Publikationsserver der Universitätsbibliothek Marburg

Titel:Zellzyklusregulation in einem strikt dimorphen Vertreter der Alphaproteobakterien
Autor:Leicht, Oliver
Weitere Beteiligte: Thanbichler, Martin (Prof. Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0046
DOI: https://doi.org/10.17192/z2017.0046
URN: urn:nbn:de:hebis:04-z2017-00464
DDC:500 Naturwissenschaften
Titel(trans.):Cell cycle regulation in a strictly dimorphic representative of the Alphaproteobacteria
Publikationsdatum:2017-01-02
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Mikrobiologie, Zellzyklus, Mikrobiologie, Bakterien, Bakterien, Zellzyklus, CtrA, Hyphomonas neptunium

Zusammenfassung:
The cell cycle is driven by a highly ordered series of events that lead to the production of two cells each containing an exact copy of the parental chromosome. Studies of the molecular mechanisms coordinating the cell cycle have revealed that the mechanisms regulating chromosome replication and segregation are highly similar in all eukaryotic cells. Until recently, it appeared that prokaryotes and eukaryotes do not share much resemblance in the mechanisms governing cell cycle progression. However, advances in prokaryotic cell biology and the analysis of the machinery orchestrating the cell cycle of the α-proteobacterium Caulobacter crescentus unveiled surprising similarities in the regulatory processes employed. The cell division of C. crescentus gives rise to two distinct cell types whose morphology is tightly coupled to their developmental cell cycle phase. While the sessile stalked cell can immediately restart a new round of replication, the motile swarmer cell has to differentiate into a sessile cell before gaining competence for chromosome replication and subsequent proliferation. The DNA-binding response regulator CtrA has been identified as the master regulator driving the cell cycle of this strictly dimorphic organism. A complex two-component signaling network regulating the activity of CtrA is critical for proper cell cycle progression and maintenance of the cellular asymmetry. Genome comparison uncovered that CtrA and the signaling network regulating its activity are highly conserved within the clade of α proteobacteria. Here, we report the functional analysis of the conserved cell cycle regulatory network in Hyphomonas neptunium, a close relative of C. crescentus, which also displays a biphasic life cycle but generates its stalk at the opposite cell pole and proliferates by an unusual budding mechanism. By employing DNA-protein interaction studies coupled to RNA-seq-based transcriptomics of conditional mutants defective in factors involved in the regulation of CtrA we were able to globally identify genes directly regulated by CtrA in H. neptunium. The subsequent analysis of this regulon unveiled a conserved role of CtrA in the transcriptional regulation of essential processes such as cell division and DNA-segregation as well as in the establishment of cellular asymmetry. By applying in vivo localization and heterologous complementation studies coupled to biochemical analysis we were able to identify a high conservation of the signaling module responsible for translating CtrA activity into asymmetry in C. crescentus. Even though transcriptional profiling suggested a role of this conserved module in the regulation of CtrA activity in H. neptunium, the inactivation of candidate genes suprisingly did not result in a pronounced cell cycle defect or a loss of asymmetry. This finding suggests that alternative regulatory factors or mechanisms are involved in the regulation of CtrA in this organism. This study highlights that even though genes are conserved between organisms, high functional variability can arise even over short evolutionary distances. A global investigation of the two-component signaling pathway of H. neptunium by a comprehensive deletion analysis led to the identification of an uncharacterized conserved single domain response regulator. Inactivation of mocR (modulator of cell cycle R) led to a severe cell cycle defect suggesting a role in the cell cycle regulation of H. neptunium. Using transcriptional profiling we were able to identify a connection to the conserved cell cycle regulatory core module. The synteny of this gene paired with heterologous complementation experiments suggests a conserved role of this protein within the α-proteobacteria. Future in depth investigation of MocR will provide valuable insights into its role in cell cycle regulation and will contribute to our understanding of the mechanisms by which single domain response regulators, which lack a dedicated output domain, participate in two-component signaling.

