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Adaptive evolution by spontaneous domain fusion and protein relocalization


Knowledge of adaptive processes encompasses understanding the emergence of new genes. Computational analyses of genomes suggest that new genes can arise by domain swapping; however, empirical evidence has been lacking. Here we describe a set of nine independent deletion mutations that arose during selection experiments with the bacterium Pseudomonas fluorescens in which the membrane-spanning domain of a fatty acid desaturase became translationally fused to a cytosolic di-guanylate cyclase, generating an adaptive ‘wrinkly spreader’ phenotype. Detailed genetic analysis of one gene fusion shows that the mutant phenotype is caused by relocalization of the di-guanylate cyclase domain to the cell membrane. The relative ease by which this new gene arose, along with its functional and regulatory effects, provides a glimpse of mutational events and their consequences that are likely to have a role in the evolution of new genes.

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  1. 1.

    Chen, S. D., Krinsky, B. H. & Long, M. Y. New genes as drivers of phenotypic evolution. Nat. Rev. Genet. 14, 645–660 (2013).

  2. 2.

    Long, M. Y., Van Kuren, N. W., Chen, S. D. & Vibranovski, M. D. New gene evolution: little did we know. Annu. Rev. Genet. 47, 307–333 (2013).

  3. 3.

    Ohno, S. Evolution by Gene Duplication (Springer, New York, 1970).

  4. 4.

    Bergthorsson, U., Andersson, D. I. & Roth, J. R. Ohno’s dilemma: evolution of new genes under continuous selection. Proc. Natl Acad. Sci. USA 104, 17004–17009 (2007).

  5. 5.

    Long, M., Betran, E., Thornton, K. & Wang, W. The origin of new genes: glimpses from the young and old. Nat. Rev. Genet. 4, 865–875 (2003).

  6. 6.

    Ding, Y., Zhou, Q. & Wang, W. Origins of new genes and evolution of their novel functions. Annu. Rev. Ecol. Evol. Syst. 43, 345–363 (2012).

  7. 7.

    Zhao, L., Saelao, P., Jones, C. D. & Begun, D. J. Origin and spread of de novo genes in Drosophila melanogaster populations. Science 343, 769–772 (2014).

  8. 8.

    Tautz, D. & Domazet-Loso, T. The evolutionary origin of orphan genes. Nat. Rev. Genet. 12, 692–702 (2011).

  9. 9.

    Ranz, J. M. & Parsch, J. Newly evolved genes: moving from comparative genomics to functional studies in model systems. Bioessays 34, 477–483 (2012).

  10. 10.

    Rogers, R. L., Bedford, T., Lyons, A. M. & Hartl, D. L. Adaptive impact of the chimeric gene Quetzalcoatl in Drosophila melanogaster. Proc. Natl Acad. Sci. USA 107, 10943–10948 (2010).

  11. 11.

    Rogers, R. L. & Hartl, D. L. Chimeric genes as a source of rapid evolution in Drosophila melanogaster. Mol. Biol. Evol. 29, 517–529 (2012).

  12. 12.

    Blount, Z. D., Barrick, J. E., Davidson, C. J. & Lenski, R. E. Genomic analysis of a key innovation in an experimental Escherichia coli population. Nature 489, 513–518 (2012).

  13. 13.

    Annala, M. J., Parker, B. C., Zhang, W. & Nykter, M. Fusion genes and their discovery using high throughput sequencing. Cancer Lett. 340, 192–200 (2013).

  14. 14.

    Bashton, M. & Chothia, C. The generation of new protein functions by the combination of domains. Structure 15, 85–99 (2007).

  15. 15.

    Peisajovich, S. G., Garbarino, J. E., Wei, P. & Lim, W. A. Rapid diversification of cell signaling phenotypes by modular domain recombination. Science 328, 368–372 (2010).

  16. 16.

    Jin, J. et al. Eukaryotic protein domains as functional units of cellular evolution. Sci. Signal 2, ra76 (2009).

  17. 17.

    Pasek, S., Risler, J. L. & Brezellec, P. Gene fusion/fission is a major contributor to evolution of multi-domain bacterial proteins. Bioinformatics 22, 1418–1423 (2006).

  18. 18.

    Zhang, J. M., Dean, A. M., Brunet, F. & Long, M. Y. Evolving protein functional diversity in new genes of Drosophila. Proc. Natl Acad. Sci. USA 101, 16246–16250 (2004).

  19. 19.

    Dai, H. Z. et al. The evolution of courtship behaviors through the origination of a new gene in Drosophila. Proc. Natl Acad. Sci. USA 105, 7478–7483 (2008).

  20. 20.

    Yeh, S. D. et al. Functional evidence that a recently evolved Drosophila sperm-specific gene boosts sperm competition. Proc. Natl Acad. Sci. USA 109, 2043–2048 (2012).

  21. 21.

