Molecular and General Genetics MGG

, Volume 242, Issue 5, pp 505–516 | Cite as

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

  • Andreas Ulrich
  • Alfred Pühler
Original Articles

Abstract

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.

Key words

Transposition Tn3 family Rhizobium leguminosarum transposon Distribution of transposon Directed mutagenesis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Appleyard RK (1954) Segregation of new lysogenic types during growth of a double lysogenic strain derived from E. coli K12. Genetics 39:440Google Scholar
  2. Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179Google Scholar
  3. Arthur M, Molinas C, Depardieu F, Courvalin P (1993) Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol 175:117–127Google Scholar
  4. Avila P, Ackroyd AJ, Halford SE (1990) DNA binding by mutants of Tn21 resolvase with DNA recognition functions from Tn3 resolvase. J Mol Biol 216:645–655Google Scholar
  5. Berg CM, Vartak NB, Wang G, Xu X, Liu L, MacNeil D, Gewain KM, Wiater LAA, Berg DE (1992) The mγδ-1 element, a small γδ (TnI000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing. Gene 113:9–16Google Scholar
  6. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198Google Scholar
  7. Brewin NJ, Beringer JE, Johnston AWB (1980) Plasmid-mediated transfer of host-range specificity between two strains of R. leguminosarum. J Gen Microbiol 120:413–420Google Scholar
  8. Dayhoff MO, Barker WC, Hunt LT (1983) Establishing homologies in protein sequences. Methods Enzymol 91:524–545Google Scholar
  9. Diver WP, Grinsted J, Fritzinger DC, Brown NL, Altenbucher J, Rogowsky P, Schmitt R (1983) DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721. Mol Gen Genet 191:189–193Google Scholar
  10. Dodd IB, Egan JB (1987) Systematic method for the detection of potential Cro-like DNA binding regions in proteins. J Mol Biol 149:557–564Google Scholar
  11. Eckhardt T (1978) A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1: 584–588Google Scholar
  12. Galas DJ, Chandler M (1989) Bacterial insertion sequences. In: Berg DE, Howe MM (eds) Mobile DNA. American Society for Microbiology, Washington DC, pp 109–162Google Scholar
  13. Gay PD, Le Coq D, Steinmetz M, Berkelman T, Kado CI (1985) Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol 164:918–921Google Scholar
  14. Grindley NDF, Reed RR (1985) Transpositional recombination in prokaryotes. Annu Rev Biochem 54:863–896Google Scholar
  15. Grinsted J, Brown NL (1984) A Tn21 terminal sequence within Tn501: complementation of tnpA gene function and transposon evolution. Mol Gen Genet 197:497–502Google Scholar
  16. Grinsted J, De La Cruz F, Schmitt R (1990) The Tn21 subgroup of bacterial transposable elements. Plasmid 24:163–189Google Scholar
  17. Grönger P, Manian SS, Reiländer H, O'Connell M, Priefer UB, Pühler A (1987) Organization and partial sequence of a DNA region of R. leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame. Nucleic Acids Res 15:31–41Google Scholar
  18. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557Google Scholar
  19. Hedges RW, Jacob A (1974) Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet 132:31–40Google Scholar
  20. Henikoff (1984) Unidirectional digestions with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359Google Scholar
  21. Hynes MF, Brucksch K, Priefer U (1988) Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain. Arch Microbiol 150:326–332Google Scholar
  22. Kiss GS, Vincze E, Kalman Z, Forrai T, Kondorosi A (1979) Genetic and biochemical analysis of mutants affected in nitrate reduction in Rhizobium meliloti. J Gen Microbiol 113:105–118Google Scholar
  23. Kleckner N (1981) Transposable elements in prokaryotes. Annu Rev Genet 15:341–404Google Scholar
  24. Koll S (1990) Standortbezogene RFLP-Analyse der hup-Genregion von Rhizobium leguminosarum bv. viciae Wildstämmen mit Pisum sativum var. Poneka oder Vicia faba var. Herz Feya als Wirtspflanze. Diploma thesis, Friedrich-Alexander-Universität Erlangen-NürnbergGoogle Scholar
  25. Kretschmer PJ, Cohen SN (1977) Selected translocation of plasmid genes: frequency and regional specificity of translocation of the Tn3 element. J Bacteriol 130:888–899Google Scholar
  26. Mahillon J, Lereclus D (1988) Structural and functional analysis of Tn4430: identification of an integrase-like protein involved in the cointegrate-resolution process. EMBO J 7:1515–1526Google Scholar
  27. Meade H, Long S, Ruvkun G, Brown S, Ausubel F (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122Google Scholar
  28. Michiels T, Cornelis G (1989) Site-specific recombinations between direct and inverted res sites of Tn2501. Plasmid 22:249–255Google Scholar
