1887

Abstract

Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance () and green fluorescence (), or lactose metabolism (), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into or strains via electroporation or natural transformation. Recombinants (Gm or Lac) were obtained at frequencies ranging from 10 to 10 c.f.u. (µg DNA). Cassettes were integrated by site-specific recombination at the integron site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of -containing recombinants revealed that expression levels from the integron-associated promoter P were five- to 10-fold higher in the plasmid-borne integron In compared with the chromosomal integrons. Integration of cassettes into integrons allowed the bacteria to grow on lactose, and the gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

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2011-12-01
2020-04-07
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References

  1. Baharoglu Z., Bikard D., Mazel D.. ( 2010;). Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation. PLoS Genet6:e1001165 [CrossRef][PubMed]
    [Google Scholar]
  2. Bikard D., Julié-Galau S., Cambray G., Mazel D.. ( 2010;). The synthetic integron: an in vivo genetic shuffling device. Nucleic Acids Res38:e153 [CrossRef][PubMed]
    [Google Scholar]
  3. Biskri L., Bouvier M., Guérout A. M., Boisnard S., Mazel D.. ( 2005;). Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities. J Bacteriol187:1740–1750 [CrossRef][PubMed]
    [Google Scholar]
  4. Blanco L., Bernad A., Lázaro J. M., Martín G., Garmendia C., Salas M.. ( 1989;). Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem264:8935–8940[PubMed]
    [Google Scholar]
  5. Boucher Y., Nesbø C. L., Joss M. J., Robinson A., Mabbutt B. C., Gillings M. R., Doolittle W. F., Stokes H. W.. ( 2006;). Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio . BMC Evol Biol6:3–17 [CrossRef][PubMed]
    [Google Scholar]
  6. Boucher Y., Labbate M., Koenig J. E., Stokes H. W.. ( 2007a;). Integrons: mobilizable platforms that promote genetic diversity in bacteria. Trends Microbiol15:301–309 [CrossRef][PubMed]
    [Google Scholar]
  7. Boucher Y., Labbate M., Koenig J. E., Stokes H. W.. ( 2007b;). Integrons: mobilizable platforms that promote genetic diversity in bacteria. Trends Microbiol15:301–309 [CrossRef][PubMed]
    [Google Scholar]
  8. Bouvier M., Demarre G., Mazel D.. ( 2005;). Integron cassette insertion: a recombination process involving a folded single strand substrate. EMBO J24:4356–4367 [CrossRef][PubMed]
    [Google Scholar]
  9. Carlson C. A., Pierson L. S., Rosen J. J., Ingraham J. L.. ( 1983;). Pseudomonas stutzeri and related species undergo natural transformation. J Bacteriol153:93–99[PubMed]
    [Google Scholar]
  10. Coleman N. V., Holmes A. J.. ( 2005;). The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes. Microbiology151:1853–1864 [CrossRef][PubMed]
    [Google Scholar]
  11. Coleman N. V., Mattes T. E., Gossett J. M., Spain J. C.. ( 2002;). Biodegradation of cis-dichloroethene as the sole carbon source by a β-proteobacterium. Appl Environ Microbiol68:2726–2730 [CrossRef][PubMed]
    [Google Scholar]
  12. Collis C. M., Hall R. M.. ( 1992;). Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol174:1574–1585[PubMed]
    [Google Scholar]
  13. Collis C. M., Grammaticopoulos G., Briton J., Stokes H. W., Hall R. M.. ( 1993;). Site-specific insertion of gene cassettes into integrons. Mol Microbiol9:41–52 [CrossRef][PubMed]
    [Google Scholar]
  14. Collis C. M., Recchia G. D., Kim M. J., Stokes H. W., Hall R. M.. ( 2001;). Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1. J Bacteriol183:2535–2542 [CrossRef][PubMed]
    [Google Scholar]
  15. Collis C. M., Kim M. J., Stokes H. W., Hall R. M.. ( 2002;). Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site. Mol Microbiol46:1415–1427 [CrossRef][PubMed]
    [Google Scholar]
  16. Cost G. J., Cozzarelli N. R.. ( 2007;). Directed assembly of DNA molecules via simultaneous ligation and digestion. Biotechniques42:84–89 [CrossRef][PubMed]
