1887

Abstract

ATP-dependent proteases play essential roles in both protein quality control and the regulation of protein activities in bacteria. ClpYQ (also known as HslVU) is one of several highly conserved ATP-dependent proteases in bacteria. The regulation and biological function of ClpYQ have been well studied in Gram-negative bacteria, but are poorly understood in Gram-positive species. In this study, we showed that in the Gram-positive bacterium Bacillus subtilis, the ΔclpYQ deletion mutant formed early and robust biofilms, while swarming motility was severely impaired. Colonies of the ΔclpYQ mutant were also much less mucoid on agar plates, indicating the loss of the production of secreted γ-poly-dl-glutamic acid (γ-PGA). Global proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ) confirmed that a number of proteins involved in motility, chemotaxis and the production of γ-PGA were less abundant in the ΔclpYQ mutant. The results from both iTRAQ and Western immunoblotting showed that levels of the biofilm master repressor SinR were modestly reduced in the ΔclpYQ mutant, but probably significantly enough to alter biofilm regulation due to the ultrasensitivity of the expression of biofilm genes to SinR protein levels. Western immunoblotting also showed that the abundance of CodY, whose gene is clustered with clpYQ in the same operon, was not impacted on by ΔclpYQ. Lastly, our results suggested that, unlike in Escherichia coli, ClpYQ does not play an essential role in heat-shock response in both B. subtilis and Bacillus cereus. In conclusion, we propose that the ClpYQ protease is primarily involved in multicellular development in B. subtilis.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000658
2018-04-09
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/5/848.html?itemId=/content/journal/micro/10.1099/mic.0.000658&mimeType=html&fmt=ahah

References

  1. Gottesman S, Wickner S, Maurizi MR. Protein quality control: triage by chaperones and proteases. Genes Dev 1997;11:815–823 [CrossRef][PubMed]
    [Google Scholar]
  2. Sauer RT, Baker TA. AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem 2011;80:587–612 [CrossRef][PubMed]
    [Google Scholar]
  3. Rohrwild M, Coux O, Huang HC, Moerschell RP, Yoo SJ et al. HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc Natl Acad Sci USA 1996;93:5808–5813 [CrossRef][PubMed]
    [Google Scholar]
  4. Frees D, Savijoki K, Varmanen P, Ingmer H. Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. Mol Microbiol 2007;63:1285–1295 [CrossRef][PubMed]
    [Google Scholar]
  5. Yepes A, Schneider J, Mielich B, Koch G, García-Betancur JC et al. The biofilm formation defect of a Bacillus subtilis flotillin-defective mutant involves the protease FtsH. Mol Microbiol 2012;86:457–471 [CrossRef][PubMed]
    [Google Scholar]
  6. Turgay K, Hahn J, Burghoorn J, Dubnau D. Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. Embo J 1998;17:6730–6738 [CrossRef][PubMed]
    [Google Scholar]
  7. Ruvolo MV, Mach KE, Burkholder WF. Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis. Mol Microbiol 2006;60:1490–1508 [CrossRef][PubMed]
    [Google Scholar]
  8. Mukherjee S, Bree AC, Liu J, Patrick JE, Chien P et al. Adaptor-mediated Lon proteolysis restricts Bacillus subtilis hyperflagellation. Proc Natl Acad Sci USA 2015;112:250–255 [CrossRef][PubMed]
    [Google Scholar]
  9. Chai Y, Kolter R, Losick R. Reversal of an epigenetic switch governing cell chaining in Bacillus subtilis by protein instability. Mol Microbiol 2010;78:218–229 [CrossRef][PubMed]
    [Google Scholar]
  10. Gerth U, Krüger E, Derré I, Msadek T, Hecker M. Stress induction of the Bacillus subtilis clpP gene encoding a homologue of the proteolytic component of the Clp protease and the involvement of ClpP and ClpX in stress tolerance. Mol Microbiol 1998;28:787–802 [CrossRef][PubMed]
    [Google Scholar]
  11. Deuerling E, Mogk A, Richter C, Purucker M, Schumann W. The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion. Mol Microbiol 1997;23:921–933 [CrossRef][PubMed]
    [Google Scholar]
  12. Kirstein J, Strahl H, Molière N, Hamoen LW, Turgay K. Localization of general and regulatory proteolysis in Bacillus subtilis cells. Mol Microbiol 2008;70:682–694 [CrossRef][PubMed]
    [Google Scholar]