Summary:
The cell cycle is driven by a highly ordered series of events that lead to the production of two cells each containing an exact copy of the parental chromosome. Studies of the molecular mechanisms coordinating the cell cycle have revealed that the mechanisms regulating chromosome replication and segregation are highly similar in all eukaryotic cells. Until recently, it appeared that prokaryotes and eukaryotes do not share much resemblance in the mechanisms governing cell cycle progression. However, advances in prokaryotic cell biology and the analysis of the machinery orchestrating the cell cycle of the α-proteobacterium Caulobacter crescentus unveiled surprising similarities in the regulatory processes employed. The cell division of C. crescentus gives rise to two distinct cell types whose morphology is tightly coupled to their developmental cell cycle phase. While the sessile stalked cell can immediately restart a new round of replication, the motile swarmer cell has to differentiate into a sessile cell before gaining competence for chromosome replication and subsequent proliferation. The DNA-binding response regulator CtrA has been identified as the master regulator driving the cell cycle of this strictly dimorphic organism. A complex two-component signaling network regulating the activity of CtrA is critical for proper cell cycle progression and maintenance of the cellular asymmetry. Genome comparison uncovered that CtrA and the signaling network regulating its activity are highly conserved within the clade of α proteobacteria. Here, we report the functional analysis of the conserved cell cycle regulatory network in Hyphomonas neptunium, a close relative of C. crescentus, which also displays a biphasic life cycle but generates its stalk at the opposite cell pole and proliferates by an unusual budding mechanism. By employing DNA-protein interaction studies coupled to RNA-seq-based transcriptomics of conditional mutants defective in factors involved in the regulation of CtrA we were able to globally identify genes directly regulated by CtrA in H. neptunium. The subsequent analysis of this regulon unveiled a conserved role of CtrA in the transcriptional regulation of essential processes such as cell division and DNA-segregation as well as in the establishment of cellular asymmetry. By applying in vivo localization and heterologous complementation studies coupled to biochemical analysis we were able to identify a high conservation of the signaling module responsible for translating CtrA activity into asymmetry in C. crescentus. Even though transcriptional profiling suggested a role of this conserved module in the regulation of CtrA activity in H. neptunium, the inactivation of candidate genes suprisingly did not result in a pronounced cell cycle defect or a loss of asymmetry. This finding suggests that alternative regulatory factors or mechanisms are involved in the regulation of CtrA in this organism. This study highlights that even though genes are conserved between organisms, high functional variability can arise even over short evolutionary distances. A global investigation of the two-component signaling pathway of H. neptunium by a comprehensive deletion analysis led to the identification of an uncharacterized conserved single domain response regulator. Inactivation of mocR (modulator of cell cycle R) led to a severe cell cycle defect suggesting a role in the cell cycle regulation of H. neptunium. Using transcriptional profiling we were able to identify a connection to the conserved cell cycle regulatory core module. The synteny of this gene paired with heterologous complementation experiments suggests a conserved role of this protein within the α-proteobacteria. Future in depth investigation of MocR will provide valuable insights into its role in cell cycle regulation and will contribute to our understanding of the mechanisms by which single domain response regulators, which lack a dedicated output domain, participate in two-component signaling.

Bibliographie / References

  1. Mann, T. H., Seth Childers, W., Blair, J. A., Eckart, M. R. & Shapiro, L. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues. Nat Commun 7, 11454 (2016).
  2. Paulovich, A. G. & Hartwell, L. H. A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage. Cell 82, 841-847 (1995).
  3. Bird, T. H. & MacKrell, A. A CtrA homolog affects swarming motility and encystment in Rhodospirillum centenum. Arch Microbiol 193, 451-459 (2011).
  4. Marczynski, G. T., Lentine, K. & Shapiro, L. A developmentally regulated chromosomal origin of replication uses essential transcription elements. Genes Dev 9, 1543-1557 (1995).
