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Volume 157, Issue 12

Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology Free

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.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Funding
This study was supported by the:
  • ARC Discovery (Award DP0450056)
© 2011 SGM
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2011-12-01
2024-04-19
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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 Genet 6:e1001165 [View Article][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 Res 38:e153 [View Article][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 Bacteriol 187:1740–1750 [View Article][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 Chem 264:8935–8940[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 Biol 6:3–17 [View Article][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 Microbiol 15:301–309 [View Article][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 Microbiol 15:301–309 [View Article][PubMed]
     [Google Scholar]
  8. Bouvier M., Demarre G., Mazel D. ( 2005). Integron cassette insertion: a recombination process involving a folded single strand substrate. EMBO J 24:4356–4367 [View Article][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 Bacteriol 153:93–99[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. Microbiology 151:1853–1864 [View Article][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 Microbiol 68:2726–2730 [View Article][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 Bacteriol 174:1574–1585[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 Microbiol 9:41–52 [View Article][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 Bacteriol 183:2535–2542 [View Article][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 Microbiol 46:1415–1427 [View Article][PubMed]
     [Google Scholar]
  16. Cost G. J., Cozzarelli N. R. ( 2007). Directed assembly of DNA molecules via simultaneous ligation and digestion. Biotechniques 42:84–89 [View Article][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 Agents 25:341–344 [View Article][PubMed]
     [Google Scholar]
  18. Datta N., Hedges R. W. ( 1972). Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae . J Gen Microbiol 72:349–355[PubMed] [CrossRef]
     [Google Scholar]
  19. Davies J., Davies D. ( 2010). Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433 [View Article][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 Res 11:1095–1099 [View Article][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 Res 35:6475–6489 [View Article][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 Bacteriol 184:1811–1815 [View Article][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 ONE 2:e1315 [View Article][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 Microbiol 9:2298–2312 [View Article][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 ONE 6:e16355 [View Article][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 Microbiol 3:722–732 [View Article][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 A 102:4419–4424 [View Article][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 Bacteriol 190:5095–5100 [View Article][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. Science 324:1034 [View Article][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 Updat 1:109–119 [View Article][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 Bacteriol 176:6286–6294[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 Microbiol 5:383–394 [View Article][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 Bacteriol 185:918–928 [View Article][PubMed]
     [Google Scholar]
  34. Johnsborg O., Eldholm V., Håvarstein L. S. ( 2007). Natural genetic transformation: prevalence, mechanisms and function. Res Microbiol 158:767–778 [View Article][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 Genet 6: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 ONE 4:e5276 [View Article][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 J 5:962–972 [View Article][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. Gene 166:175–176 [View Article][PubMed]
     [Google Scholar]
  39. Lalucat J., Bennasar A., Bosch R., García-Valdés E., Palleroni N. J. ( 2006). Biology of Pseudomonas stutzeri. . Microbiol Mol Biol Rev 70:510–547 [CrossRef]
     [Google Scholar]
  40. Léon G., Roy P. H. ( 2003). Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea . J Bacteriol 185:2036–2041 [View Article][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. Gene 142:49–54 [View Article][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 Microbiol 2:407–416 [View Article][PubMed]
     [Google Scholar]
  43. Loot C., Bikard D., Rachlin A., Mazel D. ( 2010). Cellular pathways controlling integron cassette site folding. EMBO J 29:2623–2634 [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 . Microbiology 146:3081–3090[PubMed]
     [Google Scholar]
  45. Martinez E., de la Cruz F. ( 1988). Transposon Tn21 encodes a RecA-independent site-specific integration system. Mol Gen Genet 211:320–325 [View Article][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 J 9:1275–1281[PubMed]
     [Google Scholar]
  47. Mazel D. ( 2006). Integrons: agents of bacterial evolution. Nat Rev Microbiol 4:608–620 [View Article][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 . Science 310:1824–1827 [View Article][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 Lett 195:59–65 [View Article][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 Res 26:391–406 [View Article][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 Chemother 53:952–957 [View Article][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 Biotechnol 33:410–417 [View Article]
     [Google Scholar]
  53. Recchia G. D., Hall R. M. ( 1995). Gene cassettes: a new class of mobile element. Microbiology 141:3015–3027 [View Article][PubMed]
     [Google Scholar]
  54. Recchia G. D., Hall R. M. ( 1997). Origins of the mobile gene cassettes found in integrons. Trends Microbiol 5:389–394 [View Article][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 Ecol 72:35–46 [View Article][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 Bacteriol 76:226–233 [View Article]
     [Google Scholar]
  57. Rowe-Magnus D. A., Mazel D. ( 2001). Integrons: natural tools for bacterial genome evolution. Curr Opin Microbiol 4:565–569 [View Article][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 Res 13:428–442 [View Article][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 Rev 31:449–477 [View Article][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 Biol 386:316–331 [View Article][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 Biotechnol 56:265–269 [View Article][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 Microbiol 22:393–402[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 Microbiol 68:865–873 [View Article][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 Microbiol 3:1669–1683 [View Article][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 Microbiol 26:731–745 [View Article][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 Lett 50:217–221 [View Article]
     [Google Scholar]
  69. Vaisvila R., Morgan R. D., Posfai J., Raleigh E. A. ( 2001). Discovery and distribution of super-integrons among pseudomonads. Mol Microbiol 42:587–601 [View Article][PubMed]
     [Google Scholar]
  70. Ward J. M., Grinsted J. ( 1982). Physical and genetic analysis of the Inc-W group plasmids R388, Sa, and R7K. Plasmid 7:239–250 [View Article][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 Microbiol 80:1326–1336 [View Article][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 J 2:417–428 [View Article][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 Methods 76:97–100 [View Article][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. Gene 33:103–119 [View Article][PubMed]
     [Google Scholar]
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Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology
Microbiology 157, 3349 (2011); https://doi.org/10.1099/mic.0.051623-0
/content/journal/micro/10.1099/mic.0.051623-0
/content/journal/micro/10.1099/mic.0.051623-0
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