1887

Abstract

Bacillithiol is the major low molecular mass thiol produced by many firmicutes bacteria, including the model organism and pathogens such as and . We have previously shown that four genes (, , and ) are involved in bacillithiol biosynthesis. Here, we report that these four genes are encoded within three, unlinked operons all expressed from canonical σ-dependent promoters as determined by 5′RACE (rapid amplification of cDNA ends). The and genes are embedded within a seven-gene operon additionally including , encoding methylglyoxal synthase, and the essential genes and , encoding tRNA nucleotidyltransferase (CCA transferase) and biotin-protein ligase, respectively. The gene is co-transcribed with unknown function genes, while is expressed both as part of a two-gene operon (with the upstream putative pantothenate biosynthesis gene ) and from its own promoter. All three operons are expressed at a reduced level in an null mutant, consistent with a direct role of Spx as a transcriptional activator for these operons, and all three operons are induced by the thiol oxidant diamide. In contrast with other Spx-regulated genes characterized to date, the effects of Spx on basal expression and diamide-stimulated expression appear to be independent of Cys10 in the redox centre of Spx. Consistent with the role of Spx as an activator of bacillithiol biosynthetic genes, cellular levels of bacillithiol are reduced several-fold in an null mutant.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.070482-0
2013-10-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/10/2025.html?itemId=/content/journal/micro/10.1099/mic.0.070482-0&mimeType=html&fmt=ahah

References

  1. Antelmann H., Helmann J. D. ( 2011). Thiol-based redox switches and gene regulation. Antioxid Redox Signal 14:1049–1063 [View Article][PubMed]
    [Google Scholar]
  2. Bower S., Perkins J., Yocum R. R., Serror P., Sorokin A., Rahaim P., Howitt C. L., Prasad N., Ehrlich S. D., Pero J. ( 1995). Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon. J Bacteriol 177:2572–2575[PubMed]
    [Google Scholar]
  3. Bsat N., Herbig A., Casillas-Martinez L., Setlow P., Helmann J. D. ( 1998). Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29:189–198 [View Article][PubMed]
    [Google Scholar]
  4. Chi B. K., Gronau K., Mäder U., Hessling B., Becher D., Antelmann H. ( 2011). S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics. Mol Cell Proteomics 10:009506[PubMed] [CrossRef]
    [Google Scholar]
  5. Chi B. K., Roberts A. A., Huyen T. T., Bäsell K., Becher D., Albrecht D., Hamilton C. J., Antelmann H. ( 2013). S-bacillithiolation protects conserved and essential proteins against hypochlorite stress in firmicutes bacteria. Antioxid Redox Signal 18:1273–1295 [View Article][PubMed]
    [Google Scholar]
  6. Eiamphungporn W., Helmann J. D. ( 2008). The Bacillus subtilis sigma(M) regulon and its contribution to cell envelope stress responses. Mol Microbiol 67:830–848 [View Article][PubMed]
    [Google Scholar]
  7. Fahey R. C. ( 2013). Glutathione analogs in prokaryotes. Biochim Biophys Acta 1830:3182–3198 [View Article][PubMed]
    [Google Scholar]
  8. Fahey R. C., Brown W. C., Adams W. B., Worsham M. B. ( 1978). Occurrence of glutathione in bacteria. J Bacteriol 133:1126–1129[PubMed]
    [Google Scholar]
  9. Fuangthong M., Helmann J. D. ( 2002). The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative. Proc Natl Acad Sci U S A 99:6690–6695 [View Article][PubMed]
    [Google Scholar]
  10. Gaballa A., Newton G. L., Antelmann H., Parsonage D., Upton H., Rawat M., Claiborne A., Fahey R. C., Helmann J. D. ( 2010). Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc Natl Acad Sci U S A 107:6482–6486 [View Article][PubMed]
    [Google Scholar]
  11. Helmann J. D. ( 1995). Compilation and analysis of Bacillus subtilis sigma A-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res 23:2351–2360 [View Article][PubMed]
    [Google Scholar]
  12. Helmann J. D. ( 2011). Bacillithiol, a new player in bacterial redox homeostasis. Antioxid Redox Signal 15:123–133 [View Article][PubMed]
    [Google Scholar]
  13. Helmann J. D., Wu M. F., Gaballa A., Kobel P. A., Morshedi M. M., Fawcett P., Paddon C. ( 2003). The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors. J Bacteriol 185:243–253 [View Article][PubMed]
    [Google Scholar]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. ( 1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59 [View Article][PubMed]
