The Spore Differentiation Pathway in the Enteric Pathogen
Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σF in the forespore, and σE in the mother cell control early stages of development and are replaced, at later stages, by σG and σK, respectively. Starting with σF, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σE is partially independent of σF, that σG activity is not dependent on σE, and that the activity of σK does not require σG. We also show that σK is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σF-to-σE, σE-to-σG, and σG-to-σK cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.
Vyšlo v časopise:
The Spore Differentiation Pathway in the Enteric Pathogen. PLoS Genet 9(10): e32767. doi:10.1371/journal.pgen.1003782
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1003782
Souhrn
Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σF in the forespore, and σE in the mother cell control early stages of development and are replaced, at later stages, by σG and σK, respectively. Starting with σF, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σE is partially independent of σF, that σG activity is not dependent on σE, and that the activity of σK does not require σG. We also show that σK is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σF-to-σE, σE-to-σG, and σG-to-σK cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.
Zdroje
1. NicholsonWL, MunakataN, HorneckG, MeloshHJ, SetlowP (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64: 548–572.
2. Nicholson WL (2004) Ubiquity, longevity, and ecological roles of Bacillus spores. In: Henriques AO, Ricca E, Cutting S, editors. Bacterial Spore Formers: Probiotics and Emerging Application. London: Horizon Scientific Press. pp. 1–15.
3. AngertER, LosickRM (1998) Propagation by sporulation in the guinea pig symbiont Metabacterium polyspora. Proc Natl Acad Sci U S A 95: 10218–10223.
4. FlintJF, DrzymalskiD, MontgomeryWL, SouthamG, AngertER (2005) Nocturnal production of endospores in natural populations of epulopiscium-like surgeonfish symbionts. J Bacteriol 187: 7460–7470.
5. TamNK, UyenNQ, HongHA, Duc leH, HoaTT, et al. (2006) The intestinal life cycle of Bacillus subtilis and close relatives. J Bacteriol 188: 2692–2700.
6. MockM, FouetA (2001) Anthrax. Annu Rev Microbiol 55: 647–671.
7. OlivaC, TurnboughCLJr, KearneyJF (2009) CD14-Mac-1 interactions in Bacillus anthracis spore internalization by macrophages. Proc Natl Acad Sci U S A 106: 13957–13962.
8. RupnikM, WilcoxMH, GerdingDN (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7: 526–536.
9. DeneveC, JanoirC, PoilaneI, FantinatoC, CollignonA (2009) New trends in Clostridium difficile virulence and pathogenesis. Int J Antimicrob Agents 33 Suppl 1: S24–28.
10. CarterGP, RoodJI, LyrasD (2012) The role of toxin A and toxin B in the virulence of Clostridium difficile. Trends Microbiol 20: 21–29.
11. BurnsDA, HeapJT, MintonNP (2010) The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type. Anaerobe 16: 618–622.
12. SarkerMR, Paredes-SabjaD (2012) Molecular basis of early stages of Clostridium difficile infection: germination and colonization. Future Microbiol 7: 933–943.
13. DeakinLJ, ClareS, FaganRP, DawsonLF, PickardDJ, et al. (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80: 2704–2711.
14. CartmanST, HeapJT, KuehneSA, CockayneA, MintonNP (2010) The emergence of ‘hypervirulence’ in Clostridium difficile. Int J Med Microbiol 300: 387–395.
15. HenriquesAO, MoranCPJr (2007) Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61: 555–588.
16. McKenneyPT, DriksA, EichenbergerP (2012) The Bacillus subtilis endospore: assembly and functions of the multilayered coat. Nat Rev Microbiol 11: 33–44.
17. de HoonMJ, EichenbergerP, VitkupD (2010) Hierarchical evolution of the bacterial sporulation network. Curr Biol 20: R735–745.
18. Panessa-WarrenBJ, TortoraGT, WarrenJB (2007) High resolution FESEM and TEM reveal bacterial spore attachment. Microsc Microanal 13: 251–266.
19. Paredes-SabjaD, SarkerMR (2012) Adherence of Clostridium difficile spores to Caco-2 cells in culture. J Med Microbiol 61: 1208–1218.
