Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in
Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is an emergent pathogen and the most common cause of nosocomial diarrhea. Although transmission of C. difficile is mediated by contamination of the gut by spores, the regulatory cascade controlling spore formation remains poorly characterized. During Bacillus subtilis sporulation, a cascade of four sigma factors, σF and σG in the forespore and σE and σK in the mother cell governs compartment-specific gene expression. In this work, we combined genome wide transcriptional analyses and promoter mapping to define the C. difficile σF, σE, σG and σK regulons. We identified about 225 genes under the control of these sigma factors: 25 in the σF regulon, 97 σE-dependent genes, 50 σG-governed genes and 56 genes under σK control. A significant fraction of genes in each regulon is of unknown function but new candidates for spore coat proteins could be proposed as being synthesized under σE or σK control and detected in a previously published spore proteome. SpoIIID of C. difficile also plays a pivotal role in the mother cell line of expression repressing the transcription of many members of the σE regulon and activating sigK expression. Global analysis of developmental gene expression under the control of these sigma factors revealed deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We showed that the expression of the σE regulon in the mother cell was not strictly under the control of σF despite the fact that the forespore product SpoIIR was required for the processing of pro-σE. In addition, the σK regulon was not controlled by σG in C. difficile in agreement with the lack of pro-σK processing. This work is one key step to obtain new insights about the diversity and evolution of the sporulation process among Firmicutes.
Vyšlo v časopise:
Genome-Wide Analysis of Cell Type-Specific Gene Transcription during Spore Formation in. PLoS Genet 9(10): e32767. doi:10.1371/journal.pgen.1003756
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1003756
Souhrn
Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is an emergent pathogen and the most common cause of nosocomial diarrhea. Although transmission of C. difficile is mediated by contamination of the gut by spores, the regulatory cascade controlling spore formation remains poorly characterized. During Bacillus subtilis sporulation, a cascade of four sigma factors, σF and σG in the forespore and σE and σK in the mother cell governs compartment-specific gene expression. In this work, we combined genome wide transcriptional analyses and promoter mapping to define the C. difficile σF, σE, σG and σK regulons. We identified about 225 genes under the control of these sigma factors: 25 in the σF regulon, 97 σE-dependent genes, 50 σG-governed genes and 56 genes under σK control. A significant fraction of genes in each regulon is of unknown function but new candidates for spore coat proteins could be proposed as being synthesized under σE or σK control and detected in a previously published spore proteome. SpoIIID of C. difficile also plays a pivotal role in the mother cell line of expression repressing the transcription of many members of the σE regulon and activating sigK expression. Global analysis of developmental gene expression under the control of these sigma factors revealed deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We showed that the expression of the σE regulon in the mother cell was not strictly under the control of σF despite the fact that the forespore product SpoIIR was required for the processing of pro-σE. In addition, the σK regulon was not controlled by σG in C. difficile in agreement with the lack of pro-σK processing. This work is one key step to obtain new insights about the diversity and evolution of the sporulation process among Firmicutes.
Zdroje
1. DeakinLJ, ClareS, FaganRP, DawsonLF, PickardDJ, et al. (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80: 2704–2711.
2. LawleyTD, ClareS, DeakinLJ, GouldingD, YenJL, et al. (2010) Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens. Appl Environ Microbiol 76: 6895–6900.
3. SarkerMR, Paredes-SabjaD (2012) Molecular basis of early stages of Clostridium difficile infection: germination and colonization. Future Microbiol 7: 933–943.
4. SorgJA, SonensheinAL (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190: 2505–2512.
5. HilbertDW, PiggotPJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234–262.
6. HigginsD, DworkinJ (2012) Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev 36: 131–148.
7. StragierP, LosickR (1996) Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30: 297–341.
8. MolleV, FujitaM, JensenST, EichenbergerP, Gonzalez-PastorJE, et al. (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683–1701.
9. 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.
10. WangST, SetlowB, ConlonEM, LyonJL, ImamuraD, et al. (2006) The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37.
