A novel system of bacterial cell division arrest implicated in horizontal transmission of an integrative and conjugative element

Autoři: Sotaro Takano aff001;  Kohei Fukuda aff002;  Akiko Koto aff001;  Ryo Miyazaki aff001
Působiště autorů: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan aff001;  Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Hamamatsu, Japan aff002;  Computational Bio Big Data Open Innovation Laboratory (CBBD-OIL), AIST, Tsukuba, Japan aff003;  Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan aff004
Vyšlo v časopise: A novel system of bacterial cell division arrest implicated in horizontal transmission of an integrative and conjugative element. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008445
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008445


Integrative and conjugative elements (ICEs) are widespread mobile DNA elements in the prokaryotic world. ICEs are usually retained within the bacterial chromosome, but can be excised and transferred from a donor to a new recipient cell, even of another species. Horizontal transmission of ICEclc, a prevalent ICE in proteobacteria, only occurs from developed specialized transfer competent (tc) cells in the donor population. tc cells become entirely dedicated to the ICE transmission at the cost of cell proliferation. The cell growth impairment is mediated by two ICEclc located genes, parA and shi, but the mechanistic and dynamic details of this process are unknown. To better understand the function of ParA and Shi, we followed their intracellular behavior from fluorescent protein fusions, and studied host cell division at single-cell level. Superresolution imaging revealed that ParA-mCherry colocalized with the host nucleoid while Shi-GFP was enriched at the membrane during the growth impairment. Despite being enriched at different cellular locations, the two proteins showed in vivo interactions, and mutations in the Walker A motif of ParA dislocalized both ParA and Shi. In addition, ParA mutations in the ATPase motif abolished the growth arrest on the host cell. Time-lapse microscopy revealed that ParA and Shi initially delay cell division, suggesting an extension of the S phase of cells, but eventually completely inhibit cell elongation. The parA-shi locus is highly conserved in other ICEclc-related elements, and expressing ParA-Shi from ICEclc in other proteobacterial species caused similar growth arrest, suggesting that the system functions similarly across hosts. The results of our study provide mechanistic insight into the novel and unique system on ICEs and help to understand such epistatic interaction between ICE genes and host physiology that entails efficient horizontal gene transfer.

Klíčová slova:

Adenosine triphosphatase – Cell cycle and cell division – Cell growth – DNA-binding proteins – Fluorescence imaging – Phylogenetic analysis – Pseudomonas putida – Horizontal gene transfer


1. Delavat F, Miyazaki R, Carraro N, Pradervand N, van der Meer JR. The hidden life of integrative and conjugative elements. FEMS Microbiol Rev. 2017. doi: 10.1093/femsre/fux008 28369623

2. Johnson CM, Grossman AD. Integrative and Conjugative Elements (ICEs): What They Do and How They Work. Annu Rev Genet. 2015;49:577–601. doi: 10.1146/annurev-genet-112414-055018 26473380

3. Wozniak RA, Waldor MK. Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow. Nat Rev Microbiol. 2010;8(8):552–563. doi: 10.1038/nrmicro2382 20601965

4. Bellanger X, Payot S, Leblond-Bourget N, Guedon G. Conjugative and mobilizable genomic islands in bacteria: evolution and diversity. FEMS Microbiol Rev. 2014;38(4):720–760. doi: 10.1111/1574-6976.12058 24372381

5. Guglielmini J, de la Cruz F, Rocha EP. Evolution of conjugation and type IV secretion systems. Mol Biol Evol. 2013;30(2):315–331. doi: 10.1093/molbev/mss221 22977114

6. Guglielmini J, Quintais L, Garcillan-Barcia MP, de la Cruz F, Rocha EP. The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation. PLoS Genet. 2011;7(8):e1002222. doi: 10.1371/journal.pgen.1002222 21876676

7. Roos TE, van Passel MW. A quantitative account of genomic island acquisitions in prokaryotes. BMC genomics. 2011;12:427. doi: 10.1186/1471-2164-12-427 21864345

8. Gaillard M, Pernet N, Vogne C, Hagenbuchle O, van der Meer JR. Host and invader impact of transfer of the clc genomic island into Pseudomonas aeruginosa PAO1. Proc Natl Acad Sci U S A. 2008;105(19):7058–7063. doi: 10.1073/pnas.0801269105 18448680

9. Miyazaki R, Yano H, Sentchilo V, van der Meer JR. Physiological and transcriptome changes induced by Pseudomonas putida acquisition of an integrative and conjugative element. Scientific reports. 2018;8(1):5550. doi: 10.1038/s41598-018-23858-6 29615803