    Zhang, J. M., Yang, H. Y., Long, M. Y., Li, L. M. & Dean, A. M. Evolution of enzymatic activities of testis-specific short-chain dehydrogenase/reductase in Drosophila. J. Mol. Evol. 71, 241–249 (2010).

  22. 22.

    Wang, W., Brunet, F. G., Nevo, E. & Long, M. Origin of sphinx, a young chimeric RNA gene in Drosophila melanogaster. Proc. Natl Acad. Sci. USA 99, 4448–4453 (2002).

  23. 23.

    Long, M., Wang, W. & Zhang, J. M. Origin of new genes and source for N-terminal domain of the chimerical gene, jingwei, in Drosophila. Gene 238, 135–141 (1999).

  24. 24.

    Ranz, J. M., Ponce, A. R., Hartl, D. L. & Nurminsky, D. Origin and evolution of a new gene expressed in the Drosophila sperm axoneme. Genetica 118, 233–244 (2003).

  25. 25.

    Spiers, A. J., Kahn, S. G., Bohannon, J., Travisano, M. & Rainey, P. B. Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness. Genetics 161, 33–46 (2002).

  26. 26.

    Goymer, P. et al. Adaptive divergence in experimental populations of Pseudomonas fluorescens. II. Role of the GGDEF regulator WspR in evolution and development of the wrinkly spreader phenotype. Genetics 173, 515–526 (2006).

  27. 27.

    Bantinaki, E. et al. Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity. Genetics 176, 441–453 (2007).

  28. 28.

    McDonald, M. J., Gehrig, S. M., Meintjes, P. L., Zhang, X. X. & Rainey, P. B. Adaptive divergence in experimental populations of Pseudomonas fluorescens. IV. Genetic constraints guide evolutionary trajectories in a parallel adaptive radiation. Genetics 183, 1041–1053 (2009).

  29. 29.

    Lind, P. A., Farr, A. D. & Rainey, P. B. Experimental evolution reveals hidden diversity in evolutionary pathways. Elife 4, e07074 (2015).

  30. 30.

    Rainey, P. B. & Travisano, M. Adaptive radiation in a heterogeneous environment. Nature 394, 69–72 (1998).

  31. 31.

    Spiers, A. J., Bohannon, J., Gehrig, S. M. & Rainey, P. B. Biofilm formation at the air–liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol. Microbiol. 50, 15–27 (2003).

  32. 32.

    Jenal, U., Reinders, A. & Lori, C. Cyclic di-GMP: second messenger extraordinaire. Nat. Rev. Microbiol. 15, 271–284 (2017).

  33. 33.

    Amikam, D. & Galperin, M. Y. PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22, 3–6 (2006).

  34. 34.

    Ross, P. et al. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325, 279–281 (1987).

  35. 35.

    Romling, U., Galperin, M. Y. & Gomelsky, M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52 (2013).

  36. 36.

    Schirmer, T. & Jenal, U. Structural and mechanistic determinants of c-di-GMP signalling. Nat. Rev. Microbiol. 7, 724–735 (2009).

  37. 37.

    Winsor, G. L. et al. Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res. 39, D596–D600 (2011).

  38. 38.

    Zhu, K., Choi, K. H., Schweizer, H. P., Rock, C. O. & Zhang, Y. M. Two aerobic pathways for the formation of unsaturated fatty acids in Pseudomonas aeruginosa. Mol. Microbiol. 60, 260–273 (2006).

  39. 39.

    Malone, J. G. et al. The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain. Microbiology 153, 980–994 (2007).

  40. 40.

    Mitra, A., Kesarwani, A. K., Pal, D. & Nagaraja, V. WebGeSTer DB-a transcription terminator database. Nucleic Acids Res. 39, D129–D135 (2011).

  41. 41.

    O’Connor, J. R., Kuwada, N. J., Huangyutitham, V., Wiggins, P. A. & Harwood, C. S. Surface sensing and lateral subcellular localization of WspA, the receptor in a chemosensory-like system leading to c-di-GMP production. Mol. Microbiol. 86, 720–729 (2012).

  42. 42.

    Sturtevant, A. The effects of unequal crossing over at the Bar locus in Drosophila. Genetics 10, 117–147 (1925).

  43. 43.

    Haldane, J. The time of action of genes, and its bearing on some evolutionary problems. Am. Nat. 66, 5–24 (1932).

  44. 44.

    Bridges, C. The bar “gene” a duplication. Science 83, 210–211 (1936).

  45. 45.

    Lynch, M. & Conery, J. S. The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155 (2000).

  46. 46.

    Thomson, T. M. et al. Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene. Genome Res. 10, 1743–1756 (2000).

  47. 47.

    Marques, A. C., Vinckenbosh, N., Brawand, D. & Kaessmann, H. Functional diversification of duplicate genes through subcellular adaptation of encoded proteins. Genome Biol. 9, R54 (2008).

