1887

Abstract

The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [ 59 base element (59-be)] upstream of promoterless [gentamicin (Gm) resistance] and (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in strain Q. Electroporation of pUS23 into Q gave ampicillin-resistant transformants, which yielded Gm green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at was detected by PCR in 8 % of Gm colonies and the frequency of integration was estimated as 2·0×10 per Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of in one recombinant strain (Q23-17) and a promoter (P) was localized to the 5′ end of the gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the site. An insertion sequence (IS; IS family) was discovered in the vector backbone of the reporter plasmid integrated at and also in a pUS23 derivative recovered as a plasmid in JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27854-0
2005-06-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/6/mic1511853.html?itemId=/content/journal/micro/10.1099/mic.0.27854-0&mimeType=html&fmt=ahah

References

  1. Barlow R. S., Pemberton J. M., Desmarchelier P. M., Gobius K. S. 2004; Isolation and characterization of integron-containing bacteria without antibiotic selection. Antimicrob Agents Chemother 48:838–842 [CrossRef]
    [Google Scholar]
  2. Beaber J. W., Hochhut B., Waldor M. K. 2004; SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 427:72–74 [CrossRef]
    [Google Scholar]
  3. Boyd D., Cloeckaert A., Chaslus-Dancla E., Mulvey M. R. 2002; Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46:1714–1722 [CrossRef]
    [Google Scholar]
  4. Bunny K. L., Hall R. M., Stokes H. W. 1995; New mobile gene cassettes containing an aminoglycoside resistance gene, aacA7, and a chloramphenicol resistance gene, catB3, in an integron in pBWH301. Antimicrob Agents Chemother 39:686–693 [CrossRef]
    [Google Scholar]
  5. Chablain P. A., Zgoda A. L., Sarde C. O., Truffaut N. 2001; Genetic and molecular organization of the alkylbenzene catabolism operon in the psychrotrophic strain Pseudomonas putida 01G3. Appl Environ Microbiol 67:453–458 [CrossRef]
    [Google Scholar]
  6. Chang A. C. Y., Cohen S. N. 1978; Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
    [Google Scholar]
  7. Cladera A. M., Bennasar A., Barcelo M., Lalucat J., Garcia-Valdes E. 2004; Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species. J Bacteriol 186:5239–5248 [CrossRef]
    [Google Scholar]
  8. Collis C. M., Hall R. M. 1995; Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother 39:155–162 [CrossRef]
    [Google Scholar]
  9. 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 [CrossRef]
    [Google Scholar]
  10. 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 [CrossRef]
    [Google Scholar]
  11. Collis C. M., Kim M. J., Partridge S. R., Stokes H. W., Hall R. M. 2002a; Characterization of the class 3 integron and the site-specific recombination system it determines. J Bacteriol 184:3017–3026 [CrossRef]
    [Google Scholar]
  12. Collis C. M., Kim M. J., Stokes H. W., Hall R. M. 2002b; Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site. Mol Microbiol 46:1415–1427 [CrossRef]
    [Google Scholar]
  13. Dennis J. J., Zylstra G. J. 2004; Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4. J Mol Biol 341:753–768 [CrossRef]
    [Google Scholar]
  14. Drouin F., Melancon J., Roy P. H. 2002; The intI-like tyrosine recombinase of Shewanella oneidensis is active as an integron integrase. J Bacteriol 184:1811–1815 [CrossRef]
    [Google Scholar]
  15. Francia M. V., Delacruz F., Lobo J. M. G. 1993; Secondary sites for integration mediated by the Tn21 integrase. Mol Microbiol 10:823–828 [CrossRef]
    [Google Scholar]
  16. Galas D. J., Chandler M. 1989; Bacterial insertion sequences. In Mobile DNA pp 109–162 Edited by Berg D., Howe M. Washington DC: American Society for Microbiology;
    [Google Scholar]
  17. 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 [CrossRef]
    [Google Scholar]
  18. Hall R. M., Collis C. M. 1995; Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol 15:593–600
    [Google Scholar]
  19. 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 [CrossRef]
    [Google Scholar]
  20. Hall R. M., Brookes D. E., Stokes H. W. 1991; Site-specific insertion of genes into integrons – role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol 5:1941–1959 [CrossRef]
    [Google Scholar]
  21. Hanau-Bercot B., Podglajen I., Casin I., Collatz E. 2002; An intrinsic control element for translational initiation in class 1 integrons. Mol Microbiol 44:119–130 [CrossRef]
    [Google Scholar]
  22. Hansen L. H., Ferrari B., Sorensen A. H., Veal D., Sorensen S. J. 2001; Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry. Appl Environ Microbiol 67:239–244 [CrossRef]