    [Google Scholar]
  17. Crespo O., Catalano M., Piñeiro S., Matteo M., Leanza A., Centrón D.. ( 2005;). Tn7 distribution in Helicobacter pylori: a selective paradox. Int J Antimicrob Agents25:341–344 [CrossRef][PubMed]
    [Google Scholar]
  18. Datta N., Hedges R. W.. ( 1972;). Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae . J Gen Microbiol72:349–355[PubMed][CrossRef]
    [Google Scholar]
  19. Davies J., Davies D.. ( 2010;). Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev74:417–433 [CrossRef][PubMed]
    [Google Scholar]
  20. Dean F. B., Nelson J. R., Giesler T. L., Lasken R. S.. ( 2001;). Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res11:1095–1099 [CrossRef][PubMed]
    [Google Scholar]
  21. Demarre G., Frumerie C., Gopaul D. N., Mazel D.. ( 2007;). Identification of key structural determinants of the IntI1 integron integrase that influence attC×attI1 recombination efficiency. Nucleic Acids Res35:6475–6489 [CrossRef][PubMed]
    [Google Scholar]
  22. Drouin F., Mélançon J., Roy P. H.. ( 2002;). The IntI-like tyrosine recombinase of Shewanella oneidensis is active as an integron integrase. J Bacteriol184:1811–1815 [CrossRef][PubMed]
    [Google Scholar]
  23. Dubois V., Debreyer C., Litvak S., Quentin C., Parissi V.. ( 2007;). A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity. PLoS ONE2:e1315 [CrossRef][PubMed]
    [Google Scholar]
  24. Elsaied H., Stokes H. W., Nakamura T., Kitamura K., Fuse H., Maruyama A.. ( 2007;). Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents. Environ Microbiol9:2298–2312 [CrossRef][PubMed]
    [Google Scholar]
  25. Etchuuya R., Ito M., Kitano S., Shigi F., Sobue R., Maeda S.. ( 2011;). Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone. PLoS ONE6:e16355 [CrossRef][PubMed]
    [Google Scholar]
  26. Frost L. S., Leplae R., Summers A. O., Toussaint A.. ( 2005;). Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol3:722–732 [CrossRef][PubMed]
    [Google Scholar]
  27. Gillings M. R., Holley M. P., Stokes H. W., Holmes A. J.. ( 2005;). Integrons in Xanthomonas: a source of species genome diversity. Proc Natl Acad Sci U S A102:4419–4424 [CrossRef][PubMed]
    [Google Scholar]
  28. Gillings M., Boucher Y., Labbate M., Holmes A., Krishnan S., Holley M., Stokes H. W.. ( 2008;). The evolution of class 1 integrons and the rise of antibiotic resistance. J Bacteriol190:5095–5100 [CrossRef][PubMed]
    [Google Scholar]
  29. Guerin E., Cambray G., Sanchez-Alberola N., Campoy S., Erill I., Da Re S., Gonzalez-Zorn B., Barbé J., Ploy M. C., Mazel D.. ( 2009;). The SOS response controls integron recombination. Science324:1034 [CrossRef][PubMed]
    [Google Scholar]
  30. Hall R. M., Collis C. M.. ( 1998;). Antibiotic resistance in Gram-negative bacteria: the role of gene cassettes and integrons. Drug Resist Updat1:109–119 [CrossRef][PubMed]
    [Google Scholar]
  31. Hall R. M., Brown H. J., Brookes D. E., Stokes H. W.. ( 1994;). Integrons found in different locations have identical 5′ ends but variable 3′ ends. J Bacteriol176:6286–6294[PubMed]
    [Google Scholar]
  32. Holmes A. J., Gillings M. R., Nield B. S., Mabbutt B. C., Nevalainen K. M. H., Stokes H. W.. ( 2003a;). The gene cassette metagenome is a basic resource for bacterial genome evolution. Environ Microbiol5:383–394 [CrossRef][PubMed]
    [Google Scholar]
  33. Holmes A. J., Holley M. P., Mahon A., Nield B., Gillings M., Stokes H. W.. ( 2003b;). Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil. J Bacteriol185:918–928 [CrossRef][PubMed]
    [Google Scholar]
  34. Johnsborg O., Eldholm V., Håvarstein L. S.. ( 2007;). Natural genetic transformation: prevalence, mechanisms and function. Res Microbiol158:767–778 [CrossRef][PubMed]
    [Google Scholar]
  35. Jové T., Da Re S., Denis F., Mazel D., Ploy M. C.. ( 2010;). Inverse correlation between promoter strength and excision activity in class 1 integrons. PLoS Genet6:e1000793[CrossRef]
    [Google Scholar]
