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The SNAP hypothesis: Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation


Autoři: Gerrit Brandis aff001;  Diarmaid Hughes aff001
Působiště autorů: Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden aff001
Vyšlo v časopise: The SNAP hypothesis: Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation. PLoS Genet 16(3): e32767. doi:10.1371/journal.pgen.1008615
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008615

Souhrn

The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.

Klíčová slova:

Bacterial genomics – Ecological niches – Evolutionary genetics – Frameshift mutation – Genetic drift – Natural selection – Operons – Chromosomal duplications


Zdroje

1. Woese CR. Interpreting the universal phylogenetic tree. Proc Natl Acad Sci USA. 2000;97(15):8392–6. doi: 10.1073/pnas.97.15.8392 WOS:000088273900039. 10900003

2. Koonin EV. Comparative genomics, minimal gene-sets and the last universal common ancestor. Nat Rev Microbiol. 2003;1(2):127–36. doi: 10.1038/nrmicro751 WOS:000220402500014. 15035042

3. Koonin EV. Carl Woese's vision of cellular evolution and the domains of life. RNA Biol. 2014;11(3):197–204. doi: 10.4161/rna.27673 WOS:000334999500006. 24572480

4. Forterre P. The universal tree of life: an update. Front Microbiol. 2015;6. doi: 10.3389/fmicb.2015.00717 WOS:000358717800001. 26257711

5. Booth A, Mariscal C, Doolittle WF. The Modern Synthesis in the Light of Microbial Genomics. Annu Rev Microbiol. 2016;70:279–97. doi: 10.1146/annurev-micro-102215-095456 WOS:000383052200016. 27482743

6. Weiss MC, Preiner M, Xavier JC, Zimorski V, Martin WF. The last universal common ancestor between ancient Earth chemistry and the onset of genetics. PLoS Genet. 2018;14(8). doi: 10.1371/journal.pgen.1007518 WOS:000443389100009. 30114187

7. Touchon M, Rocha EPC. Coevolution of the Organization and Structure of Prokaryotic Genomes. CSH Perspect Biol. 2016;8(1). doi: 10.1101/cshperspect.a018168 WOS:000371181000007. 26729648

8. Tamames J. Evolution of gene order conservation in prokaryotes. Genome Biol. 2001;2(6). WOS:000207584100012.

9. Wachtershauser G. Towards a reconstruction of ancestral genomes by gene cluster alignment. Syst Appl Microbiol. 1998;21(4):473–7. WOS:000078011000001.

10. Coenye T, Vandamme P. Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes. Fems Microbiol Lett. 2005;242(1):117–26. doi: 10.1016/j.femsle.2004.10.050 WOS:000226264100016. 15621428

11. Barloy-Hubler F, Lelaure V, Galibert F. Ribosomal protein gene cluster analysis in eubacterium genomics: homology between Sinorhizobium meliloti strain 1021 and Bacillus subtilis. Nucleic Acids Res. 2001;29(13):2747–56. doi: 10.1093/nar/29.13.2747 PubMed Central PMCID: PMC55768. 11433019

12. Brocks JJ, Schaeffer P. Okenane, a biomarker for purple sulfur bacteria (Chromatiaceae), and other new carotenoid derivatives from the 1640 Ma Barney Creek Formation. Geochim Cosmochim Ac. 2008;72(5):1396–414. doi: 10.1016/j.gca.2007.12.006 WOS:000254198000010.

13. Marin J, Battistuzzi FU, Brown AC, Hedges SB. The Timetree of Prokaryotes: New Insights into Their Evolution and Speciation. Mol Biol Evol. 2017;34(2):437–46. doi: 10.1093/molbev/msw245 WOS:000396511300012. 27965376

14. Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA, Bowden SA. Biomarker evidence for green and purple sulphur bacteria in a stratified Palaeoproterozoic sea. Nature. 2005;437(7060):866–70. doi: 10.1038/nature04068 16208367.

15. McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature. 2001;413(6858):852–6. doi: 10.1038/35101614 11677609.

16. Brandis G, Cao S, Hughes D. Operon concatenation is an ancient feature that restricts the potential to rearrange bacterial chromosomes. Mol Biol Evol. 2019;36(9):1990–2000. doi: 10.1093/molbev/msz129 31132113.

