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.

Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.McsB | YwlE | ClpC | HSP100/Clp | phosphagen kinase

Summary In eukaryotes, lysine acetylation is a well-established posttranslational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologs of the enzymes that catalyze protein acetylation are widely conserved and distributed amongst bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase (RNAP) subunits: β, β’ and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Since the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression.

The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2-D gel-based approach combining Pro-Q Diamond staining and [(33)P]-labeling. In exponentially growing B. subtilis cells 27 proteins could be identified after staining with Pro-Q Diamond and/or [(33)P]-labeling and one additional protein was labeled solely by [(33)P] resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor sigma(B). We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C-termini were verified by MS (T171, T186, T181, respectively).

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pKa of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14–Cys49′ intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8–9 Å apart from Cys49′. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4′ helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Summary The Bacillus subtilis stressosome is a 1.8 MDa complex that orchestrates activation of the σB transcription factor by environmental stress. The complex comprises members of the RsbR co-antagonist family and the RsbS antagonist, which together form an icosahedral core that sequesters the RsbT serine-threonine kinase. Phosphorylation of this core by RsbT is associated with RsbT release, which activates downstream signaling. RsbRA, the prototype co-antagonist, is phosphorylated on T171 and T205 in vitro. In unstressed cells T171 is already phosphorylated; this is a prerequisite but not the trigger for activation, which correlates with stress-induced phosphorylation of RsbS on S59. By contrast, phosphorylation of RsbRA T205 has not been detected in vivo. Here we find (i) RsbRA is additionally phosphorylated on T205 following strong stresses; (ii) this modification requires RsbT; and (iii) the phosphorylation-deficient T205A substitution greatly increases post-stress activation of σB. We infer that T205 phosphorylation constitutes a second feedback mechanism to limit σB activation, operating in addition to the RsbX feedback phosphatase. Loss of RsbX function increases the fraction of phosphorylated RsbS and doubly phosphorylated RsbRA in unstressed cells. We propose that RsbX both maintains the ready state of the stressosome prior to stress, and restores it post-stress.

The MarR/DUF24-type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox-sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6-Ala mutant that the conserved Cys6 apparently contributes to the DNA-binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox-sensing in response to diamide and 2-methylhydroquinone (MHQ) in vivo. DNA-binding experiments indicate that YodB is regulated by a reversible thiol-modification in response to diamide and MHQ in vitro. Redox-regulation of YodB involves Cys6-Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C-terminal Cys101' or Cys108' residues. This study reveals a thiol-disulfide switch model of redox-regulation for the YodB repressor to sense electrophilic compounds in vivo.

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.