    [Google Scholar]
  23. Herrero M., de Lorenzo V., Timmis K. N. 1990; Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172:6557–6567
    [Google Scholar]
  24. Hochhut B., Lotfi Y., Mazel D., Faruque S. M., Woodgate R., Waldor M. K. 2001; Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob Agents Chemother 45:2991–3000 [CrossRef]
    [Google Scholar]
  25. 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 [CrossRef]
    [Google Scholar]
  26. 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 [CrossRef]
    [Google Scholar]
  27. Kovach M. E., Elzer P. H., Hill D. S., Robertson G. T., Farris M. A., Roop R. M., Peterson K. M. 1995; Four new derivatives of the broad-host-range cloning vector pBBRMCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176 [CrossRef]
    [Google Scholar]
  28. Leon G., Roy P. H. 2003; Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea . J Bacteriol 185:2036–2041 [CrossRef]
    [Google Scholar]
  29. Levesque 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 [CrossRef]
    [Google Scholar]
  30. Liebert C. A., Hall R. M., Summers A. O. 1999; Transposon Tn21, flagship of the floating genome. Microbiol Mol Biol Rev 63:507–522
    [Google Scholar]
  31. Lupski J. R., Projan S. J., Ozaki L. S., Godson G. N. 1986; A temperature-dependent pBR322 copy number mutant resulting from a Tn5 position effect. Proc Natl Acad Sci U S A 83:7381–7385 [CrossRef]
    [Google Scholar]
  32. Mahillon J., Chandler M. 1998; Insertion sequences. Microbiol Mol Biol Rev 62:725–774
    [Google Scholar]
  33. Michael C. A., Gillings M. R., Holmes A. J., Hughes L., Andrew N. R., Holley M. P., Stokes H. W. 2004; Mobile gene cassettes: a fundamental resource for bacterial evolution. Am Nat 164:1–12 [CrossRef]
    [Google Scholar]
  34. Nemergut D. R., Martin A. P., Schmidt S. K. 2004; Integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution. Appl Environ Microbiol 70:1160–1168 [CrossRef]
    [Google Scholar]
  35. 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 [CrossRef]
    [Google Scholar]
  36. Nojiri H., Sekiguchi H., Maeda K., Urata M., Nakai S. I., Yoshida T., Habe H., Omori T. 2001; Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10. J Bacteriol 183:3663–3679 [CrossRef]
    [Google Scholar]
  37. Pemberton J. M., Penfold R. J. 1992; High-frequency electroporation and maintenance of pUC-based and pBR-based cloning vectors in Pseudomonas stutzeri . Curr Microbiol 25:25–29 [CrossRef]
    [Google Scholar]
  38. Recchia G. D., Stokes H. W., Hall R. M. 1994; Characterization of specific and secondary recombination sites recognized by the integron DNA integrase. Nucleic Acids Res 22:2071–2078 [CrossRef]
    [Google Scholar]
  39. Rowe-Magnus D. A., Mazel D. 2001; Integrons: natural tools for bacterial genome evolution. Curr Opin Microbiol 4:565–569 [CrossRef]
    [Google Scholar]
  40. Rowe-Magnus D. A., Guerout A. M., Ploncard P., Dychinco B., Davies J., Mazel D. 2001; The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc Natl Acad Sci U S A 98:652–657 [CrossRef]
    [Google Scholar]
  41. Rowe-Magnus D. A., Guerout A. M., Mazel D. 2002; Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol Microbiol 43:1657–1669 [CrossRef]
    [Google Scholar]
  42. 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 [CrossRef]
    [Google Scholar]
  43. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  44. Segal H., Elisha B. G. 1999; Characterization of the Acinetobacter plasmid, pRAY, and the identification of regulatory sequences upstream of an aadB gene cassette on this plasmid. Plasmid 42:60–66 [CrossRef]
    [Google Scholar]
  45. Simon R., Priefer U., Puhler A. 1983; A broad hostrange mobilization system for in vivo genetic-engineering – transposon mutagenesis in gram-negative bacteria. BioTechnology 1:784–791 [CrossRef]
    [Google Scholar]
  46. 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 [CrossRef]
    [Google Scholar]
  47. Stokes H. W., Holmes A. J., Nield B. S., Holley M. P., Nevalainen K. M., Mabbutt B. C., Gillings M. R. 2001; Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA. Appl Environ Microbiol 67:5240–5246 [CrossRef]
    [Google Scholar]
  48. Szczepanowski R., Krahn I., Linke B., Goesmann A., Puhler A., Schluter A. 2004; Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system. Microbiology 150:3613–3630 [CrossRef]
    [Google Scholar]
  49. Vaisvila R., Morgan R. D., Posfai J., Raleigh E. A. 2001; Discovery and distribution of super-integrons among pseudomonads. Mol Microbiol 42:587–601
    [Google Scholar]
  50. 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 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27854-0
Loading
/content/journal/micro/10.1099/mic.0.27854-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error