  13. Kain J, He GG, Losick R. Polar localization and compartmentalization of ClpP proteases during growth and sporulation in Bacillus subtilis. J Bacteriol 2008;190:6749–6757 [CrossRef][PubMed]
    [Google Scholar]
  14. Simmons LA, Grossman AD, Walker GC. Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis. J Bacteriol 2008;190:6758–6768 [CrossRef][PubMed]
    [Google Scholar]
  15. Kang MS, Lim BK, Seong IS, Seol JH, Tanahashi N et al. The ATP-dependent CodWX (HslVU) protease in Bacillus subtilis is an N-terminal serine protease. Embo J 2001;20:734–742 [CrossRef][PubMed]
    [Google Scholar]
  16. Frees D, Thomsen LE, Ingmer H. Staphylococcus aureus ClpYQ plays a minor role in stress survival. Arch Microbiol 2005;183:286–291 [CrossRef][PubMed]
    [Google Scholar]
  17. Yan F, Yu Y, Gozzi K, Chen Y, Guo JH et al. Genome-wide investigation of biofilm formation in Bacillus cereus. Appl Environ Microbiol 2017;83:e00561-17 [CrossRef][PubMed]
    [Google Scholar]
  18. Bochtler M, Hartmann C, Song HK, Bourenkov GP, Bartunik HD et al. The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature 2000;403:800–805 [CrossRef][PubMed]
    [Google Scholar]
  19. Hamon MA, Lazazzera BA. The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis. Mol Microbiol 2001;42:1199–1209 [CrossRef][PubMed]
    [Google Scholar]
  20. Branda SS, González-Pastor JE, Ben-Yehuda S, Losick R, Kolter R. Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci USA 2001;98:11621–11626 [CrossRef][PubMed]
    [Google Scholar]
  21. Stanley NR, Britton RA, Grossman AD, Lazazzera BA. Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays. J Bacteriol 2003;185:1951–1957 [CrossRef][PubMed]
    [Google Scholar]
  22. Vlamakis H, Chai Y, Beauregard P, Losick R, Kolter R. Sticking together: building a biofilm the Bacillus subtilis way. Nat Rev Microbiol 2013;11:157–168 [CrossRef][PubMed]
    [Google Scholar]
  23. Aguilar C, Vlamakis H, Losick R, Kolter R. Thinking about Bacillus subtilis as a multicellular organism. Curr Opin Microbiol 2007;10:638–643 [CrossRef][PubMed]
    [Google Scholar]
  24. Shemesh M, Chai Y. A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling. J Bacteriol 2013;195:2747–2754 [CrossRef][PubMed]
    [Google Scholar]
  25. Gozzi K, Ching C, Paruthiyil S, Zhao Y, Godoy-Carter V et al. Bacillus subtilisutilizes the DNA damage response to manage multicellular development. NPJ Biofilms Microbiomes 2017;3:8 [CrossRef][PubMed]
    [Google Scholar]
  26. Chai Y, Norman T, Kolter R, Losick R. An epigenetic switch governing daughter cell separation in Bacillus subtilis. Genes Dev 2010;24:754–765 [CrossRef][PubMed]
    [Google Scholar]
  27. Kearns DB, Losick R. Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev 2005;19:3083–3094 [CrossRef][PubMed]
    [Google Scholar]
  28. Kobayashi K. Gradual activation of the response regulator DegU controls serial expression of genes for flagellum formation and biofilm formation in Bacillus subtilis. Mol Microbiol 2007;66:395–409 [CrossRef][PubMed]
    [Google Scholar]
  29. Diehl A, Roske Y, Ball L, Chowdhury A, Hiller M et al. Structural changes of TasA in biofilm formation ofBacillus subtilis. Proc Natl Acad Sci USA 2018;115:3237–3242 [CrossRef][PubMed]
    [Google Scholar]
  30. Erskine E, Morris R, Schor M, Earl C, Gillespie RMC et al. Formation of functional non-amyloidogenic fibres by recombinant Bacillus subtilis TasA. bioRxiv 2017
    [Google Scholar]
  31. Kearns DB, Chu F, Branda SS, Kolter R, Losick R. A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol 2005;55:739–749 [CrossRef][PubMed]
    [Google Scholar]
  32. Branda SS, Chu F, Kearns DB, Losick R, Kolter R. A major protein component of the Bacillus subtilis biofilm matrix. Mol Microbiol 2006;59:1229–1238 [CrossRef][PubMed]
    [Google Scholar]
  33. Yu Y, Yan F, Chen Y, Jin C, Guo J-H et al. Poly-γ-glutamic acids contribute to biofilm formation and plant root colonization in selected environmental isolates of Bacillus subtilis. Frontiers in Microbiology 1811;2016:7
    [Google Scholar]
  34. Stanley NR, Lazazzera BA. Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-γ-dl-glutamic acid production and biofilm formation. Mol Microbiol 2005;57:1143–1158 [CrossRef]
    [Google Scholar]
  35. Morikawa M, Kagihiro S, Haruki M, Takano K, Branda S et al. Biofilm formation by a Bacillus subtilis strain that produces γ-polyglutamate. Microbiology 2006;152:2801–2807 [CrossRef][PubMed]