17. Tamames J, Casari G, Ouzounis C, Valencia A. Conserved clusters of functionally related genes in two bacterial genomes. J Mol Evol. 1997;44(1):66–73. doi: 10.1007/pl00006122 WOS:A1997WD35900007. 9010137

18. Ermolaeva MD, White O, Salzberg SL. Prediction of operons in microbial genomes. Nucleic Acids Res. 2001;29(5):1216–21. doi: 10.1093/nar/29.5.1216 WOS:000167240500024. 11222772

19. Moreno-Hagelsieb G, Trevino V, Perez-Rueda E, Smith TF, Collado-Vides J. Transcription unit conservation in the three domains of life: a perspective from Escherichia coli. Trends Genet. 2001;17(4):175–7. doi: 10.1016/s0168-9525(01)02241-7 WOS:000168718300004. 11275307

20. Lawrence JG, Roth JR. Selfish operons: Horizontal transfer may drive the evolution of gene clusters. Genetics. 1996;143(4):1843–60. WOS:A1996VA24400030. 8844169

21. Itoh T, Takemoto K, Mori H, Gojobori T. Evolutionary instability of operon structures disclosed by sequence comparisons of complete microbial genomes. Mol Biol Evol. 1999;16(3):332–46. doi: 10.1093/oxfordjournals.molbev.a026114 WOS:000079160500003. 10331260

22. Ochman H, Groisman EA. The origin and evolution of species differences in Escherichia coli and Salmonella typhimurium. EXS. 1994;69:479–93. doi: 10.1007/978-3-0348-7527-1_27 7994120.

23. Tatusov RL, Mushegian AR, Bork P, Brown NP, Hayes WS, Borodovsky M, et al. Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli. Curr Biol. 1996;6(3):279–91. doi: 10.1016/s0960-9822(02)00478-5 WOS:A1996UC44000022. 8805245

24. Koonin EV, Mushegian AR, Rudd KE. Sequencing and analysis of bacterial genomes. Curr Biol. 1996;6(4):404–16. doi: 10.1016/s0960-9822(02)00508-0 WOS:A1996UH68400022. 8723345

25. Rocha EPC. Inference and analysis of the relative stability of bacterial chromosomes. Mol Biol Evol. 2006;23(3):513–22. doi: 10.1093/molbev/msj052 WOS:000235610300005. 16280545

26. Brandis G, Cao S, Hughes D. Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella. Mol Microbiol. 2018: doi: 10.1111/mmi.13959 29603442.

27. Hughes D. Co-evolution of the tuf genes links gene conversion with the generation of chromosomal inversions. J Mol Biol. 2000;297(2):355–64. doi: 10.1006/jmbi.2000.3587 10715206.

28. Hughes D. Evaluating genome dynamics: the constraints on rearrangements within bacterial genomes. Genome Biol. 2000;1(6). WOS:000207583400002.

29. Hughes D. Impact of homologous recombination on genome organization and stability. In: Charlebois RL, editor. Organization of the prokaryotic genome. Washington DC, USA: ASM Press; 1999. p. 109–28.

30. Anderson P, Roth J. Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc Natl Acad Sci U S A. 1981;78(5):3113–7. doi: 10.1073/pnas.78.5.3113 6789329.

31. Straus DS, Hoffmann GR. Selection for a large genetic duplication in Salmonella typhimurium. Genetics. 1975;80(2):227–37. 1093939.

32. Sonti RV, Roth JR. Role of gene duplications in the adaptation of Salmonella typhimurium to growth on limiting carbon sources. Genetics. 1989;123(1):19–28. 2680755.

33. Andersson DI, Hughes D. Gene amplification and adaptive evolution in bacteria. Annu Rev Genet. 2009;43:167–95. doi: 10.1146/annurev-genet-102108-134805 19686082.

34. Sandegren L, Andersson DI. Bacterial gene amplification: implications for the evolution of antibiotic resistance. Nat Rev Microbiol. 2009;7(8):578–88. doi: 10.1038/nrmicro2174 19609259.

35. Sun S, Berg OG, Roth JR, Andersson DI. Contribution of gene amplification to evolution of increased antibiotic resistance in Salmonella typhimurium. Genetics. 2009;182(4):1183–95. doi: 10.1534/genetics.109.103028 19474201; PubMed Central PMCID: PMC2728858.