10. Auchtung JM, Lee CA, Garrison KL, Grossman AD. Identification and characterization of the immunity repressor (ImmR) that controls the mobile genetic element ICEBs1 of Bacillus subtilis. Mol Microbiol. 2007;64(6):1515–1528. doi: 10.1111/j.1365-2958.2007.05748.x 17511812

11. Pradervand N, Sulser S, Delavat F, Miyazaki R, Lamas I, van der Meer JR. An operon of three transcriptional regulators controls horizontal gene transfer of the integrative and conjugative element ICEclc in Pseudomonas knackmussii B13. PLoS Genet. 2014;10(6):e1004441. doi: 10.1371/journal.pgen.1004441 24945944

12. Miyazaki R, Bertelli C, Benaglio P, Canton J, De Coi N, Gharib WH, et al. Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds. Environ Microbiol. 2015;17(1):91–104. doi: 10.1111/1462-2920.12498 24803113

13. Obi CC, Vayla S, de Gannes V, Berres ME, Walker J, Pavelec D, et al. The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2. Front Microbiol. 2018;9:1532. doi: 10.3389/fmicb.2018.01532 30050515

14. Reinhard F, Miyazaki R, Pradervand N, van der Meer JR. Cell differentiation to "mating bodies" induced by an integrating and conjugative element in free-living bacteria. Curr Biol. 2013;23(3):255–259. doi: 10.1016/j.cub.2012.12.025 23333318

15. Minoia M, Gaillard M, Reinhard F, Stojanov M, Sentchilo V, van der Meer JR. Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas. Proc Natl Acad Sci U S A. 2008;105(52):20792–20797. doi: 10.1073/pnas.0806164106 19098098

16. Miyazaki R, Minoia M, Pradervand N, Sulser S, Reinhard F, van der Meer JR. Cellular variability of RpoS expression underlies subpopulation activation of an integrative and conjugative element. PLoS Genet. 2012;8(7):e1002818. doi: 10.1371/journal.pgen.1002818 22807690

17. Delavat F, Mitri S, Pelet S, van der Meer JR. Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element. Proc Natl Acad Sci U S A. 2016;113(24):E3375–3383. doi: 10.1073/pnas.1604479113 27247406

18. Reinhard F, van der Meer JR. Life history analysis of integrative and conjugative element activation in growing microcolonies of Pseudomonas. J Bacteriol. 2014;196(7):1425–1434. doi: 10.1128/JB.01333-13 24464463

19. Vecchiarelli AG, Han YW, Tan X, Mizuuchi M, Ghirlando R, Biertumpfel C, et al. ATP control of dynamic P1 ParA-DNA interactions: a key role for the nucleoid in plasmid partition. Mol Microbiol. 2010;78(1):78–91. doi: 10.1111/j.1365-2958.2010.07314.x 20659294

20. Vecchiarelli AG, Havey JC, Ing LL, Wong EO, Waples WG, Funnell BE. Dissection of the ATPase active site of P1 ParA reveals multiple active forms essential for plasmid partition. J Biol Chem. 2013;288(24):17823–17831. doi: 10.1074/jbc.M113.469981 23632076

21. Yamaguchi Y, Inouye M. Regulation of growth and death in Escherichia coli by toxin-antitoxin systems. Nat Rev Microbiol. 2011;9(11):779–790. doi: 10.1038/nrmicro2651 21927020

22. Baltrus DA. Exploring the costs of horizontal gene transfer. Trends in ecology & evolution. 2013;28(8):489–495. doi: 10.1016/j.tree.2013.04.002 23706556

23. Banos RC, Vivero A, Aznar S, Garcia J, Pons M, Madrid C, et al. Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS. PLoS Genet. 2009;5(6):e1000513. doi: 10.1371/journal.pgen.1000513 19521501

24. Doyle M, Fookes M, Ivens A, Mangan MW, Wain J, Dorman CJ. An H-NS-like stealth protein aids horizontal DNA transmission in bacteria. Science. 2007;315(5809):251–252. doi: 10.1126/science.1137550 17218529

25. Mruk I, Kobayashi I. To be or not to be: regulation of restriction-modification systems and other toxin-antitoxin systems. Nucleic Acids Res. 2014;42(1):70–86. doi: 10.1093/nar/gkt711 23945938

26. Chen Y, Golding I, Sawai S, Guo L, Cox EC. Population fitness and the regulation of Escherichia coli genes by bacterial viruses. PLoS Biol. 2005;3(7):e229. doi: 10.1371/journal.pbio.0030229 15984911

27. Feiner R, Argov T, Rabinovich L, Sigal N, Borovok I, Herskovits AA. A new perspective on lysogeny: prophages as active regulatory switches of bacteria. Nat Rev Microbiol. 2015;13(10):641–650. doi: 10.1038/nrmicro3527 26373372