20. Paredes-SabjaD, Cofre-AranedaG, Brito-SilvaC, Pizarro-GuajardoM, SarkerMR (2012) Clostridium difficile spore-macrophage interactions: spore survival. PLoS One 7: e43635.
21. HilbertDW, PiggotPJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234–262.
22. HigginsD, DworkinJ (2012) Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev 36: 131–148.
23. PiggotPJ, HilbertDW (2004) Sporulation of Bacillus subtilis. Curr Opin Microbiol 7: 579–586.
24. GalperinMY, MekhedovSL, PuigboP, SmirnovS, WolfYI, et al. (2012) Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes. Environ Microbiol 14: 2870–2890.
25. TraagBA, PuglieseA, EisenJA, LosickR (2012) Gene conservation among endospore-forming bacteria reveals additional sporulation genes in Bacillus subtilis. J Bacteriol 195(2): 253–60.
26. MillerDA, SuenG, ClementsKD, AngertER (2012) The genomic basis for the evolution of a novel form of cellular reproduction in the bacterium Epulopiscium. BMC Genomics 13: 265.
27. AbecasisA, SerranoM, AlvesL, QuintaisL, Pereira-LealJB, et al. (2013) A genomic signature and the identification of new endosporulaion genes. J Bacteriol 195(9): 2101–15.
28. ParedesCJ, AlsakerKV, PapoutsakisET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3: 969–978.
29. HarryKH, ZhouR, KroosL, MelvilleSB (2009) Sporulation and enterotoxin (CPE) synthesis are controlled by the sporulation-specific sigma factors SigE and SigK in Clostridium perfringens. J Bacteriol 191: 2728–2742.
30. LiJ, McClaneBA (2010) Evaluating the involvement of alternative sigma factors SigF and SigG in Clostridium perfringens sporulation and enterotoxin synthesis. Infect Immun 78: 4286–4293.
31. TracyBP, JonesSW, PapoutsakisET (2011) Inactivation of sigmaE and sigmaG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis. J Bacteriol 193: 1414–1426.
32. JonesSW, ParedesCJ, TracyB, ChengN, SillersR, et al. (2008) The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol 9: R114.
33. JonesSW, TracyBP, GaidaSM, PapoutsakisET (2011) Inactivation of sigmaF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes sigmaE and sigmaG protein expression but does not block solvent formation. J Bacteriol 193: 2429–2440.
34. KirkDG, DahlstenE, ZhangZ, KorkealaH, LindstromM (2012) Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 78: 4590–4596.
35. BiC, JonesSW, HessDR, TracyBP, PapoutsakisET (2011) SpoIIE is necessary for asymmetric division, sporulation, and expression of sigmaF, sigmaE, and sigmaG but does not control solvent production in Clostridium acetobutylicum ATCC 824. J Bacteriol 193: 5130–5137.
36. Stragier P (2002) A gene odyssey: exploring the genomes of endospore-forming bacteria. In: AL S, editor. Bacillus subtilis and its closest relatives: from genes to cells. Washington ASM. pp. 519–525.
37. PiggotPJ, CooteJG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40: 908–962.
38. BurnsDA, HeegD, CartmanST, MintonNP (2011) Reconsidering the sporulation characteristics of hypervirulent Clostridium difficile BI/NAP1/027. PLoS One 6: e24894.
39. HaraldsenJD, SonensheinAL (2003) Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene. Mol Microbiol 48: 811–821.
40. BurnsDA, MintonNP (2011) Sporulation studies in Clostridium difficile. J Microbiol Methods 87: 133–138.
41. WilsonKH, KennedyMJ, FeketyFR (1982) Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J Clin Microbiol 15: 443–446.
42. PutnamEE, NockAM, LawleyTD, ShenA (2013) SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins. J Bacteriol 195(6): 1214–25.
43. PoglianoJ, OsborneN, SharpMD, Abanes-De MelloA, PerezA, et al. (1999) A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation. Mol Microbiol 31: 1149–1159.
