Residue substitutions near the redox center of Bacillus subtilis Spx affect RNA polymerase interaction, redox control, and Spx-DNA contact at a conserved cis-acting element.

Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP.

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP.

Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx(DD) (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxΔC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis.

Spx activates transcription initiation in Bacillus subtilis by directly interacting with the C-terminal domain of the RNA polymerase (RNAP) holoenzyme α subunit, which generates a complex that recognizes the promoter regions of genes within the Spx regulon. Many Gram-positive species possess multiple paralogs of Spx, suggesting that two paralogous forms of Spx could simultaneously contact RNAP. The composition of Spx/RNAP was examined in vitro using an Spx variant (SpxΔCHA) bearing a 12-amino-acid deletion of the C terminus (SpxΔC) and a hemagglutinin (HA) epitope tag and Spxc-Myc, a full-length Spx with a C-terminal myelocytomatosis oncoprotein (c-Myc) epitope tag. All Spx/RNAP complexes bearing deletion or C-terminal-tagged variants were transcriptionally active in vivo and in vitro. Reaction mixtures containing SpxΔCHA and Spxc-Myc combined with RNAP were applied to either anti-HA or anti-c-Myc affinity columns. Eluted fractions contained RNAP with only one of the epitope-tagged Spx derivatives. The resin-bound RNAP complex bearing a single epitope-tagged Spx derivative was transcriptionally active. In vivo production of SpxΔC and SpxΔCHA followed by anti-HA affinity column chromatography of a cleared lysate resulted in retrieval of Spx/RNAP with only the SpxΔCHA derivative. Binding reactions that combined active Spxc-Myc, inactive Spx(R60E)ΔCHA, and RNAP, when applied to the anti-HA affinity column, yielded only inactive Spx(R60E)ΔCHA/RNAP complexes. The results strongly argue for a model in which a single Spx monomer engages RNAP to generate an active transcriptional complex.

Phenotype enhancement screen of a regulatory spx mutant unveils a role for the ytpQ gene in the control of iron homeostasis.

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis.