S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics

Abstract

Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indicative of disulfide stress as shown by the induction of the thiol-and oxidative stress-specific Spx, CtsR, and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thioloxidations in response to NaOCl stress that include GapA and MetE. Shotgun-liquid chromatography-tandem MS analyses revealed that GapA, Spx, and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-D-phosphoglycerate dehydrogenase SerA, and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to upregulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. Sbacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the bacillithiol redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…Sites-The ␣, ␤, and ␤Ј bands were excised and subjected to tryptic digestion, as described previously (24). Tryptic peptides were separated and measured online by ESI-mass spectrometry using a nano-ACQUITY UPLC TM system (Waters, Milford, MA) coupled to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA).…”

Mycothiol protects <i>Corynebacterium glutamicum</i> against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and <i>S</i>-mycothiolating MetE

“…As the main low-molecular-weight thiol in C. glutamicum, we speculated that the protective role of MSH against acid stress is correlated to its ability to scavenge reactive oxygen species (ROS). The formation of ROS upon acid stress has previous been experimentally verified in B. subtilis (Chi et al, 2011). In addition, data is accumulating to demonstrate that the exposure of microorganisms to various stresses, such as heavy metals, antibiotics, xenobiotics, heat and salt stress, can also increase the production of ROS and induce secondary oxidative stress (Kohanski et al, 2007;Mols and Abee, 2011a).…”

“…S-glutathionylation, a well-documented protein S-thiolation modification formed between cysteine residues and low-molecularweight thiols such as glutathione, is induced in response to oxidative stress in eukaryotic and most GSH containing Gram-negative bacteria cells, and plays important roles in various biological processes, including cell signaling, metabolism and energy, redox homeostasis and protein degradation (Dalle-Donne et al, 2009;Mieyal and Chock, 2012;Shenton and Grant, 2003). Recently, six Sbacillithiolated proteins and 25 S-mycothiolated proteins, formed between cysteine residues and low-molecularweight thiols BSH (S-bacillithiolation) or MSH (Smycothiolation), respectively, were identified upon oxidative stress in B. subtilis and C. glutamicum (Chi et al, 2011(Chi et al, , 2014. MetE, one of the proteins most susceptible to oxidation, is subjected to all 3 kinds of S-thiolation modification, and plays a key role in linking oxidative stress and methionine availability (Chi et al, 2011(Chi et al, , 2013(Chi et al, , 2014Hondorp and Matthews, 2004).…”

“…Recently, six Sbacillithiolated proteins and 25 S-mycothiolated proteins, formed between cysteine residues and low-molecularweight thiols BSH (S-bacillithiolation) or MSH (Smycothiolation), respectively, were identified upon oxidative stress in B. subtilis and C. glutamicum (Chi et al, 2011(Chi et al, , 2014. MetE, one of the proteins most susceptible to oxidation, is subjected to all 3 kinds of S-thiolation modification, and plays a key role in linking oxidative stress and methionine availability (Chi et al, 2011(Chi et al, , 2013(Chi et al, , 2014Hondorp and Matthews, 2004). Both Sglutathionylation and S-bacillithiolation lead to MetE inactivation and Met auxotrophy in oxidative stressed E. coli and B. subtilis cells (Hondorp and Matthews, 2004;Chi et al, 2011).…”

“…The methionine starvation phenotype has been well studied in oxidative stressed E. coli and B. subtilis cells, resulting from the S-thiolation modification of MetE, one of the methionine synthesis enzymes vulnerable to oxidation (Chi et al, 2011;Hondorp and Matthews, 2004). Recently, S-mycothiolation of MetE was also observed as an important protection mechanism under oxidative stress in C. glutamicum (Chi et al, 2014).…”