44. HeapJT, PenningtonOJ, CartmanST, CarterGP, MintonNP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70: 452–464.
45. HeapJT, PenningtonOJ, CartmanST, MintonNP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78: 79–85.
46. EldarA, CharyVK, XenopoulosP, FontesME, LosonOC, et al. (2009) Partial penetrance facilitates developmental evolution in bacteria. Nature 460: 510–514.
47. EichenbergerP, FawcettP, LosickR (2001) A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis. Mol Microbiol 42: 1147–1162.
48. Karmazyn-CampelliC, BonamyC, SavelliB, StragierP (1989) Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis. Genes Dev 3: 150–157.
49. LuS, HalbergR, KroosL (1990) Processing of the mother-cell sigma factor, sigma K, may depend on events occurring in the forespore during Bacillus subtilis development. Proc Natl Acad Sci U S A 87: 9722–9726.
50. CuttingS, OkeV, DriksA, LosickR, LuS, et al. (1990) A forespore checkpoint for mother cell gene expression during development in B. subtilis. Cell 62: 239–250.
51. WangKH, IsidroAL, DominguesL, EskandarianHA, McKenneyPT, et al. (2009) The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis. Mol Microbiol 74: 634–649.
52. ReidBG, FlynnGC (1997) Chromophore formation in green fluorescent protein. Biochemistry 36: 6786–6791.
53. DonovanC, BramkampM (2009) Characterization and subcellular localization of a bacterial flotillin homologue. Microbiology 155: 1786–1799.
54. NicolleO, RouillonA, GuyodoH, Tamanai-ShacooriZ, ChandadF, et al. (2010) Development of SNAP-tag-mediated live cell labeling as an alternative to GFP in Porphyromonas gingivalis. FEMS Immunol Med Microbiol 59: 357–363.
55. PermpoonpattanaP, TollsEH, NademR, TanS, BrissonA, et al. (2011) Surface layers of Clostridium difficile endospores. J Bacteriol 193: 6461–6470.
56. PermpoonpattanaP, PhetcharaburaninJ, MikelsoneA, DembekM, TanS, et al. (2013) Functional characterization of Clostridium difficile spore coat proteins. J Bacteriol 195: 1492–1503.
57. VasudevanP, WeaverA, ReichertED, LinnstaedtSD, PophamDL (2007) Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K. Mol Microbiol 65: 1582–1594.
58. MaggeA, SetlowB, CowanAE, SetlowP (2009) Analysis of dye binding by and membrane potential in spores of Bacillus species. J Appl Microbiol 106: 814–824.
59. FaganRP, FairweatherNF (2011) Clostridium difficile has two parallel and essential Sec secretion systems. J Biol Chem 286: 27483–27493.
60. RosenbuschKE, BakkerD, KuijperEJ, SmitsWK (2012) C. difficile 630Deltaerm Spo0A regulates sporulation, but does not contribute to toxin production, by direct high-affinity binding to target DNA. PLoS One 7: e48608.
61. SaujetL, MonotM, DupuyB, SoutourinaO, Martin-VerstraeteI (2011) The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile. J Bacteriol 193: 3186–3196.
62. SaujetL, PereiraFC, SerranoM, SoutourinaO, MonotM, et al. (2013) Genome-wide analysis of cell type-specific gene expression during spore formation in Clostridium difficile. PLOS Gen 9: e1003756.
63. SunDX, Cabrera-MartinezRM, SetlowP (1991) Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G. J Bacteriol 173: 2977–2984.
64. PartridgeSR, ErringtonJ (1993) The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis. Mol Microbiol 8: 945–955.
65. SerranoM, RealG, SantosJ, CarneiroJ, MoranCPJr, et al. (2011) A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of Bacillus subtilis. PLoS Genet 7: e1002220.
66. Londono-VallejoJA, StragierP (1995) Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis. Genes Dev 9: 503–508.
67. KarowML, GlaserP, PiggotPJ (1995) Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A 92: 2012–2016.
68. WangST, SetlowB, ConlonEM, LyonJL, ImamuraD, et al. (2006) The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37.
69. SteilL, SerranoM, HenriquesAO, VolkerU (2005) Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiology 151: 399–420.
70. EichenbergerP, FujitaM, JensenST, ConlonEM, RudnerDZ, et al. (2004) The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2: e328.