    [Google Scholar]
  36. Arnaud M, Chastanet A, Débarbouillé M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol 2004;70:6887–6891 [CrossRef][PubMed]
    [Google Scholar]
  37. Yasbin RE, Young FE. Transduction in Bacillus subtilis by bacteriophage SPP1. J Virol 1974;14:1343–1348[PubMed]
    [Google Scholar]
  38. Kearns DB, Chu F, Rudner R, Losick R. Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol 2004;52:357–369 [CrossRef][PubMed]
    [Google Scholar]
  39. Slack FJ, Serror P, Joyce E, Sonenshein AL. A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol 1995;15:689–702 [CrossRef][PubMed]
    [Google Scholar]
  40. Pohl K, Francois P, Stenz L, Schlink F, Geiger T et al. CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression. J Bacteriol 2009;191:2953–2963 [CrossRef][PubMed]
    [Google Scholar]
  41. Roux A, Todd DA, Velázquez JV, Cech NB, Sonenshein AL. CodY-mediated regulation of the Staphylococcus aureus Agr system integrates nutritional and population density signals. J Bacteriol 2014;196:1184–1196 [CrossRef][PubMed]
    [Google Scholar]
  42. Belitsky BR, Sonenshein AL. Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution. Proc Natl Acad Sci USA 2013;110:7026–7031 [CrossRef][PubMed]
    [Google Scholar]
  43. Mirel DB, Chamberlin MJ. The Bacillus subtilis flagellin gene (hag) is transcribed by the sigma 28 form of RNA polymerase. J Bacteriol 1989;171:3095–3101 [CrossRef][PubMed]
    [Google Scholar]
  44. Ashiuchi M, Misono H. Biochemistry and molecular genetics of poly-γ-glutamate synthesis. Appl Microbiol Biotechnol 2002;59:9–14 [CrossRef][PubMed]
    [Google Scholar]
  45. Stanley NR, Lazazzera BA. Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-γ-dl-glutamic acid production and biofilm formation. Mol Microbiol 2005;57:1143–1158 [CrossRef][PubMed]
    [Google Scholar]
  46. Ashiuchi M, Soda K, Misono H. A poly-γ-glutamate synthetic system of Bacillus subtilis IFO 3336: gene cloning and biochemical analysis of poly-γ-glutamate produced by Escherichia coli clone cells. Biochem Biophys Res Commun 1999;263:6–12 [CrossRef][PubMed]
    [Google Scholar]
  47. Gao T, Greenwich J, Li Y, Wang Q, Chai Y. The bacterial tyrosine kinase activator TkmA contributes to biofilm formation largely independent of the cognate kinase PtkA in Bacillus subtilis. J Bac 2015;197:3421–3432 [CrossRef]
    [Google Scholar]
  48. Ross PL, Huang YN, Marchese JN, Williamson B, Parker K et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 2004;3:1154–1169 [CrossRef][PubMed]
    [Google Scholar]
  49. Bernhardt J, Völker U, Völker A, Antelmann H, Schmid R et al. Specific and general stress proteins in Bacillus subtilis - a two-deimensional protein electrophoresis study. Microbiology 1997;143:999–1017 [CrossRef][PubMed]
    [Google Scholar]
  50. Brinsmade SR, Alexander EL, Livny J, Stettner AI, Segrè D et al. Hierarchical expression of genes controlled by the Bacillus subtilis global regulatory protein CodY. Proc Natl Acad Sci USA 2014;111:8227–8232 [CrossRef][PubMed]
    [Google Scholar]
  51. Chai Y, Norman T, Kolter R, Losick R. Evidence that metabolism and chromosome copy number control mutually exclusive cell fates in Bacillus subtilis. Embo J 2011;30:1402–1413 [CrossRef][PubMed]
    [Google Scholar]
  52. Subramaniam AR, Deloughery A, Bradshaw N, Chen Y, O'Shea E et al. A serine sensor for multicellularity in a bacterium. Elife 2013;2:e01501 [CrossRef][PubMed]
    [Google Scholar]
  53. Ducret A, Quardokus EM, Brun YV. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol 2016;1:16077 [CrossRef]
    [Google Scholar]
  54. Mordini S, Osera C, Marini S, Scavone F, Bellazzi R et al. The role of SwrA, DegU and P(D3) in fla/che expression in B. subtilis. PLoS One 2013;8:e85065 [CrossRef][PubMed]
    [Google Scholar]
  55. Youngman P, Perkins JB, Losick R. Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid 1984;12:1–9 [CrossRef][PubMed]
    [Google Scholar]
  56. Niu DD, Liu HX, Jiang CH, Wang YP, Wang QY et al. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways. Mol Plant Microbe Interact 2011;24:533–542 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000658
Loading
/content/journal/micro/10.1099/mic.0.000658
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
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