  36. Koenig J. E., Sharp C., Dlutek M., Curtis B., Joss M., Boucher Y., Doolittle W. F.. ( 2009;). Integron gene cassettes and degradation of compounds associated with industrial waste: the case of the Sydney tar ponds. PLoS ONE4:e5276 [CrossRef][PubMed]
    [Google Scholar]
  37. Koenig J. E., Bourne D. G., Curtis B., Dlutek M., Stokes H. W., Doolittle W. F., Boucher Y.. ( 2011;). Coral-mucus-associated Vibrio integrons in the Great Barrier Reef: genomic hotspots for environmental adaptation. ISME J5:962–972 [CrossRef][PubMed]
    [Google Scholar]
  38. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M. II, Peterson K. M.. ( 1995;). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene166:175–176 [CrossRef][PubMed]
    [Google Scholar]
  39. Lalucat J., Bennasar A., Bosch R., García-Valdés E., Palleroni N. J.. ( 2006;). Biology of Pseudomonas stutzeri. . Microbiol Mol Biol Rev70:510–547[CrossRef]
    [Google Scholar]
  40. Léon G., Roy P. H.. ( 2003;). Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea . J Bacteriol185:2036–2041 [CrossRef][PubMed]
    [Google Scholar]
  41. Lévesque C., Brassard S., Lapointe J., Roy P. H.. ( 1994;). Diversity and relative strength of tandem promoters for the antibiotic-resistance genes of several integrons. Gene142:49–54 [CrossRef][PubMed]
    [Google Scholar]
  42. Lewis T. A., Cortese M. S., Sebat J. L., Green T. L., Lee C. H., Crawford R. L.. ( 2000;). A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid). Environ Microbiol2:407–416 [CrossRef][PubMed]
    [Google Scholar]
  43. Loot C., Bikard D., Rachlin A., Mazel D.. ( 2010;). Cellular pathways controlling integron cassette site folding. EMBO J29:2623–2634[CrossRef]
    [Google Scholar]
  44. Lorenz M. G., Sikorski J.. ( 2000;). The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri . Microbiology146:3081–3090[PubMed]
    [Google Scholar]
  45. Martinez E., de la Cruz F.. ( 1988;). Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet211:320–325 [CrossRef][PubMed]
    [Google Scholar]
  46. Martinez E., de la Cruz F.. ( 1990;). Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J9:1275–1281[PubMed]
    [Google Scholar]
  47. Mazel D.. ( 2006;). Integrons: agents of bacterial evolution. Nat Rev Microbiol4:608–620 [CrossRef][PubMed]
    [Google Scholar]
  48. Meibom K. L., Blokesch M., Dolganov N. A., Wu C. Y., Schoolnik G. K.. ( 2005;). Chitin induces natural competence in Vibrio cholerae . Science310:1824–1827 [CrossRef][PubMed]
    [Google Scholar]
  49. Nield B. S., Holmes A. J., Gillings M. R., Recchia G. D., Mabbutt B. C., Nevalainen K. M., Stokes H. W.. ( 2001;). Recovery of new integron classes from environmental DNA. FEMS Microbiol Lett195:59–65 [CrossRef][PubMed]
    [Google Scholar]
  50. Nunes-Düby S. E., Kwon H. J., Tirumalai R. S., Ellenberger T., Landy A.. ( 1998;). Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res26:391–406 [CrossRef][PubMed]
    [Google Scholar]
  51. O’Halloran F., Lucey B., Cryan B., Buckley T., Fanning S.. ( 2004;). Molecular characterization of class 1 integrons from Irish thermophilic Campylobacter spp. J Antimicrob Chemother53:952–957 [CrossRef][PubMed]
    [Google Scholar]
  52. Pries A., Steinbuchel A., Schlegel H. G.. ( 1990;). Lactose-utilizing and galactose-utilizing strains of poly(hydroxyalkanoic acid)-accumulating Alcaligenes eutrophus and Pseudomonas saccharophila obtained by recombinant-DNA technology. Appl Microbiol Biotechnol33:410–417 [CrossRef]
    [Google Scholar]
  53. Recchia G. D., Hall R. M.. ( 1995;). Gene cassettes: a new class of mobile element. Microbiology141:3015–3027 [CrossRef][PubMed]
    [Google Scholar]
  54. Recchia G. D., Hall R. M.. ( 1997;). Origins of the mobile gene cassettes found in integrons. Trends Microbiol5:389–394 [CrossRef][PubMed]
    [Google Scholar]
  55. Rosewarne C. P., Pettigrove V., Stokes H. W., Parsons Y. M.. ( 2010;). Class 1 integrons in benthic bacterial communities: abundance, association with Tn402-like transposition modules and evidence for coselection with heavy-metal resistance. FEMS Microbiol Ecol72:35–46 [CrossRef][PubMed]
    [Google Scholar]
