Global Characterization of Disulfide Stress inBacillus subtilis

Abstract

We used DNA macroarray and proteome analysis to analyze the regulatory networks in Bacillus subtilis that are affected by disulfide stress. To induce disulfide stress, we used the specific thiol oxidant diamide. After addition of 1 mM diamide to an exponentially growing culture, cell growth stopped until the medium was cleared of diamide. Global analysis of the mRNA expression pattern during growth arrest revealed 350 genes that were induced by disulfide stress by greater than threefold. Strongly induced genes included known oxidative stress genes that are under the control of the global repressor PerR and heat shock genes controlled by the global repressor CtsR. Other genes that were strongly induced encode putative regulators of gene expression and proteins protecting against toxic elements and heavy metals. Many genes were substantially repressed by disulfide stress, among them most of the genes belonging to the negative stringent response. Two-dimensional gels of radioactively labeled protein extracts allowed us to visualize and quantitate the massive changes in the protein expression pattern that occurred in response to disulfide stress. The observed dramatic alteration in the protein pattern reflected the changes found in the transcriptome experiments. The response to disulfide stress seems to be a complex combination of different regulatory networks, indicating that redox-sensing cysteines play a key role in different signaling pathways sensing oxidative stress, heat stress, toxic element stress, and growth inhibition.

Role of a Cysteine Synthase in Staphylococcus aureus

“…Glucose limitation leads to the development of a stable survival state in 0.1 to 1% of S. aureus cells (66), and in these conditions, J96 (cysM) apparently obtains enough cysteine from the medium to enable it to remain viable. A recent study has shown that a number of B. subtilis genes are induced by diamide stress, including cysK (40). Although the S. aureus cysM gene is involved in resistance to diamide and other stresses, expression of cysM was not affected by addition of diamide, methyl viologen, or hydrogen peroxide, indicating differences in the stress responses of B. subtilis and S. aureus.…”

“…The observation that plasmid pJIM80 could complement the sensitivity of J96 (cysM) to diamide, a specific thiol oxidant, suggests that the increased sensitivity of J96 (cysM) is not due to any polar effects on genes downstream of cysM. Tellurite, hydrogen peroxide, acid, and diamide are all substances that can cause imbalance in the thiol redox status of the cytoplasm, or oxidative stress (6,34,40,58). Cysteine residues in the cytoplasm are normally kept in a reduced state, but under oxidative conditions they form disulfide bonds, causing misfolding and inactivation of proteins (2).…”

Differential Gene Expression in Response to Hydrogen Peroxide and the Putative PerR Regulon ofSynechocystissp. Strain PCC 6803

“…One objective for future studies is to determine the response of Synechocystis sp. strain PCC 6803 to disulfide stress and the relationship to peroxide stress, as was accomplished recently for B. subtilis (34).…”

Three Different Systems Participate in l -Cystine Uptake in Bacillus subtilis

“…E. coli cells were grown in Luria-Bertani broth (31). B. subtilis was grown in SP medium or in minimal medium (6 mM K 2 HPO 4 , 4.4 mM KH 2 PO 4 , 0.3 mM trisodium citrate, 4 mM MgCl 2 , 250 M CaCl 2 , 10 M MnCl 2 , 0.5% glucose, 50 mg of L-tryptophan liter Ϫ1 , 11 mg of ferric ammonium citrate liter Ϫ1 , 0.1% L-glutamine) supplemented with one of the following sulfur sources: 1 mM K 2 SO 4 , 1 mM L-methionine, 1 mM DL-homocysteine, 20 M to 1 mM L-cystine, 0.1 to 1 mM DL-cystathionine, 0.1 to 1 mM L-djenkolic acid, or 0.1 to 1 mM S-methylcysteine. In this minimal medium, residual growth in the absence of any added sulfur source was observed.…”