  29. Michiels T, Cornelis G, Ellis K, Grinsted J (1987) Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements. J Bacteriol 169:624–631Google Scholar
  30. Miksch G, Lentzsch P (1988) Transfer of Rhizobium leguminosarum Sym plasmids to R. meliloti and stability of resident and transferred plasmids. J Bas Microbiol 28:445–455Google Scholar
  31. Murphy E (1989) Transposable elements in gram-positive bacteria. In: Berg DE, Howe MM (eds) Mobile DNA. American Society for Microbiology, Washington DC, pp 269–288Google Scholar
  32. Nakatsu C, Ng J, Singh R, Straus N, Wyndham C (1991) Chlorobenzoate catabolic transposon Tn5271 is a composite class I element with flanking class II insertion sequences. Proc Natl Acad Sci USA 88:8312–8316Google Scholar
  33. Pabo C, Sauer R (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321Google Scholar
  34. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448Google Scholar
  35. Plasterk RHA, Brinkman A, Van De Putte P (1983) DNA inversions in the chromosome of Escherichia coli and in bacteriophage Mu: relationship to other site-specific recombination systems. Proc Natl Acad Sci USA 80:5355–5358Google Scholar
  36. Post LE, Nomura M (1980) DNA sequences from the str operon of E. coli. J Biol Chem 255:4660–4666Google Scholar
  37. Raabe T, Jenny E, Meyer J (1988) A selection cartridge for rapid detection and analysis of spontaneous mutations including insertions of transposable elements in Enterobacteriaceae. Mol Gen Genet 215:176–180Google Scholar
  38. Rogowsky P, Halford SE, Schmitt R (1985) Definition of the three resolvase binding sites at the res loci of Tn21 and Tn1721. EMBO J 4:2135–2141Google Scholar
  39. Rowland SJ, Dyke KGH (1989) Characterization of the staphylococcal β-lactamase transposon Tn552. EMBO J 8:2761–2773Google Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  41. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  42. Shaw JH, Clewell DB (1985) Complete nucleotide sequence of macrolide-lincosamide-streptogramin B resistance transposon Tn917 in Streptococcus faecalis. J Bacteriol 164:782–796Google Scholar
  43. Sherrat D (1989) Tn3 and other related transposable elements: site-specific recombination and transposition. In: Berg DE, Howe MM (eds) Mobile DNA. American Society for Microbiology, Washington DC, pp 163–184Google Scholar
  44. Siemieniak DR, Slightom JL, Chung ST (1990) Nucleotide sequence of Streptomyces fradiae transposable element Tn4556: a class-II transposon related to Tn3. Gene 86:1–9Google Scholar
  45. Simon R (1984) High frequency mobilization of gram-negative bacterial replicons using the in vitro constructed Tn5-Mob transposon. Mol Gen Genet 196:413–420Google Scholar
  46. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology 1: 784–791Google Scholar
  47. Simon R, O'Connell M, Labes M, Pühler A (1986) Plasmid vectors for the genetic analysis and manipulation of Rhizobia and other gram-negative bacteria. Methods Enzymol 118:640–659Google Scholar
  48. Simon R, Klauke B, Hötte B (1988) The use of IS-elements for the characterization of gram-negative bacteria. In: Klingmüller W (ed) Risk assessment for deliberate releases. Springer, Berlin-Heidelberg, pp 121–126Google Scholar
  49. Simon R, Hötte B, Klauke B, Kosier B (1991) Isolation and characterization of insertion sequence elements from gram-negative bacteria using new broad host range, positive selection vectors. J Bacteriol 173:1502–1508Google Scholar
  50. Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acid Res 14:217–232Google Scholar
  51. Steinmetz M, Le Coq D, Aymerich S, Gonzy-Tréboul G, Gay P (1985) The DNA sequence of the gene for secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet 200:220–228Google Scholar
  52. Strausbaugh LD, Bourke MT, Sommer MT, Coon ME (1990) Probe mapping to facilitate transposon-based DNA sequencing. Proc Natl Acad Sci USA 87:6213–6217Google Scholar
  53. Tsuda M, Mianegishi K-I, Iino T (1989) Toluene transposons Tn4651 and Tn4653 are class II transposons. J Bacteriol 171:1386–1393Google Scholar
  54. Tu C-P D, Cohen SN (1980) Translocation specificity of the Tn3 element: characterization of sites of multiple insertions. Cell 19:151–160Google Scholar
  55. Ubben D, Schmitt R (1986) Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. Gene 41:145–152Google Scholar
  56. Wells RG, Grindley NDF (1984) Analysis of the γδ res site: sites required for site-specific recombination and gene expression. J Mol Biol 179:667–687Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Andreas Ulrich
    • 1
  • Alfred Pühler
    • 2
  1. 1.Abteilung Mikrobiologie, Institut für Ökophysiologie der PrimärproduktionZentrum für Agrarlandschafts- und LandnutzungsforschungMünchebergGermany
  2. 2.Lehrstuhl für Genetik, Fakultät für BiologieUniversität BielefeldBielefeldGermany

Personalised recommendations