Cyclic di-GMP Activates Adenylate Cyclase A and Protein Kinase A to Induce Stalk Formation in Dictyostelium

  • Zhi-hui Chen
  • Christina Schilde
  • Pauline SchaapEmail author


Cyclic di-GMP, an important prokaryote second messenger is used by the eukaryote Dictyostelium discoideum as a secreted signal to trigger stalk formation in fruiting bodies. Cyclic di-GMP is synthesized by a prokaryote-type diguanylate cyclase DgcA, but its mode of action was unknown. Transcriptional profiling yielded several target genes for cyclic di-GMP, which were tested for cyclic di-GMP induced expression in mutants with similar phenotypes as dgca-. A mutant with reduced PKA activity showed defective cyclic di-GMP induced stalk gene expression. Cyclic di-GMP increased cAMP levels in wild-type cells, but not in a mutant that lacked adenylate cyclase A (ACA) activity in slugs. This mutant also did not show cyclic di-GMP induced stalk gene expression. The stalk-less dgca- mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is the intermediate for cyclic di-GMP in normal development. ACA is expressed at the tip of emerging fruiting bodies, where it produces the cAMP pulses that organize morphogenetic movement. The tip is also the site where stalk differentiation initiates. Our finding that cyclic di-GMP acts on tip-expressed ACA explains why the Dictyostelium stalk is always formed at the morphogenetic organizer.


Dictyostelium discoideum Cyclic di-GMP Fruiting bodies Stalk formation Protein kinase A Adenylate cyclase A Organizer 



Research reported in this study was funded by Leverhulme Trust grants RPG-2012-746 and RPG-2016-220, Wellcome Trust grant 100293/Z/12/Z and European Research Council grant 742288.