71. EichenbergerP, JensenST, ConlonEM, van OoijC, SilvaggiJ, et al. (2003) The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis. J Mol Biol 327: 945–972.
72. DoanT, MorlotC, MeisnerJ, SerranoM, HenriquesAO, et al. (2009) Novel secretion apparatus maintains spore integrity and developmental gene expression in Bacillus subtilis. PLoS Genet 5: e1000566.
73. SerranoM, VieiraF, MoranCPJr, HenriquesAO (2008) Processing of a membrane protein required for cell-to-cell signaling during endospore formation in Bacillus subtilis. J Bacteriol 190: 7786–7796.
74. MeisnerJ, WangX, SerranoM, HenriquesAO, MoranCPJr (2008) A channel connecting the mother cell and forespore during bacterial endospore formation. Proc Natl Acad Sci U S A 105: 15100–15105.
75. CampAH, LosickR (2008) A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels. Mol Microbiol 69: 402–417.
76. CampAH, LosickR (2009) A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis. Genes Dev 23: 1014–1024.
77. FujitaM, LosickR (2003) The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division. Genes Dev 17: 1166–1174.
78. TracyBP, JonesSW, FastAG, IndurthiDC, PapoutsakisET (2011) Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr Opin Biotechnol 23: 364–381.
79. CharyVK, XenopoulosP, PiggotPJ (2007) Expression of the sigmaF-directed csfB locus prevents premature appearance of sigmaG activity during sporulation of Bacillus subtilis. J Bacteriol 189: 8754–8757.
80. Karmazyn-CampelliC, RhayatL, Carballido-LopezR, DuperrierS, FrandsenN, et al. (2008) How the early sporulation sigma factor sigmaF delays the switch to late development in Bacillus subtilis. Mol Microbiol 67: 1169–1180.
81. BroderDH, PoglianoK (2006) Forespore engulfment mediated by a ratchet-like mechanism. Cell 126: 917–928.
82. DahlstenE, KirkD, LindstromM, KorkealaH (2013) Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502. Appl Environ Microbiol 79: 3867–3869.
83. JiangX, RubioA, ChibaS, PoglianoK (2005) Engulfment-regulated proteolysis of SpoIIQ: evidence that dual checkpoints control sigma activity. Mol Microbiol 58: 102–115.
84. HenriquesAO, BeallBW, RolandK, MoranCPJr (1995) Characterization of cotJ, a sigma E-controlled operon affecting the polypeptide composition of the coat of Bacillus subtilis spores. J Bacteriol 177: 3394–3406.
85. HussainHA, RobertsAP, MullanyP (2005) Generation of an erythromycin-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn916DeltaE enters the genome of this strain at multiple sites. J Med Microbiol 54: 137–141.
86. KepplerA, GendreizigS, GronemeyerT, PickH, VogelH, et al. (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21: 86–89.
87. VidaTA, EmrSD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128: 779–792.
88. GeorgeWL, SutterVL, CitronD, FinegoldSM (1979) Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol 9: 214–219.
89. JoshiLT, PhillipsDS, WilliamsCF, AlyousefA, BaillieL (2012) Contribution of spores to the ability of Clostridium difficile to adhere to surfaces. Appl Environ Microbiol 78: 7671–7679.
90. BalomenouS, FouetA, TzanodaskalakiM, Couture-TosiE, BouriotisV, et al. (2013) Distinct functions of polysaccharide deacetylases in cell shape, neutral polysaccharide synthesis and virulence of Bacillus anthracis. Mol Microbiol 87(4): 867–83.
91. MonotM, Boursaux-EudeC, ThibonnierM, VallenetD, MoszerI, et al. (2011) Reannotation of the genome sequence of Clostridium difficile strain 630. J Med Microbiol 60: 1193–1199.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2013 Číslo 10
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Dominant Mutations in Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway
- Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer
- The Histone H3 K27 Methyltransferase KMT6 Regulates Development and Expression of Secondary Metabolite Gene Clusters
- A Mutation in the Gene in Labrador Retrievers with Hereditary Nasal Parakeratosis (HNPK) Provides Insights into the Epigenetics of Keratinocyte Differentiation