11. Londono-VallejoJA, StragierP (1995) Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis. Genes Dev 9: 503–508.
12. 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.
13. FeuchtA, EvansL, ErringtonJ (2003) Identification of sporulation genes by genome-wide analysis of the sigmaE regulon of Bacillus subtilis. Microbiology 149: 3023–3034.
14. 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.
15. AbecassisA, SerranoM, AlvesR, QuintaisL, Pereira-LealJB, et al. (2013) A genomic signature and the identification of new endosporulation genes. J Bacteriol 195: 2101–2115.
16. de HoonMJ, EichenbergerP, VitkupD (2010) Hierarchical evolution of the bacterial sporulation network. Curr Biol 20: R735–745.
17. 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.
18. ParedesCJ, AlsakerKV, PapoutsakisET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3: 969–978.
19. PereiraFC, SaujetL, ToméAR, SerranoM, MonotM, et al. (2013) The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9: e1003782.
20. Stragier P (2002) A gene odyssey: exploring the genomes of endospore-forming bacteria; In: Sonenshein AL, Hoch JA, Losick R, editors. Bacillus subtilis: from cells to genes and from genes to cells. Washington, D. C.: ASM Press.
21. 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.
22. 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.
23. 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.
24. 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.
25. HaraldsenJD, SonensheinAL (2003) Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene. Mol Microbiol 48: 811–821.
26. JonesSW, ParedesCJ, TracyB, ChengN, SillersR, et al. (2008) The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol 9: R114.
27. 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.
28. UnderwoodS, GuanS, VijayasubhashV, BainesSD, GrahamL, et al. (2009) Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 191: 7296–7305.
29. SteinerE, DagoAE, YoungDI, HeapJT, MintonNP, et al. (2011) Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum. Mol Microbiol 80: 641–654.
30. 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.
31. SoutourinaO, MonotM, BoudryP, SaujetL, PichonC, et al. (2013) Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS Genet 9: e1003493.
32. TattiKM, ShulerMF, MoranCPJr (1995) Sequence-specific interactions between promoter DNA and the RNA polymerase sigma factor E. J Mol Biol 253: 8–16.
33. SantangeloJD, KuhnA, Treuner-LangeA, DurreP (1998) Sporulation and time course expression of sigma-factor homologous genes in Clostridium acetobutylicum. FEMS Microbiol Lett 161: 157–164.
34. ZhaoY, MelvilleSB (1998) Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens. J Bacteriol 180: 136–142.
35. EichenbergerP, FawcettP, LosickR (2001) A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis. Mol Microbiol 42: 1147–1162.
36. MorlotC, UeharaT, MarquisKA, BernhardtTG, RudnerDZ (2010) A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis. Genes Dev 24: 411–422.
37. 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.
38. MeisnerJ, MaehigashiT, AndreI, DunhamCM, MoranCPJr (2012) Structure of the basal components of a bacterial transporter. Proc Natl Acad Sci U S A 109: 5446–5451.
39. LawleyTD, CroucherNJ, YuL, ClareS, SebaihiaM, et al. (2009) Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores. J Bacteriol 191: 5377–5386.
40. PutmanE, NockA, LawleyT, ShenA (2013) SpoIVA and SipL are Clostridium difficile spore morphogenic proteins. J Bacteriol 195: 1214–1225.
41. PermpoonpattanaP, TollsEH, NademR, TanS, BrissonA, et al. (2011) Surface layers of Clostridium difficile endospores. J Bacteriol 193: 6461–6470.
42. GilmoreM, BandyopadhyayD, DeanAM, LinnstaedtSD, DLP (2004) Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan. J Bacteriol 186: 80–89.
43. Paredes-SabjaD, SarkerN, SetlowB, SetlowP, SarkerMR (2008) Roles of DacB and spm proteins in Clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation. Appl Environ Microbiol 74: 3730–3738.
44. FigueiredoMC, LoboSA, CaritaJN, NobreLS, SaraivaLM (2012) Bacterioferritin protects the anaerobe Desulfovibrio vulgaris Hildenborough against oxygen. Anaerobe 18: 454–458.
