Microbial Ecology

, Volume 21, Issue 1, pp 21–33 | Cite as

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

  • Suresh D. Pillai
  • Ian L. Pepper


Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.


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  1. 1.
    Allan E, Kelman (1987) Immunofluorescent stain procedures for detection and identification ofErwinia carotovora var.atroseptica. Phytopathology 67:1305–1312Google Scholar
  2. 2.
    Amy PS, Schulke JW, Frazier LM, Seidler RJ (1985) Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid. Appl Environ Microbiol 49:1237–1245PubMedGoogle Scholar
  3. 3.
    Berry JO, Atherly AG (1984) Induced plasmid-genome rearrangements inRhizobium japonicum. J Bacteriol 157:218–224PubMedGoogle Scholar
  4. 4.
    de Bruijn FJ, Lupiski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multiple copy plasmids—A review, Gene 27:131–149CrossRefPubMedGoogle Scholar
  5. 5.
    Calos PC, Miller JH (1980) Transposable elements. Cell 20:579–595CrossRefPubMedGoogle Scholar
  6. 6.
    Chang FN, Flaks JG (1972) Binding of dihydrostreptomycin toEscherichia coli ribosomes; characteristics and equilibrium of the reaction. Antimicrobial Agents and Chemotherapy 2:294–307PubMedGoogle Scholar
  7. 7.
    Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM (1985) Viable but non-culturableVibrio cholerae and related pathogens in the environment: Implications for release of genetically engineered microorganisms. Biotechnology 3:817–820CrossRefGoogle Scholar
  8. 8.
    Compeau G, Al-Achi BJ, Platsouka E, Levy SB (1988) Survival of rifampicin resistant mutants ofPseudomonas fluorescens andPseudomonas putida in soil systems. Appl Environ Microbiol 30:279–284Google Scholar
  9. 9.
    Curtis RL (1976) Genetic manipulations of microorganisms: Potential benefits and hazards. Ann Rev Microbiol 30:279–294CrossRefGoogle Scholar
  10. 10.
    Davison J (1988) Plant beneficial bacteria. Biotechnology 6:282–286CrossRefGoogle Scholar
  11. 11.
    Devanas MA, Stotzky G (1986) Fate in soil of a recombinant plasmid carrying aDrosophila gene. Curr Microbiol 13:279–283CrossRefGoogle Scholar
  12. 12.
    Drahos DJ, Hemming BC, McPherson S (1986) Tracking recombinant organisms in the environment: β-galactosidase as a selectable non-antibiotic marker for fluorescent pseudomonads. Biotechnology 4:439–44CrossRefGoogle Scholar
  13. 13.
    Egner C, Berg DE (1981) Excision of transposon Tn5 is dependent on the inverted repeats but not on the transposase function of Tn5. Proc Natl Acad Sci (USA) 78:459–463CrossRefGoogle Scholar
  14. 14.
    Flores M, Gonzalez V, Pardo MA, Leija A, Martinez E, Romero D, Pinero D, Davilla G, Palacios R (1988) Genomic instability inRhizobium phaseoli. J Bacteriol 170:1191–1196PubMedGoogle Scholar
  15. 15.
    Fredrickson JK, Bezdicek DF, Brockman FE, Li SW (1988) Enumeration of Tn5 mutant bacteria in soil by most-probable-number-DNA hybridization procedure and antibiotic resistance. Appl Environ Microbiol 54:446–453PubMedGoogle Scholar
  16. 16.
    Holben WE, Jansson JK, Chelm BK, Tiedje JM (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl Environ Microbiol 54:703–711PubMedGoogle Scholar
  17. 17.
    Jorgensen RA, Rothstein SJ, Reznikoff WS (1979) A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Molec Gen Genet 177:65–72CrossRefPubMedGoogle Scholar
  18. 18.
    Khalil TA, Gealt MA (1987) Temperature, pH and cations affect the ability ofEscherichia coli to mobilize plasmids in L broth and synthetic waste water. Can J Microbiol 33:733–737PubMedCrossRefGoogle Scholar
  19. 19.
    Klute A (1986) In: Methods of soil analysis. Part 1. Physical and mineralogical methods. 2nd Ed. SSSA, Madison, Wisconsin, Ch. 15Google Scholar
  20. 20.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning—A laboratory handbook. NY Cold Spring Harbor Press, Cold Spring Harbor, New YorkGoogle Scholar
  21. 21.
    Page AL, Miller RH, Keeney DR (1982) In: Methods of soil analysis. Part 2. Chemical and microbiological properties. 2nd Ed. SSSA, Madison, Wisconsin, Ch. 29–31Google Scholar
  22. 22.
    Pillai SD, Pepper IL (1990) Survival of Tn5 mutant bean rhizobia in desert soils: Phenotypic expression of Tn5 under moisture stress. Soil Biol Biochem 22:265–270CrossRefGoogle Scholar
  23. 23.
    Rissler JF (1984) Research needs for biotic environmental effects of GEMs. Recomb DNA Tech Bull 7:20–30PubMedGoogle Scholar
  24. 24.
    Rosenberg C, Hughet T (1984) The pAtC58 plasmid ofAgrobacterium tumefaciens is not essential for tumor induction. Mol Gen Genet 196:533–536CrossRefGoogle Scholar
  25. 25.
    Saylor GS, Shields MS, Tedford ET, Breen A, Hopper SW, Sirotkin KM, Davis JW (1985) Application of DNA:DNA colony hybridization to detection of catabolic genotypes in environmental samples. Appl Environ Microbiol 49:1295–1307Google Scholar
  26. 26.
    Selvaraj G, Iyer VN (1983) Suicide plasmid vehicles for insertion mutagenesis inRhizobium meliloti and related bacteria. J Bacteriol 156:1292–1300PubMedGoogle Scholar
  27. 27.
    Selvaraj G, Iyer VN (1984) Transposon Tn5 specifies streptomycin resistance inRhizobium spp. J Bacteriol 158:580–589PubMedGoogle Scholar
  28. 28.
    Van Elsas JD, Pereira MTPRR (1986) Occurrence of antibiotic resistance among bacilli in Brazilian soils and the possible involvement of resistance plasmids. Plant Soil 94:213–226CrossRefGoogle Scholar
  29. 29.
    Van Elsas JD, Trevors JT, Starodub ME (1988) Plasmid transfer in soil and rhizosphere. In: Klingmuller W (ed) Risk assessment for deliberate releases. Springer-Verlag, Berlin, pp. 88–89Google Scholar
  30. 30.
    Vincent JM (1970) In: A manual for the practical study of the root nodule bacteria. International Biological Programme Handbook 15. Blackwell, Oxford, pp. 3–4Google Scholar
  31. 31.
    Wollum AG, Miller RH (1980) Density centrifugation method for recoveringRhizobium spp. from soil for fluorescent-antibody studies. Appl Environ Microbiol 39:466–469PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • Suresh D. Pillai
    • 1
  • Ian L. Pepper
    • 2
  1. 1.Department of Microbiology and ImmunologyUniversity of ArizonaTucsonUSA
  2. 2.Soil and Water ScienceUniversity of ArizonaTucsonUSA

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