  56. Rosselló-Mora R. A., Lalucat J., Dott W., Kampfer P.. ( 1994;). Biochemical and chemotaxonomic characterization of Pseudomonas stutzeri genomovars. J Appl Bacteriol76:226–233 [CrossRef]
    [Google Scholar]
  57. Rowe-Magnus D. A., Mazel D.. ( 2001;). Integrons: natural tools for bacterial genome evolution. Curr Opin Microbiol4:565–569 [CrossRef][PubMed]
    [Google Scholar]
  58. Rowe-Magnus D. A., Guerout A. M., Biskri L., Bouige P., Mazel D.. ( 2003;). Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res13:428–442 [CrossRef][PubMed]
    [Google Scholar]
  59. Sambrook J., Russell D. W.. ( 2001;). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  60. Schlüter A., Szczepanowski R., Pühler A., Top E. M.. ( 2007;). Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol Rev31:449–477 [CrossRef][PubMed]
    [Google Scholar]
  61. Shearer J. E. S., Summers A. O.. ( 2009;). Intracellular steady-state concentration of integron recombination products varies with integrase level and growth phase. J Mol Biol386:316–331 [CrossRef][PubMed]
    [Google Scholar]
  62. Shim H., Ryoo D., Barbieri P., Wood T. K.. ( 2001;). Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Appl Microbiol Biotechnol56:265–269 [CrossRef][PubMed]
    [Google Scholar]
  63. Sikorski J., Rosselló-Mora R., Lorenz M. G.. ( 1999;). Analysis of genotypic diversity and relationships among Pseudomonas stutzeri strains by PCR-based genomic fingerprinting and multilocus enzyme electrophoresis. Syst Appl Microbiol22:393–402[PubMed][CrossRef]
    [Google Scholar]
  64. Sikorski J., Teschner N., Wackernagel W.. ( 2002;). Highly different levels of natural transformation are associated with genomic subgroups within a local population of Pseudomonas stutzeri from soil. Appl Environ Microbiol68:865–873 [CrossRef][PubMed]
    [Google Scholar]
  65. Stokes H. W., Hall R. M.. ( 1989;). A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons. Mol Microbiol3:1669–1683 [CrossRef][PubMed]
    [Google Scholar]
  66. Stokes H. W., O’Gorman D. B., Recchia G. D., Parsekhian M., Hall R. M.. ( 1997;). Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Mol Microbiol26:731–745 [CrossRef][PubMed]
    [Google Scholar]
  67. Tetu S.. ( 2007;). Gene Cassettes as an Evolutionary Resource for Pseudomonas Species PhD thesis: University of Sydney;
    [Google Scholar]
  68. Utsumi R., Noda M., Kawamukai M., Komano T.. ( 1988;). Isolation of a cAMP-requiring mutant in Escherichia-coli K12: evidence of growth-regulation via N-acetylglucosamine metabolism controlled by cAMP. FEMS Microbiol Lett50:217–221 [CrossRef]
    [Google Scholar]
  69. Vaisvila R., Morgan R. D., Posfai J., Raleigh E. A.. ( 2001;). Discovery and distribution of super-integrons among pseudomonads. Mol Microbiol42:587–601 [CrossRef][PubMed]
    [Google Scholar]
  70. Ward J. M., Grinsted J.. ( 1982;). Physical and genetic analysis of the Inc-W group plasmids R388, Sa, and R7K. Plasmid7:239–250 [CrossRef][PubMed]
    [Google Scholar]
  71. Wei Q., Jiang X., Li M., Chen X., Li G., Li R., Lu Y.. ( 2011;). Transcription of integron-harboured gene cassette impacts integration efficiency in class 1 integron. Mol Microbiol80:1326–1336 [CrossRef][PubMed]
    [Google Scholar]
  72. Wilson N. L.. ( 2007;). Integrons in Pseudomonads are Associated with Hotspots of Genomic Diversity PhD thesis: University of Sydney;
    [Google Scholar]
  73. Wright M. S., Baker-Austin C., Lindell A. H., Stepanauskas R., Stokes H. W., McArthur J. V.. ( 2008;). Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities. ISME J2:417–428 [CrossRef][PubMed]
    [Google Scholar]
  74. Yang Z. H., Jiang X. F., Wei Q. H., Chen N., Lu Y.. ( 2009;). A novel and rapid method for determining integration frequency catalyzed by integron integrase intI1. J Microbiol Methods76:97–100 [CrossRef][PubMed]
    [Google Scholar]
  75. Yanisch-Perron C., Vieira J., Messing J.. ( 1985;). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119 [CrossRef][PubMed]
    [Google Scholar]
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