36. Anderson RP, Roth JR. Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol. 1977;31:473–505. doi: 10.1146/annurev.mi.31.100177.002353 334045.

37. Roller BR, Stoddard SF, Schmidt TM. Exploiting rRNA operon copy number to investigate bacterial reproductive strategies. Nat Microbiol. 2016;1(11):16160. doi: 10.1038/nmicrobiol.2016.160 27617693; PubMed Central PMCID: PMC5061577.

38. Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol. 2000;66(4):1328–33. doi: 10.1128/aem.66.4.1328-1333.2000 10742207; PubMed Central PMCID: PMC91988.

39. Gyorfy Z, Draskovits G, Vernyik V, Blattner FF, Gaal T, Posfai G. Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number. Nucleic Acids Res. 2015;43(3):1783–94. doi: 10.1093/nar/gkv040 25618851; PubMed Central PMCID: PMC4330394.

40. Valdivia-Anistro JA, Eguiarte-Fruns LE, Delgado-Sapien G, Marquez-Zacarias P, Gasca-Pineda J, Learned J, et al. Variability of rRNA Operon Copy Number and Growth Rate Dynamics of Bacillus Isolated from an Extremely Oligotrophic Aquatic Ecosystem. Front Microbiol. 2015;6:1486. doi: 10.3389/fmicb.2015.01486 26779143; PubMed Central PMCID: PMC4700252.

41. Yano K, Masuda K, Akanuma G, Wada T, Matsumoto T, Shiwa Y, et al. Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon. Microbiol. 2016;162(1):35–45. doi: 10.1099/mic.0.000207 26518335.

42. Kacar B, Garmendia E, Tuncbag N, Andersson DI, Hughes D. Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs. mBio. 2017;8(4):e01276–17. ARTN e01276-17 doi: 10.1128/mBio.01276-17 WOS:000409384300045. 28851849

43. Garmendia E, Brandis G, Hughes D. Transcriptional Regulation Buffers Gene Dosage Effects on a Highly Expressed Operon in Salmonella. mBio. 2018;9(5). doi: 10.1128/mBio.01446-18 30206172; PubMed Central PMCID: PMC6134099.

44. Tubulekas I, Hughes D. Growth and translation elongation rate are sensitive to the concentration of EF-Tu. Mol Microbiol. 1993a;8(4):761–70. doi: 10.1111/j.1365-2958.1993.tb01619.x 8332067.

45. Adler M, Anjum M, Berg OG, Andersson DI, Sandegren L. High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms. Mol Biol Evol. 2014;31(6):1526–35. doi: 10.1093/molbev/msu111 24659815.

46. Andersson DI, Hughes D, Roth JR. The origin of mutants under selection: Interactions of mutation, growth and selection. 2011. In: EcoSal-Escherichia coli and Salmonella: Cellular and Molecular Biology [Internet]. Washington, DC.: ASM Press. Available from: http://www.ecosal.org.

47. Praski Alzrigat L, Huseby DL, Brandis G, Hughes D. Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli. J Antimicrob Chemother. 2017;72(11):3016–24. doi: 10.1093/jac/dkx270 28962020; PubMed Central PMCID: PMC5890708.

48. Goodall ECA, Robinson A, Johnston IG, Jabbari S, Turner KA, Cunningham AF, et al. The Essential Genome of Escherichia coli K-12. mBio. 2018;9(1). doi: 10.1128/mBio.02096-17 29463657; PubMed Central PMCID: PMC5821084.

49. Chaudhuri RR, Morgan E, Peters SE, Pleasance SJ, Hudson DL, Davies HM, et al. Comprehensive assignment of roles for Salmonella typhimurium genes in intestinal colonization of food-producing animals. PLoS Genet. 2013;9(4):e1003456. doi: 10.1371/journal.pgen.1003456 23637626; PubMed Central PMCID: PMC3630085.

50. Vohra P, Chaudhuri RR, Mayho M, Vrettou C, Chintoan-Uta C, Thomson NR, et al. Retrospective application of transposon-directed insertion-site sequencing to investigate niche-specific virulence of Salmonella Typhimurium in cattle. BMC Genomics. 2019;20(1):20. doi: 10.1186/s12864-018-5319-0 30621582; PubMed Central PMCID: PMC6325888.