    [Google Scholar]
  15. Hong M., Fuangthong M., Helmann J. D., Brennan R. G. ( 2005). Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of the MarR family. Mol Cell 20:131–141 [View Article][PubMed]
    [Google Scholar]
  16. Huang C. S., Moore W. R., Meister A. ( 1988). On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation. Proc Natl Acad Sci U S A 85:2464–2468 [View Article][PubMed]
    [Google Scholar]
  17. Jothivasan V. K., Hamilton C. J. ( 2008). Mycothiol: synthesis, biosynthesis and biological functions of the major low molecular weight thiol in actinomycetes. Nat Prod Rep 25:1091–1117 [View Article][PubMed]
    [Google Scholar]
  18. Kobayashi K., Ehrlich S. D., Albertini A., Amati G., Andersen K. K., Arnaud M., Asai K., Ashikaga S., Aymerich S. & other authors ( 2003). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100:4678–4683 [View Article][PubMed]
    [Google Scholar]
  19. Lamers A. P., Keithly M. E., Kim K., Cook P. D., Stec D. F., Hines K. M., Sulikowski G. A., Armstrong R. N. ( 2012). Synthesis of bacillithiol and the catalytic selectivity of FosB-type fosfomycin resistance proteins. Org Lett 14:5207–5209 [View Article][PubMed]
    [Google Scholar]
  20. Lee J. W., Helmann J. D. ( 2006). The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Nature 440:363–367 [View Article][PubMed]
    [Google Scholar]
  21. Lee E. J., Karoonuthaisiri N., Kim H. S., Park J. H., Cha C. J., Kao C. M., Roe J. H. ( 2005). A master regulator sigmaB governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor . Mol Microbiol 57:1252–1264 [View Article][PubMed]
    [Google Scholar]
  22. Lee J. W., Soonsanga S., Helmann J. D. ( 2007). A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR. Proc Natl Acad Sci U S A 104:8743–8748 [View Article][PubMed]
    [Google Scholar]
  23. Masip L., Veeravalli K., Georgiou G. ( 2006). The many faces of glutathione in bacteria. Antioxid Redox Signal 8:753–762 [View Article][PubMed]
    [Google Scholar]
  24. Miller J. H. ( 1972). Experiments in Molecular Genetics Cold Spring Harbor: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Nakano S., Küster-Schöck E., Grossman A. D., Zuber P. ( 2003). Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis . Proc Natl Acad Sci U S A 100:13603–13608 [View Article][PubMed]
    [Google Scholar]
  26. Nakano S., Erwin K. N., Ralle M., Zuber P. ( 2005). Redox-sensitive transcriptional control by a thiol/disulphide switch in the global regulator, Spx. Mol Microbiol 55:498–510 [View Article][PubMed]
    [Google Scholar]
  27. Nakano M. M., Lin A., Zuber C. S., Newberry K. J., Brennan R. G., Zuber P. ( 2010). Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS ONE 5:e8664 [View Article][PubMed]
    [Google Scholar]
  28. Newton G. L., Koledin T., Gorovitz B., Rawat M., Fahey R. C., Av-Gay Y. ( 2003). The glycosyltransferase gene encoding the enzyme catalyzing the first step of mycothiol biosynthesis (mshA). J Bacteriol 185:3476–3479 [View Article][PubMed]
    [Google Scholar]
  29. Newton G. L., Buchmeier N., Fahey R. C. ( 2008). Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria . Microbiol Mol Biol Rev 72:471–494 [View Article][PubMed]
    [Google Scholar]
  30. Newton G. L., Rawat M., La Clair J. J., Jothivasan V. K., Budiarto T., Hamilton C. J., Claiborne A., Helmann J. D., Fahey R. C. ( 2009). Bacillithiol is an antioxidant thiol produced in Bacilli . Nat Chem Biol 5:625–627 [View Article][PubMed]
    [Google Scholar]
  31. Newton G. L., Fahey R. C., Rawat M. ( 2012). Detoxification of toxins by bacillithiol in Staphylococcus aureus . Microbiology 158:1117–1126 [View Article][PubMed]
    [Google Scholar]
  32. Nicely N. I., Parsonage D., Paige C., Newton G. L., Fahey R. C., Leonardi R., Jackowski S., Mallett T. C., Claiborne A. ( 2007). Structure of the type III pantothenate kinase from Bacillus anthracis at 2.0 A resolution: implications for coenzyme A-dependent redox biology. Biochemistry 46:3234–3245 [View Article][PubMed]
    [Google Scholar]
  33. Nicolas P., Mäder U., Dervyn E., Rochat T., Leduc A., Pigeonneau N., Bidnenko E., Marchadier E., Hoebeke M. & other authors ( 2012). Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis . Science 335:1103–1106 [View Article][PubMed]
    [Google Scholar]
  34. Park J. H., Roe J. H. ( 2008). Mycothiol regulates and is regulated by a thiol-specific antisigma factor RsrA and sigma(R) in Streptomyces coelicolor . Mol Microbiol 68:861–870 [View Article][PubMed]