45. AdamsCM, EckenrothBE, PutnamEE, DoublieS, ShenA (2013) Structural and Functional Analysis of the CspB Protease Required for Clostridium Spore Germination. PLoS Pathog 9: e1003165.
46. CartmanST, MintonNP (2010) A mariner-Based Transposon System for In Vivo Random Mutagenesis of Clostridium difficile. Appl Environ Microbiol 76: 1103–1109.
47. Paredes-SabjaD, SetlowP, SarkerMR (2011) Germination of spores of Bacillales and Clostridiales species: mechanisms and proteins involved. Trends Microbiol 19: 85–94.
48. FrancisMB, AllenCA, ShresthaR, SorgJA (2013) Bile Acid Recognition by the Clostridium difficile Germinant Receptor, CspC, Is Important for Establishing Infection. PLoS Pathog 9: e1003356.
49. ChenFC, ShenLF, TsaiMC, ChakKF (2003) The IspA protease's involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis. Biochem Biophys Res Commun 312: 708–715.
50. RothenbacherFP, SuzukiM, HurleyJM, MontvilleTJ, KirnTJ, et al. (2012) Clostridium difficile MazF toxin exhibits selective, not global, mRNA cleavage. J Bacteriol 194: 3464–3474.
51. AdlerE, BarakI, StragierP (2001) Bacillus subtilis locus encoding a killer protein and its antidote. J Bacteriol 183: 3574–3581.
52. SetlowP (2007) I will survive: DNA protection in bacterial spores. Trends Microbiol 15: 172–180.
53. Tovar-RojoF, ChanderM, SetlowB, SetlowP (2002) The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis. J Bacteriol 184: 584–587.
54. PermpoonpattanaP, PhetcharaburaninJ, MikelsoneA, DembekM, TanS, et al. (2013) Functional characterization of Clostridium difficile spore coat proteins. J Bacteriol 195: 1492–1503.
55. BurnsDA, HeapJT, MintonNP (2010) Clostridium difficile spore germination: an update. Res Microbiol 161: 730–734.
56. XiaoY, FranckeC, AbeeT, Wells-BennikMH (2011) Clostridial spore germination versus bacilli: genome mining and current insights. Food Microbiol 28: 266–274.
57. HenriquesAO, MoranCPJr (2007) Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61: 555–588.
58. SteichenCT, KearneyJF, TurnboughCLJr (2007) Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth. Mol Microbiol 64: 359–367.
59. NugrohoFA, YamamotoH, KobayashiY, SekiguchiJ (1999) Characterization of a new sigma-K-dependent peptidoglycan hydrolase gene that plays a role in Bacillus subtilis mother cell lysis. J Bacteriol 181: 6230–6237.
60. BurnsDA, HeapJT, MintonNP (2010) SleC is essential for germination of Clostridium difficile spores in nutrient-rich medium supplemented with the bile salt taurocholate. J Bacteriol 192: 657–664.
61. GerdesK, ChristensenSK, Lobner-OlesenA (2005) Prokaryotic toxin-antitoxin stress response loci. Nat Rev Microbiol 3: 371–382.
62. HarrisLM, WelkerNE, PapoutsakisET (2002) Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 184: 3586–3597.
63. RhayatL, DuperrierS, Carballido-LopezR, PellegriniO, StragierP (2009) Genetic dissection of an inhibitor of the sporulation sigma factor sigma(G). J Mol Biol 390: 835–844.
64. 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.
65. BagyanI, HobotJ, CuttingS (1996) A compartmentalized regulator of developmental gene expression in Bacillus subtilis. J Bacteriol 178: 4500–4507.
66. Ramirez-PeraltaA, StewartKA, ThomasSK, SetlowB, ChenZ, et al. (2012) Effects of the SpoVT regulatory protein on the germination and germination protein levels of spores of Bacillus subtilis. J Bacteriol 194: 3417–3425.