51. Lawley TD, Chan K, Thompson LJ, Kim CC, Govoni GR, Monack DM. Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse. PLoS Pathog. 2006;2(2):e11. doi: 10.1371/journal.ppat.0020011 16518469; PubMed Central PMCID: PMC1383486.

52. Campo N, Dias MJ, Daveran-Mingot ML, Ritzenthaler P, Le Bourgeois P. Chromosomal constraints in Gram-positive bacteria revealed by artificial inversions. Mol Microbiol. 2004;51(2):511–22. doi: 10.1046/j.1365-2958.2003.03847.x 14756790.

53. Liu GR, Liu WQ, Johnston RN, Sanderson KE, Li SX, Liu SL. Genome plasticity and ori-ter rebalancing in Salmonella typhi. Mol Biol Evol. 2006;23(2):365–71. doi: 10.1093/molbev/msj042 16237205.

54. Savic DJ, Nguyen SV, McCullor K, McShan WM. Biological impact of a large-scale genomic inversion that grossly disrupts the relative positions of the origin and terminus loci of the Streptococcus pyogenes chromosome. J Bacteriol. 2019;201(17). doi: 10.1128/JB.00090-19 31235514; PubMed Central PMCID: PMC6689312.

55. Lesterlin C, Pages C, Dubarry N, Dasgupta S, Cornet F. Asymmetry of chromosome replichores renders the DNA translocase activity of FtsK essential for cell division and cell shape maintenance in Escherichia coli. PLoS Genet. 2008;4(12):e1000288. doi: 10.1371/journal.pgen.1000288 19057667; PubMed Central PMCID: PMC2585057.

56. Darling AE, Miklos I, Ragan MA. Dynamics of genome rearrangement in bacterial populations. PLoS Genet. 2008;4(7):e1000128. doi: 10.1371/journal.pgen.1000128 18650965; PubMed Central PMCID: PMC2483231.

57. Esnault E, Valens M, Espeli O, Boccard F. Chromosome structuring limits genome plasticity in Escherichia coli. PLoS Genet. 2007;3(12):e226. doi: 10.1371/journal.pgen.0030226 18085828; PubMed Central PMCID: PMC2134941.

58. Taddei F, Radman M, Maynard-Smith J, Toupance B, Gouyon PH, Godelle B. Role of mutator alleles in adaptive evolution. Nature. 1997;387(6634):700–2. doi: 10.1038/42696 9192893.

59. LeClerc JE, Li B, Payne WL, Cebula TA. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science. 1996;274(5290):1208–11. doi: 10.1126/science.274.5290.1208 8895473.

60. Gross MD, Siegel EC. Incidence of mutator strains in Escherichia coli and coliforms in nature. Mutat Res. 1981;91(2):107–10. doi: 10.1016/0165-7992(81)90081-6 7019693.

61. Ellington MJ, Livermore DM, Pitt TL, Hall LM, Woodford N. Mutators among CTX-M beta-lactamase-producing Escherichia coli and risk for the emergence of fosfomycin resistance. J Antimicrob Chemother. 2006;58(4):848–52. doi: 10.1093/jac/dkl315 16891630.

62. Marinus MG. DNA Mismatch Repair. EcoSal Plus. 2012;5(1). doi: 10.1128/ecosalplus.7.2.5 26442827; PubMed Central PMCID: PMC4231543.

63. Petit MA, Dimpfl J, Radman M, Echols H. Control of large chromosomal duplications in Escherichia coli by the mismatch repair system. Genetics. 1991;129(2):327–32. 1743481; PubMed Central PMCID: PMC1204626.

64. Bzymek M, Saveson CJ, Feschenko VV, Lovett ST. Slipped misalignment mechanisms of deletion formation: in vivo susceptibility to nucleases. J Bacteriol. 1999;181(2):477–82. 9882661; PubMed Central PMCID: PMC93401.

65. Xie G, Keyhani NO, Bonner CA, Jensen RA. Ancient origin of the tryptophan operon and the dynamics of evolutionary change. Microbiol Mol Biol Rev. 2003;67(3):303–42. doi: 10.1128/MMBR.67.3.303-342.2003 12966138.