    [Google Scholar]
  35. Parsonage D., Newton G. L., Holder R. C., Wallace B. D., Paige C., Hamilton C. J., Dos Santos P. C., Redinbo M. R., Reid S. D., Claiborne A. ( 2010). Characterization of the N-acetyl-α-d-glucosaminyl l-malate synthase and deacetylase functions for bacillithiol biosynthesis in Bacillus anthracis . Biochemistry 49:8398–8414 [View Article][PubMed]
    [Google Scholar]
  36. Rasmussen S., Nielsen H. B., Jarmer H. ( 2009). The transcriptionally active regions in the genome of Bacillus subtilis . Mol Microbiol 73:1043–1057 [View Article][PubMed]
    [Google Scholar]
  37. Rawat M., Av-Gay Y. ( 2007). Mycothiol-dependent proteins in actinomycetes. FEMS Microbiol Rev 31:278–292 [View Article][PubMed]
    [Google Scholar]
  38. Raynal L. C., Krisch H. M., Carpousis A. J. ( 1998). The Bacillus subtilis nucleotidyltransferase is a tRNA CCA-adding enzyme. J Bacteriol 180:6276–6282[PubMed]
    [Google Scholar]
  39. Reyes D. Y., Zuber P. ( 2008). Activation of transcription initiation by Spx: formation of transcription complex and identification of a Cis-acting element required for transcriptional activation. Mol Microbiol 69:765–779 [View Article][PubMed]
    [Google Scholar]
  40. Roberts A. A., Sharma S. V., Strankman A. W., Duran S. R., Rawat M., Hamilton C. J. ( 2013). Mechanistic studies of FosB: a divalent-metal-dependent bacillithiol-S-transferase that mediates fosfomycin resistance in Staphylococcus aureus . Biochem J 451:69–79 [View Article][PubMed]
    [Google Scholar]
  41. Rochat T., Nicolas P., Delumeau O., Rabatinová A., Korelusová J., Leduc A., Bessières P., Dervyn E., Krásny L., Noirot P. ( 2012). Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis . Nucleic Acids Res 40:9571–9583 [View Article][PubMed]
    [Google Scholar]
  42. Sambrook J., Russell D. W. ( 2001). Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  43. Sareen D., Steffek M., Newton G. L., Fahey R. C. ( 2002). ATP-dependent l-cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases. Biochemistry 41:6885–6890 [View Article][PubMed]
    [Google Scholar]
  44. Sharma S. V., Jothivasan V. K., Newton G. L., Upton H., Wakabayashi J. I., Kane M. G., Roberts A. A., Rawat M., La Clair J. J., Hamilton C. J. ( 2011). Chemical and chemoenzymatic syntheses of bacillithiol: a unique low-molecular-weight thiol amongst low G + C Gram-positive bacteria. Angew Chem Int Ed Engl 50:7101–7104 [View Article][PubMed]
    [Google Scholar]
  45. Soonsanga S., Fuangthong M., Helmann J. D. ( 2007). Mutational analysis of active site residues essential for sensing of organic hydroperoxides by Bacillus subtilis OhrR. J Bacteriol 189:7069–7076 [View Article][PubMed]
    [Google Scholar]
  46. Soonsanga S., Lee J. W., Helmann J. D. ( 2008). Oxidant-dependent switching between reversible and sacrificial oxidation pathways for Bacillus subtilis OhrR. Mol Microbiol 68:978–986 [View Article][PubMed]
    [Google Scholar]
  47. Stülke J., Hanschke R., Hecker M. ( 1993). Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol 139:2041–2045 [View Article][PubMed]
    [Google Scholar]
  48. Upton H., Newton G. L., Gushiken M., Lo K., Holden D., Fahey R. C., Rawat M. ( 2012). Characterization of BshA, bacillithiol glycosyltransferase from Staphylococcus aureus and Bacillus subtilis . FEBS Lett 586:1004–1008 [View Article][PubMed]
    [Google Scholar]
  49. Vagner V., Dervyn E., Ehrlich S. D. ( 1998). A vector for systematic gene inactivation in Bacillus subtilis . Microbiology 144:3097–3104 [View Article][PubMed]
    [Google Scholar]
  50. Van Laer K., Hamilton C. J., Messens J. ( 2013). Low-molecular-weight thiols in thiol-disulfide exchange. Antioxid Redox Signal 18:1642–1653 [View Article][PubMed]
    [Google Scholar]
  51. Wetzstein M., Völker U., Dedio J., Löbau S., Zuber U., Schiesswohl M., Herget C., Hecker M., Schumann W. ( 1992). Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis . J Bacteriol 174:3300–3310[PubMed]
    [Google Scholar]
  52. Zuber P. ( 2004). Spx-RNA polymerase interaction and global transcriptional control during oxidative stress. J Bacteriol 186:1911–1918 [View Article][PubMed]
    [Google Scholar]
  53. Zuber P. ( 2009). Management of oxidative stress in Bacillus . Annu Rev Microbiol 63:575–597 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.070482-0
Loading
/content/journal/micro/10.1099/mic.0.070482-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error