67. CangianoG, MazzoneA, BaccigalupiL, IsticatoR, EichenbergerP, et al. (2010) Direct and indirect control of late sporulation genes by GerR of Bacillus subtilis. J Bacteriol 192: 3406–3413.
68. HimesP, McBryantSJ, KroosL (2010) Two regions of Bacillus subtilis transcription factor SpoIIID allow a monomer to bind DNA. J Bacteriol 192: 1596–1606.
69. KunkelB, LosickR, StragierP (1990) The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene. Genes Dev 4: 525–535.
70. ErringtonJ (1993) Bacillus subtilis sporulation: regulation of gene expression and control of morphogenesis. Microbiol Rev 57: 1–33.
71. YoshisueH, IharaK, NishimotoT, SakaiH, KomanoT (1995) Cloning and characterization of a Bacillus thuringiensis homolog of the spoIIID gene from Bacillus subtilis. Gene 154: 23–29.
72. OkeV, LosickR (1993) Multilevel regulation of the sporulation transcription factor sigma K in Bacillus subtilis. J Bacteriol 175: 7341–7347.
73. KirkDG, DahlstenE, ZhangZ, KorkealaH, LindstromM (2012) Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 78: 4590–4606.
74. FawcettP, EichenbergerP, LosickR, YoungmanP (2000) The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc Natl Acad Sci U S A 97: 8063–8068.
75. JonasRM, HaldenwangWG (1989) Influence of spo mutations on sigma E synthesis in Bacillus subtilis. J Bacteriol 171: 5226–5228.
76. 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.
77. EldarA, CharyVK, XenopoulosP, FontesME, LosonOC, et al. (2009) Partial penetrance facilitates developmental evolution in bacteria. Nature 460: 510–514.
78. ZhangL, HigginsML, PiggotPJ, KarowML (1996) Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis. J Bacteriol 178: 2813–2817.
79. GuillotC, MoranCPJr (2007) Essential internal promoter in the spoIIIA locus of Bacillus subtilis. J Bacteriol 189: 7181–7189.
80. 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.
81. TraagB, PuglieseA, EisenJ, LosickR (2013) Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis. J Bacteriol 195: 253–260.
82. DworkinJ, LosickR (2005) Developmental commitment in a bacterium. Cell 121: 401–409.
83. JiangX, RubioA, ChibaS, PoglianoK (2005) Engulfment-regulated proteolysis of SpoIIQ: evidence that dual checkpoints control sigma activity. Mol Microbiol 58: 102–115.
84. OkeV, ShchepetovM, CuttingS (1997) SpoIVB has two distinct functions during spore formation in Bacillus subtilis. Mol Microbiol 23: 223–230.
85. WangY, LiX, MaoY, BlaschekHP (2012) Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genomics 13: 102.
86. 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.
87. HeapJT, PenningtonOJ, CartmanST, CarterGP, MintonNP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70: 452–464.
88. 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.
89. HeapJT, PenningtonOJ, CartmanST, MintonNP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78: 79–85.
90. LangmeadB, TrapnellC, PopM, SalzbergSL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.
91. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079.
92. LivakKJ, SchmittgenTD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25: 402–408.
93. SmythGK, SpeedT (2003) Normalization of cDNA microarray data. Methods 31: 265–273.
94. BenjaminiY, HochbergY (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statist Soc Ser 289–300.
95. MironovA, VinokurovaN, GelfandM (2000) GenomeExplorer: software for analysis of complete bacterial genomes. Mol Biol (Mosk) 34: 253–262.
96. SierroN, MakitaY, de HoonM, NakaiK (2008) DBTBS: a database of transcriptional regulation in Bacillus subtilis containing upstream intergenic conservation information. Nucleic Acids Res 36: D93–96.
97. SerranoM, ZilhaoR, RiccaE, OzinAJ, MoranCPJr, et al. (1999) A Bacillus subtilis secreted protein with a role in endospore coat assembly and function. J Bacteriol 181: 3632–3643.
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