66. Fondi M, Emiliani G, Fani R. Origin and evolution of operons and metabolic pathways. Res Microbiol. 2009;160(7):502–12. doi: 10.1016/j.resmic.2009.05.001 19465116.

67. Reams AB, Neidle EL. Selection for gene clustering by tandem duplication. Annu Rev Microbiol. 2004;58:119–42. doi: 10.1146/annurev.micro.58.030603.123806 15487932.

68. Romero D, Palacios R. Gene amplification and genomic plasticity in prokaryotes. Annu Rev Genet. 1997;31:91–111. doi: 10.1146/annurev.genet.31.1.91 9442891.

69. Hooper SD, Berg OG. On the nature of gene innovation: duplication patterns in microbial genomes. Mol Biol Evol. 2003;20(6):945–54. doi: 10.1093/molbev/msg101 12716994.

70. Jordan IK, Makarova KS, Spouge JL, Wolf YI, Koonin EV. Lineage-specific gene expansions in bacterial and archaeal genomes. Genome Res. 2001;11(4):555–65. doi: 10.1101/gr.166001 11282971; PubMed Central PMCID: PMC311027.

71. Bosserman RE, Thompson CR, Nicholson KR, Champion PA. Esx paralogs are functionally equivalent to ESX-1 proteins but are dispensable for virulence in Mycobacterium marinum. J Bacteriol. 2018;200(11). doi: 10.1128/JB.00726-17 29555701; PubMed Central PMCID: PMC5952400.

72. Saier MH Jr., Paulsen IT, Sliwinski MK, Pao SS, Skurray RA, Nikaido H. Evolutionary origins of multidrug and drug-specific efflux pumps in bacteria. FASEB J. 1998;12(3):265–74. doi: 10.1096/fasebj.12.3.265 9506471.

73. Perrin E, Fondi M, Bosi E, Mengoni A, Buroni S, Scoffone VC, et al. Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance. BMC Genomics. 2017;18(1):834. doi: 10.1186/s12864-017-4222-4 29084524; PubMed Central PMCID: PMC5663151.

74. Shah S, Cannon JR, Fenselau C, Briken V. A duplicated ESAT-6 region of ESX-5 is involved in protein export and virulence of Mycobacteria. Infect Immun. 2015;83(11):4349–61. doi: 10.1128/IAI.00827-15 26303392; PubMed Central PMCID: PMC4598393.

75. Brosch R, Gordon SV, Buchrieser C, Pym AS, Garnier T, Cole ST. Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast. 2000;17(2):111–23. doi: 10.1002/1097-0061(20000630)17:2<111::AID-YEA17>3.0.CO;2-G 10900457; PubMed Central PMCID: PMC2448323.

76. Galamba A, Soetaert K, Wang XM, De Bruyn J, Jacobs P, Content J. Disruption of adhC reveals a large duplication in the Mycobacterium smegmatis mc(2)155 genome. Microbiol. 2001;147(Pt 12):3281–94. doi: 10.1099/00221287-147-12-3281 11739760.

77. Domenech P, Rog A, Moolji JU, Radomski N, Fallow A, Leon-Solis L, et al. Origins of a 350-kilobase genomic duplication in Mycobacterium tuberculosis and its impact on virulence. Infect Immun. 2014;82(7):2902–12. doi: 10.1128/IAI.01791-14 24778110; PubMed Central PMCID: PMC4097636.

78. Andersson SG, Zomorodipour A, Winkler HH, Kurland CG. Unusual organization of the rRNA genes in Rickettsia prowazekii. J Bacteriol. 1995;177(14):4171–5. doi: 10.1128/jb.177.14.4171-4175.1995 7608097; PubMed Central PMCID: PMC177156.

79. Bercovier H, Kafri O, Sela S. Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. Biochem Biophys Res Commun. 1986;136(3):1136–41. doi: 10.1016/0006-291x(86)90452-3 3013168.

80. Monod J. The Growth of Bacterial Cultures. Annu Rev Microbiol. 1949;3:371–94. doi: 10.1146/annurev.mi.03.100149.002103 WOS:A1949XS94800016.


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