divergently oriented yrvN genes are shown with associated ribosome-binding sites (rbs) and a putative transcriptional start site for cymR (bent arrow).

Spx controls cymR indirectly. A. Western analysis of CymR protein levels in wild-type (WT) and spx mutant cells. Strains ORB5691 (ytlI-lacZ) and ORB4871 (ytl-lacZ spx::aphA3) were grown in TSS medium containing MgSO₄ or cysteine as the sulfur source. Cleared lysates from mid-log-phase cells were obtained after lysis by French press and centrifugation. Lysate from ORB5724 (cymR::aphA) was used as a negative control. B. In vitro runoff transcription reactions of the cymR promoter fragment (5 nM) in the reaction mixtures containing purified RNAP in the presence and absence of Spx. Concentrations of Spx in the reaction mixtures are indicated. A positive control is the trxB promoter fragment (10 nM), which shows activation by Spx. The rpsD template DNA (20 nM) containing the ribosomal protein S4 gene promoter shows no effect by Spx. C. The CymR repressor, supplied with the Pspac promoter in the spx mutant, restores the repression of ytlI. Cells of WT and spx mutant strains (indicated) bearing the ytlI-lacZ fusion were transformed with plasmid pSY20, in which the cymR gene was placed under the control of P_spank-hy. Cultures were grown to mid-log phase in sulfate medium in presence or absence of IPTG. Samples were collected and assayed for β-galactosidase activity.

Transcription from the yrrT promoter is activated by Spx in vitro. A. Organization of the yrrT operon. Dotted arrows indicate regions of dyad symmetry. The −10 TG promoter is indicated, as is the −35 sequence. The dashed arrow indicates the complementary sequence of the downstream primer used to construct the transcription promoter fragment by PCR. The bent arrow indicates the approximate location of the transcription start site as deduced from the size of the in vitro transcript. B. Gel profiles of transcription reactions containing promoter fragments trxB or yrrT (10 nM) and RNAP (50 nM) in the presence and absence of Spx and DTT. Concentrations of Spx and DTT are indicated. C. Repression of yrrT transcription by CymR. In vitro runoff transcription reaction mixtures contained RNAP (25 nM) and either trxB promoter (20 nM) or yrrT promoter (10 nM) DNA. Spx and CymR were added to the reaction mixtures at the indicated concentrations. The lower panel shows a plot of band intensities of the in vitro runoff transcription of yrrT shown in the upper panel. Band intensities were quantified using ImageQuant version 5.2, and error bars were derived from two repeats of the experiment. D. DNase I footprinting of the yrrT promoter with CymR. The yrrT promoter prepared by PCR using oyrrT-1 and oyrrT-2 is shown. Reaction mixtures contained CymR and promoter DNA with a radiolabeled coding strand. The concentrations of CymR used were 0.5, 1, 2, and 4 μM. The positions relative to the transcription start site are shown on the left. The −10 and +1 are shown next to the dideoxy sequencing ladders. The protected region is indicated by the solid line on the right side.