  5. Wang, S. P., Sharma, P. L., Schoenlein, P. V. & Ely, B. A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus. Proc Natl Acad Sci U S A 90, 630-634 (1993).
  6. Norbury, C. & Nurse, P. Animal cell cycles and their control. Annu Rev Biochem 61, 441-470 (1992).
  7. Motokura, T. et al. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature 350, 512-515 (1991).
  8. Ishige, K., Nagasawa, S., Tokishita, S. & Mizuno, T. A novel device of bacterial signal transducers. EMBO J 13, 5195-5202 (1994).
  9. Wolfel, T. et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281-1284 (1995).
  10. Bhargava, H. N. & Leonard, P. A. Triclosan: Applications and safety. American Journal of Infection 17, 209-218 (1996).
  11. Hung, D. Y. & Shapiro, L. A signal transduction protein cues proteolytic events critical to Caulobacter cell cycle progression. Proc Natl Acad Sci U S A 99, 13160-13165 (2002).
  12. Lois, A. F., Weinstein, M., Ditta, G. S. & Helinski, D. R. Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen. J Biol Chem 268, 4370-4375 (1993).
  13. Yueh, M. F. & Tukey, R. H. Triclosan: A widespread environmental toxicant with many biological effects. Annu Rev Pharmacol Toxicol 56, 251-272 (2016).
  14. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J Mol Biol 215, 403-410 (1990).
  15. Gao, R. & Stock, A. M. Biological insights from structures of two-component proteins. Annu Rev Microbiol 63, 133-154 (2009).
  16. Poindexter, J. S. Biological properties and classification of the Caulobacter group. Bacteriol Rev 28, 231-295 (1964).
  17. Sherr, C. J. Cancer cell cycles. Science 274, 1672-1677 (1996).
  18. Quon, K. C., Marczynski, G. T. & Shapiro, L. Cell cycle control by an essential bacterial twocomponent signal transduction protein. Cell 84, 83-93 (1996).
  19. Collier, J. Cell cycle control in Alphaproteobacteria. Curr Opin Microbiol 30, 107-113 (2016).
  20. Domian I. J., Quon, K. C. & Shapiro, L. Cell Type-Specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell 90, 415- 424 (1997).
  21. Degnen, S. T. & Newton, A. Chromosome replication during development in Caulobacter crescentus. J Mol Biol 64, 671-680 (1972).
  22. Parkinson, J. S. & Kofoid, E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet 26, 71-112 (1992).
  23. Brown, P. J. B., Hardy, G. G., Trimble, M. J. & Brun, Y. V. Complex regulatory pathways coordinate cell-cycle progression and development in Caulobacter crescentus. Adv Microb Physiol 54, 1-77 (2008).
  24. Murray, S. M., Panis, G., Fumeaux, C., Viollier, P. H. & Howard, M. Computational and genetic reduction of a cell cycle to its simplest, primordial components. PLoS Biol 11 (2013).
  25. Imyanitov, E. N. et al. Construction of a broad host range cloning vector conferring Triclosan resistance. BioTechniques 33, 490-492 (2002).
  26. Bertoli, C., Skotheim, J. M. & de Bruin, R. A. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 14, 518-528 (2013).
  27. Stephens, C. M., Zweiger, G. & Shapiro, L. Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy. J Bacteriol 177, 1662-1669 (1995).
  28. Kim, J., Heindl, J. E. & Fuqua, C. Coordination of division and development influences complex multicellular behavior in Agrobacterium tumefaciens. PLoS One 8 (2013).
  29. Ninfa, A. J. & Magasanik, B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A 83, 5909-5913 (1986).
  30. Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D. & Hunt, T. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33, 389-396 (1983).