  1. 1.
    Romling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77(1):1–52. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jenal U, Reinders A, Lori C (2017) Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol 15(5):271–284. CrossRefGoogle Scholar
  3. 3.
    Chou SH, Galperin MY (2016) Diversity of cyclic di-GMP-binding proteins and mechanisms. J Bacteriol 198(1):32–46. CrossRefGoogle Scholar
  4. 4.
    Fahmi T, Port GC, Cho KH (2017) c-di-AMP: an essential molecule in the signaling pathways that regulate the viability and virulence of gram-positive bacteria. Genes (Basel) 8(8):197. CrossRefGoogle Scholar
  5. 5.
    Davies BW, Bogard RW, Young TS, Mekalanos JJ (2012) Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell 149(2):358–370. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Nelson JW, Sudarsan N, Phillips GE, Stav S, Lünse CE, McCown PJ, Breaker RR (2015) Control of bacterial exoelectrogenesis by c-AMP-GMP. Proc Natl Acad Sci U S A 112(17):5389–5394. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Margolis SR, Wilson SC, Vance RE (2017) Evolutionary origins of cGAS-STING signaling. Trends Immunol 38(10):733–743. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chen ZH, Schaap P (2012) The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium. Nature 488(7413):680–683. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Morris HR, Taylor GW, Masento MS, Jermyn KA, Kay RR (1987) Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum. Nature 328(6133):811–814CrossRefGoogle Scholar
  10. 10.
    Saito T, Kato A, Kay RR (2008) DIF-1 induces the basal disc of the Dictyostelium fruiting body. Dev Biol 317(2):444–453. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Neumann CS, Walsh CT, Kay RR (2010) A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1. Proc Natl Acad Sci U S A 107(13):5798–5803. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Thompson CR, Kay RR (2000) The role of DIF-1 signaling in Dictyostelium development. Mol Cell 6(6):1509–1514CrossRefGoogle Scholar
  13. 13.
    Song Y, Luciani MF, Giusti C, Golstein P (2015) c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1. Mol Biol Cell 26:651–658. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Chen ZH, Singh R, Cole C, Lawal HM, Schilde C, Febrer M, Barton GJ, Schaap P (2017) Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer. Proc Natl Acad Sci U S A 114(3):516–521. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Jermyn KA, Williams JG (1991) An analysis of culmination in Dictyostelium using prestalk and stalk-specific cell autonomous markers. Development 111:779–787PubMedGoogle Scholar
  16. 16.
    Robinson V, Williams J (1997) A marker of terminal stalk cell terminal differentiation in Dictyostelium. Differentiation 61:223–228CrossRefGoogle Scholar
  17. 17.
    Harwood AJ, Hopper NA, Simon M-N, Driscoll DM, Veron M, Williams JG (1992) Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase. Cell 69:615–624CrossRefGoogle Scholar
  18. 18.
    Raffelberg S, Wang L, Gao S, Losi A, Gartner W, Nagel G (2013) A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420. Biochem J 455(3):359–365. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Chen ZH, Raffelberg S, Losi A, Schaap P, Gartner W (2014) A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant. J Biotechnol 191:246–249. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Pitt GS, Milona N, Borleis J, Lin KC, Reed RR, Devreotes PN (1992) Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development. Cell 69:305–315CrossRefGoogle Scholar
  21. 21.
    Pitt GS, Brandt R, Lin KC, Devreotes PN, Schaap P (1993) Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA). Genes Dev 7:2172–2180CrossRefGoogle Scholar
  22. 22.
    Siegert F, Weijer CJ (1992) Three-dimensional scroll waves organize Dictyostelium slugs. Proc Natl Acad Sci U S A 89:6433–6437CrossRefGoogle Scholar
  23. 23.
    Kriebel PW, Parent CA (2004) Adenylyl cyclase expression and regulation during the differentiation of Dictyostelium discoideum. IUBMB Life 56(9):541–546CrossRefGoogle Scholar
  24. 24.
    Verkerke-van Wijk I, Fukuzawa M, Devreotes PN, Schaap P (2001) Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression. Dev Biol 234(1):151–160CrossRefGoogle Scholar
  25. 25.
    Siegert F, Weijer CJ (1995) Spiral and concentric waves organize multicellular Dictyostelium mounds. Curr Biol 5:937–943CrossRefGoogle Scholar
  26. 26.
    Raper KB (1940) Pseudoplasmodium formation and organization in Dictyostelium discoideum. J Elisha Mitchell Sci Soc 56:241–282Google Scholar
  27. 27.
    Anderson C, Stern CD (2016) Organizers in development. Curr Top Dev Biol 117:435–454. CrossRefPubMedGoogle Scholar
  28. 28.
    Katoh-Kurasawa M, Santhanam B, Shaulsky G (2016) The GATA transcription factor gene gtaG is required for terminal differentiation in Dictyostelium. J Cell Sci 129(8):1722–1733. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Fukuzawa M, Hopper N, Williams J (1997) cudA: a Dictyostelium gene with pleiotropic effects on cellular differentiation and slug behaviour. Development 124:2719–2728PubMedGoogle Scholar
  30. 30.
    Mohanty S, Jermyn KA, Early A, Kawata T, Aubry L, Ceccarelli A, Schaap P, Williams JG, Firtel RA (1999) Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis. Development 126(15):3391–3405PubMedGoogle Scholar
  31. 31.
    Hopper NA, Harwood AJ, Bouzid S, Véron M, Williams JG (1993) Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia. EMBO J 12:2459–2466CrossRefGoogle Scholar
  32. 32.
    Kay RR, Thompson CR (2001) Cross-induction of cell types in Dictyostelium: evidence that DIF-1 is made by prespore cells. Development 128(24):4959–4966PubMedGoogle Scholar
  33. 33.
    Hillmann F, Forbes G, Novohradska S, Ferling I, Riege K, Groth M, Westermann M, Marz M, Spaller T, Winckler T, Schaap P, Glockner G (2018) Multiple roots of fruiting body formation in Amoebozoa. Genome Biol Evol 10(2):591–606. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Schaap P, Barrantes I, Minx P, Sasaki N, Anderson RW, Benard M, Biggar KK, Buchler NE, Bundschuh R, Chen X, Fronick C, Fulton L, Golderer G, Jahn N, Knoop V, Landweber LF, Maric C, Miller D, Noegel AA, Peace R, Pierron G, Sasaki T, Schallenberg-Rudinger M, Schleicher M, Singh R, Spaller T, Storey KB, Suzuki T, Tomlinson C, Tyson JJ, Warren WC, Werner ER, Werner-Felmayer G, Wilson RK, Winckler T, Gott JM, Glockner G, Marwan W (2015) The Physarum polycephalum genome reveals extensive use of prokaryotic two-component and metazoan-type tyrosine kinase signaling. Genome Biol Evol 8(1):109–125. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Clarke M, Lohan AJ, Liu B, Lagkouvardos I, Roy S, Zafar N, Bertelli C, Schilde C, Kianianmomeni A, Burglin TR, Frech C, Turcotte B, Kopec KO, Synnott JM, Choo C, Paponov I, Finkler A, Soon Heng Tan C, Hutchins AP, Weinmeier T, Rattei T, Chu JS, Gimenez G, Irimia M, Rigden DJ, Fitzpatrick DA, Lorenzo-Morales J, Bateman A, Chiu CH, Tang P, Hegemann P, Fromm H, Raoult D, Greub G, Miranda-Saavedra D, Chen N, Nash P, Ginger ML, Horn M, Schaap P, Caler L, Loftus B (2013) Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling. Genome Biol 14(2):R11. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, Amedeo P, Roncaglia P, Berriman M, Hirt RP, Mann BJ, Nozaki T, Suh B, Pop M, Duchene M, Ackers J, Tannich E, Leippe M, Hofer M, Bruchhaus I, Willhoeft U, Bhattacharya A, Chillingworth T, Churcher C, Hance Z, Harris B, Harris D, Jagels K, Moule S, Mungall K, Ormond D, Squares R, Whitehead S, Quail MA, Rabbinowitsch E, Norbertczak H, Price C, Wang Z, Guillen N, Gilchrist C, Stroup SE, Bhattacharya S, Lohia A, Foster PG, Sicheritz-Ponten T, Weber C, Singh U, Mukherjee C, El-Sayed NM, Petri WA Jr, Clark CG, Embley TM, Barrell B, Fraser CM, Hall N (2005) The genome of the protist parasite Entamoeba histolytica. Nature 433(7028):865–868. CrossRefPubMedGoogle Scholar
  37. 37.
    Gloeckner G, Lawal HM, Felder M, Singh R, Singer G, Weijer CJ, Schaap P (2016) The multicellularity genes of dictyostelid social amoebas. Nat Commun 7:12085. CrossRefGoogle Scholar
  38. 38.
    Anjard C, Loomis WF (2008) Cytokinins induce sporulation in Dictyostelium. Development 135:819–827CrossRefGoogle Scholar
  39. 39.
    Schaap P (2013) Cyclic di-nucleotide signaling enters the eukaryote domain. IUBMB Life 65(11):897–903. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  • Zhi-hui Chen
    • 1
  • Christina Schilde
    • 1
  • Pauline Schaap
    • 1
    Email author
  1. 1.School of Life SciencesUniversity of DundeeDundeeUK

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