  48. 48.

    Wang, X. J., Huang, Y., Lavrov, D. V. & Gu, X. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications. BMC Evol. Biol. 9, 275 (2009).

  49. 49.

    Rogers, R. L., Bedford, T. & Hartl, D. L. Formation and longevity of chimeric and duplicate genes in Drosophila melanogaster. Genetics 181, 313–322 (2009).

  50. 50.

    Pawson, T. & Nash, P. Assembly of cell regulatory systems through protein interaction domains. Science 300, 445–452 (2003).

  51. 51.

    Huangyutitham, V., Guvener, Z. T. & Harwood, C. S. Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity. mBio 4, e00242-13 (2013).

  52. 52.

    Aldridge, P., Paul, R., Goymer, P., Rainey, P. & Jenal, U. Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol. Microbiol. 47, 1695–1708 (2003).

  53. 53.

    Paul, R. et al. Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization. J. Biol. Chem. 282, 29170–29177 (2007).

  54. 54.

    Massie, J. P. et al. Quantification of high-specificity cyclic diguanylate signaling. Proc. Natl Acad. Sci. USA 109, 12746–12751 (2012).

  55. 55.

    Blanka, A. et al. Constitutive production of c-di-GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane composition. Sci. Signal. 8, RA36 (2015).

  56. 56.

    Jenal, U. & Malone, J. Mechanisms of cyclic-di-GMP signaling in bacteria. Annu. Rev. Genet. 40, 385–407 (2006).

  57. 57.

    Galperin, M. Y. Bacterial signal transduction network in a genomic perspective. Environ. Microbiol. 6, 552–567 (2004).

  58. 58.

    Hammerschmidt, K., Rose, C. J., Kerr, B. & Rainey, P. B. Life cycles, fitness decoupling and the evolution of multicellularity. Nature 515, 75–79 (2014).

  59. 59.

    Lind, P. A., Farr, A. D. & Rainey, P. B. Evolutionary convergence in experimental Pseudomonas populations. ISME J. 11, 589–600 (2017).

  60. 60.

    Bailey, S. F. & Bataillon, T. Can the experimental evolution programme help us elucidate the genetic basis of adaptation in nature? Mol. Ecol. 25, 203–218 (2016).

  61. 61.

    Silby, M. W. et al. Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens. Genome Biol. 10, R51 (2009).

  62. 62.

    King, E. O., Ward, M. K. & Raney, D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44, 301–307 (1954).

  63. 63.

    Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557–580 (1983).

  64. 64.

    Bertani, G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293–300 (1951).

  65. 65.

    Sambrook, J. & Russell, D. W. Molecular Cloning. A Laboratory Manual 3rd edn (Cold Spring Harbour Laboratory Press, New York, 2001).

  66. 66.

    Rainey, P. B. Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ. Microbiol. 1, 243–257 (1999).

  67. 67.

    Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

  68. 68.

    Kitten, T., Kinscherf, T. G., McEvoy, J. L. & Willis, D. K. A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae. Mol. Microbiol. 28, 917–929 (1998).

  69. 69.

    Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Sayers, E. W. GenBank. Nucleic Acids Res. 44, D67–D72 (2016).

  70. 70.

    Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–2246 (1998).

  71. 71.

    Choi, K.-H. & Schweizer, H. P. mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa. Nat. Protocols 1, 153–161 (2006).

  72. 72.

    Livak, K. S. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408 (2001).

  73. 73.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  74. 74.

    Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

  75. 75.

    Van Steensel, B. et al. Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J. Cell Sci. 109, 787–792 (1996).

  76. 76.

    Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

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This work was supported in part by Marsden Fund Council from New Zealand Government funding (administered by the Royal Society of New Zealand) and the New Zealand Institute for Advanced Study. We thank J. Gallie and P. Lind for discussion and comments on the manuscript and H. Hendrickson for assistance with microscopy.

Author information

A.D.F. and P.B.R. conceived and designed the study. A.D.F. and P.R. acquired the data. All authors analysed and interpreted the data. A.D.F. and P.B.R. drafted and revised the paper.

Competing interests

The authors declare no competing financial interests.

Correspondence to Paul B. Rainey.

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Fig. 1: Numerous mutational routes activating cellulose over-production underpin the WS phenotype.
Fig. 2: Arrangement of pflu0184 and pflu0183 in the ancestral smooth genotype and following gene fusion.
Fig. 3: Transcription of fwsR from the fadA promoter is insufficient to cause FWS.
Fig. 4: The FWS phenotype requires translational fusion of the fadA and fwsR genes.
Fig. 5: Fluorescence microscopy depicting the distribution of GFP-tagged proteins encoded from the fwsR locus.
Fig. 6: Translational fusion of the transmembrane regions of mwsR to fwsR causes the FWS phenotype.