  31. Pines, J. Cyclins and cyclin-dependent kinases: a biochemical view. Biochem J 308 697-711 (1995).
  32. Pines, J. Cyclins and cyclin-dependent kinases: theme and variations. Adv Cancer Res 66, 181-212 (1995).
  33. Pines, J. Cyclins: wheels within wheels. Cell Growth Differ 2, 305-310 (1991).
  34. Easton, J., Wei, T., Lahti, J. M. & Kidd, V. J. Disruption of the cyclin D/cyclin-dependent kinase/INK4/retinoblastoma protein regulatory pathway in human neuroblastoma. Cancer Res 58, 2624-2632 (1998).
  35. Galperin, M. Y. Diversity of structure and function of response regulator output domains. Curr Opin Microbiol 13, 150-159 (2010).
  36. Chen, Y. E., Tsokos, C. G., Biondi, E. G., Perchuk, B. S. & Laub, M. T. Dynamics of two Phosphorelays controlling cell cycle progression in Caulobacter crescentus. J Bacteriol 191, 7417- 7429 (2009).
  37. Laub, M. T., Chen, S. L., Shapiro, L. & McAdams, H. H. Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc Natl Acad Sci U S A 99, 4632-4637 (2002).
  38. Willett, J. W. & Kirby, J. R. Genetic and biochemical dissection of a HisKA domain identifies residues required exclusively for kinase and phosphatase activities. PLoS Genet 8 (2012).
  39. Curtis, P. D. & Brun, Y. V. Getting in the loop: regulation of development in Caulobacter crescentus. Microbiol Mol Biol Rev 74, 13-41 (2010).
  40. Fujita, M., Gonzalez-Pastor, J. E. & Losick, R. High- and low-threshold genes in the Spo0A regulon of Bacillus subtilis. J Bacteriol 187, 1357-1368 (2005).
  41. Margolin, W. & Bernander, R. How do prokaryotic cells cycle? Curr Biol 14, 768-770 (2004).
  42. Tsung, K., Brissette, R. E. & Inouye, M. Identification of the DNA-binding domain of the OmpR protein required for transcriptional activation of the ompF and ompC genes of Escherichia coli by in vivo DNA footprinting. J Biol Chem 264, 10104-10109 (1989).
  43. Meisenzahl, A. C., Shapiro, L. & Jenal, U. Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus. J Bacteriol 179, 592-600 (1997).
  44. Ninfa, E. G., Atkinson, M. R., Kamberov, E. S. & Ninfa, A. J. Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits. J Bacteriol 175, 7024-7032 (1993).
  45. Jeffrey, P. D. et al. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature 376, 313-320 (1995).
  46. Wolanin, P. M., Webre, D. J. & Stock, J. B. Mechanism of phosphatase activity in the chemotaxis response regulator CheY. Biochemistry 42, 14075-14082 (2003).
  47. Weiss, V., Kramer, G., Dunnebier, T. & Flotho, A. Mechanism of regulation of the bifunctional histidine kinase NtrB in Escherichia coli. J Mol Microbiol Biotechnol 4, 229-233 (2002).
  48. Li, S. Mechanisms of cellular signal transduction. Int J Biol Sci 1, 152 (2005).
  49. Levy, C. W. et al. Molecular basis of triclosan activity. Nature 398, 383-384 (1999).
  50. Tzeng, Y. L. & Hoch, J. A. Molecular recognition in signal transduction: the interaction surfaces of the Spo0F response regulator with its cognate phosphorelay proteins revealed by alanine scanning mutagenesis. J Mol Biol 272, 200-212 (1997).
  51. Jiang, M., Shao, W., Perego, M. & Hoch, J. A. Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol Microbiol 38, 535-542 (2000).
  52. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671-675 (2012).
  53. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell 88, 323-331 (1997).
  54. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell 53, 79-87 (1988).
  55. Sanders, D. A., Gillece-Castro, B. L., Burlingame, A. L. & Koshland, D. E. Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. J Bacteriol 174, 5117-5122 (1992).
  56. Tsokos, C. G. & Laub, M. T. Polarity and cell fate asymmetry in Caulobacter crescentus. Curr Opin Microbiol 15, 744-750 (2012).