28. Paul JH. Prophages in marine bacteria: dangerous molecular time bombs or the key to survival in the seas? The ISME journal. 2008;2(6):579–589. doi: 10.1038/ismej.2008.35 18521076

29. Trempy JE, Kirby JE, Gottesman S. Alp Suppression of Lon—Dependence on the Slpa Gene. Journal of Bacteriology. 1994;176(7):2061–2067. doi: 10.1128/jb.176.7.2061-2067.1994 7511582

30. Kirby JE, Trempy JE, Gottesman S. Excision of a P4-Like Cryptic Prophage Leads to Alp Protease Expression in Escherichia-Coli. Journal of Bacteriology. 1994;176(7):2068–2081. doi: 10.1128/jb.176.7.2068-2081.1994 7511583

31. Ravatn R, Studer S, Zehnder AJ, van der Meer JR. Int-B13, an unusual site-specific recombinase of the bacteriophage P4 integrase family, is responsible for chromosomal insertion of the 105-kilobase clc element of Pseudomonas sp. Strain B13. J Bacteriol. 1998;180(21):5505–5514. 9791097

32. Baxter JC, Funnell BE. Plasmid Partition Mechanisms. Microbiol Spectr. 2014;2(6). doi: 10.1128/microbiolspec.PLAS-0023-2014 26104442

33. Brooks AC, Hwang LC. Reconstitutions of plasmid partition systems and their mechanisms. Plasmid. 2017;91:37–41. doi: 10.1016/j.plasmid.2017.03.004 28322855

34. Le Gall A, Cattoni DI, Guilhas B, Mathieu-Demaziere C, Oudjedi L, Fiche JB, et al. Bacterial partition complexes segregate within the volume of the nucleoid. Nature communications. 2016;7:12107. doi: 10.1038/ncomms12107 27377966

35. Vecchiarelli AG, Hwang LC, Mizuuchi K. Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proc Natl Acad Sci U S A. 2013;110(15):E1390–1397. doi: 10.1073/pnas.1302745110 23479605

36. Zhang H, Schumacher MA. Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation. Genes Dev. 2017;31(5):481–492. doi: 10.1101/gad.296319.117 28373206

37. Fung E, Bouet JY, Funnell BE. Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis. EMBO J. 2001;20(17):4901–4911. doi: 10.1093/emboj/20.17.4901 11532954

38. Jorgensen MG, Pandey DP, Jaskolska M, Gerdes K. HicA of Escherichia coli defines a novel family of translation-independent mRNA interferases in bacteria and archaea. J Bacteriol. 2009;191(4):1191–1199. doi: 10.1128/JB.01013-08 19060138

39. Murray H, Errington J. Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell. 2008;135(1):74–84. doi: 10.1016/j.cell.2008.07.044 18854156

40. Scholefield G, Whiting R, Errington J, Murray H. Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation. Mol Microbiol. 2011;79(4):1089–1100. doi: 10.1111/j.1365-2958.2010.07507.x 21235642

41. Sambrook J, Russell DW. Molecular Cloning: a laboratory manual. Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory Press; 2001.

42. Bachmann BJ. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972;36(4):525–557. 4568763

43. Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, et al. Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat Biotechnol. 2006;24(10):1257–1262. doi: 10.1038/nbt1244 16964242

44. McClure NC, Weightman AJ, Fry JC. Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit. Appl Environ Microbiol. 1989;55(10):2627–2634. 2604401

45. Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Kreig NR, et al. Manual of Methods for General Bacteriology. Washington, DC: American Society for Microbiology; 1981.

46. Miyazaki R, van der Meer JR. A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13. Mol Microbiol. 2011;79(3):743–758. doi: 10.1111/j.1365-2958.2010.07484.x 21255116

47. Jaspers MC, Meier C, Zehnder AJ, Harms H, van der Meer JR. Measuring mass transfer processes of octane with the help of an alkSalkB::gfp-tagged Escherichia coli. Environ Microbiol. 2001;3(8):512–524. 11578312

48. Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, et al. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods. 2005;2(6):443–448. doi: 10.1038/nmeth765 15908923

49. Sentchilo V, Czechowska K, Pradervand N, Minoia M, Miyazaki R, van der Meer JR. Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13. Mol Microbiol. 2009;72(5):1293–1306. doi: 10.1111/j.1365-2958.2009.06726.x 19432799

50. Young JW, Locke JC, Altinok A, Rosenfeld N, Bacarian T, Swain PS, et al. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat Protoc. 2011;7(1):80–88. doi: 10.1038/nprot.2011.432 22179594

51. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–1874. doi: 10.1093/molbev/msw054 27004904

52. Reinhard F, van der Meer JR. Improved statistical analysis of low abundance phenomena in bimodal bacterial populations. PLoS One. 2013;8(10):e78288. doi: 10.1371/journal.pone.0078288 24205184

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 10

Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se