  57. Wickner, S., Maurizi, M. R. & Gottesman, S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286, 1888-1893 (1999).
  58. Keener, J. & Kustu, S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A 85, 4976-4980 (1988).
  59. Anderson, B. M., Cordes, E. H. & Jencks, W. P. Reactivity and catalysis in reactions of the serine hydroxyl group and of O-acyl serines. J Biol Chem 236, 455-463 (1961).
  60. Oren, M. Regulation of the p53 tumor suppressor protein. J Biol Chem 274, 36031-36034 (1999).
  61. Porter, S. L., Wadhams, G. H. & Armitage, J. P. Signal processing in complex chemotaxis pathways. Nat Rev Microbiol 9, 153-165 (2011).
  62. Stein, S. S. & Koshland, D. E. Solubilization of certain L- and D-Peptidases of hog kidney particulates. Arch Biochem Biophys 39, 230-231 (1952).
  63. Tomomori, C. et al. Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat Struct Biol 6, 729-734 (1999).
  64. Ausmees, N. & Jacobs-Wagner, C. Spatial and temporal control of differentiation and cell cycle progression in Caulobacter crescentus. Annu Rev Microbiol 57, 225-247 (2003).
  65. Laub, M. T. & Goulian, M. Specificity in two-component signal transduction pathways. Annu Rev Genet 41, 121-145 (2007).
  66. Mascher, T., Helmann, J. D. & Unden, G. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev 70, 910-938 (2006).
  67. Volz, K. Structural conservation in the CheY superfamily. Biochemistry 32, 11741-11753 (1993).
  68. Wolfe, A. J. The acetate switch. Microbiol Mol Biol Rev 69, 12-50 (2005).
  69. Vermeulen, K., Van Bockstaele, D. R. & Berneman, Z. N. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 36, 131-149 (2003).
  70. Ryan, K. R., Judd, E. M. & Shapiro, L. The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally controlled proteolysis. J Mol Biol 324, 443-455 (2002).
  71. Reisenauer, A., Quon, K. & Shapiro, L. The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle. J Bacteriol 181, 2430-2439 (1999).
  72. Radhakrishnan, S. K., Thanbichler, M. & Viollier, P. H. The dynamic interplay between a cell fate determinant and a lysozyme homolog drives the asymmetric division cycle of Caulobacter crescentus. Genes Dev 22, 212-225 (2008).
  73. Heath, R. J., Su, N., Murphy, C. K. & Rock, C. O. The enoyl-[acyl-carrier-protein] reductases FabI and FabL from Bacillus subtilis. J Biol Chem 275, 40128-40133 (2000).
  74. Carnero, A. & Hannon, G. J. The INK4 family of CDK inhibitors. Curr Top Microbiol Immunol 227, 43-55 (1998).
  75. Casino, P., Rubio, V. & Marina, A. The mechanism of signal transduction by two-component systems. Curr Opin Struct Biol 20, 763-771 (2010).
  76. Johnson, D. R., Czechowska, K., Chevre, N. & van der Meer, J. R. Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry. Environ Microbiol 11, 1682-1691 (2009).
  77. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 Hoch, J. A. Two-component and phosphorelay signal transduction. Curr Opin Microbiol 3, 165- 170 (2000).
  78. Nixon, B. T., Ronson, C. W. & Ausubel, F. M. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci U S A 83, 7850-7854 (1986).
  79. Stock, A. M., Robinson, V. L. & Goudreau, P. N. Two-component signal transduction. Annu Rev Biochem 69, 183-215 (2000).
  80. Skerker, J. M., Prasol, M. S., Perchuk, B. S., Biondi, E. G. & Laub, M. T. Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a systemlevel analysis. PLoS Biol 3 (2005).


* Das Dokument ist im Internet frei zugänglich - Hinweise zu den Nutzungsrechten