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The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres, and implications for gene flow

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Abstract

The distribution of two genetically modified Rhizobium leguminosarum strains was investigated in the field. One, RSM2004, released in 1987, carries a Tn5 marker on its conjugative symbiotic plasmid (pSym). The second, CT0370, released at the same site in 1994, has a gusA gene integrated into its chromosome but no pSym. Plate counts indicated that the CT0370 population became established at a higher level than RSM2004. However, when peas, alfalfa and barley were grown, RSM2004 was found to outnumber CT0370 on all roots and by 100-fold on pea. Although the transfer of pSym from RSM2004 to CT0370 could be detected on plates and in microcosm studies with high inoculum densities, no transfer was detected in the field. Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it conferred an advantage in the rhizosphere. In addition to increasing host fitness, plasmids may transfer, or mobilise other genetic elements, to other bacteria. This is more likely in sites such as the rhizosphere, where cells are active and numbers are high. The distribution of pSym and other genetic elements associated with rhizobia, in bacterial sub-populations from the soil and roots of the different plants, was investigated using PCR. The genetic elements studied were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB102, a broad host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid replication origins repCI and repCII. As expected, ISRm3 was detected in rhizoflora cultured from alfalfa but not the other plants. The mer gene was ubiquitous but the transfer region of pSB102 was not detected. The nodC and both repC primers amplified products from all the plants, giving further evidence for the occurrence of plasmids originating from Rhizobium in the rhizoflora of non-host plants. Despite the abundance of elements associated with transferable plasmids in rhizobia, none was detected in either inoculant strain.

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References

  • Berrah G & Konetzka WA (1961) Selective and reversible inhibition of the synthesis of bacterial deoxyribonulceic acid by phenethyl alcohol. J. Bacteriol. 23: 738–744.

    Google Scholar 

  • Cullen DW, Nicholson PS, Mendum TA & Hirsch PR (1998) Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction. J. Appl. Microbiol. 84: 1025–1034.

    Article  PubMed  CAS  Google Scholar 

  • Drahos DJ, Barry GF, Hemming BC, Brandt EJ, Skipper HD, Kline EL, Kluepfel DA, Hughes TA & Gooden DT (1988) Pre-release testing procedures: US field test of a lacZY-engineered soil bacterium. In: Sussman M, Collins CH, Skinner FA & Stewart-Tull DE (Eds) The Release of Genetically-Engineered Micro-organisms (pp 181–191). Academic Press, London UK.

    Google Scholar 

  • Gustafsson K (2000) Regulatory Aspects. In: Jansson JK, van Elsas JD & Bailey MJ (Eds) Tracking Genetically Engineered Microorganisms (pp 153–162). Landes Bioscience, Georgetown, USA.

    Google Scholar 

  • Hirsch PR (1996) Population dynamics of indigenous and genetically modified rhizobia in the field. New Phytol. 133: 159–171.

    Article  Google Scholar 

  • Hirsch PR & Skinner FA (1992) The identification and classsification of Rhizobium and Bradyrhizobium. In: Board RG, Jones D & Skinner FA (Eds) Identification Methods in Applied and Environmental Microbiology (pp 45–65). Blackwell Scientific Publications, Oxford, UK.

    Google Scholar 

  • Hirsch PR & Spokes JD (1994) Survival and dispersion of genetically modified rhizobia in the field and genetic interactions with native strains. FEMS Microbiol. Ecol. 15: 147–160.

    Article  CAS  Google Scholar 

  • Hirsch PR, Mendum TA, Pühler A & Selbitschka W (2000) The field release and monitoring of GUS-marked rhizobial strain CT0370. In: Jansson JK, van Elsas JD & Bailey MJ (Eds) Tracking Genetically Engineered Microorganisms (pp 145–151). Landes Bioscience, Georgetown, USA.

    Google Scholar 

  • Kaiser O, Puhler A & Selbitschka W (2001) Phylogenetic analysis of microbial diversity in the rhizoplane of oilseed rape (Brassica napus cv. Westar) employing cultivation-dependent and cultivation-independent approaches. Microbial Ecol. 42: 136–49.

    CAS  Google Scholar 

  • Laguerre G, Nour SM, Macheret V, Sanjuan J, Drouin P & Amarger N (2001) Classification of rhizobia based on nodC andnifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiol. UK 147: 981–993.

    CAS  Google Scholar 

  • Liebert CA, Wireman J, Smith T & Summers AO (1997) Phylogeny of mercury resistance (mer) operons of Gram-negative bacteria isolated from the fecal flora of primates. Appl. Environ. Microbiol. 63: 1066–1076.

    PubMed  CAS  Google Scholar 

  • Lilley AK & Bailey MJ (1997) The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions. Appl. Environ. Microbiol. 63: 1577–1583.

    PubMed  CAS  Google Scholar 

  • Lindow SE & Panopoulos N (1988) Field tests of recombinant Ice- Pseudomonas syringae for biological frost control in potato. In: Sussman M, Collins CH, Skinner FA & Stewart-Tull DE (Eds) The Release of Genetically-Engineered Micro-organisms (pp 121–138). Academic Press, London UK.

    Google Scholar 

  • Mendum TA, Clark IM & Hirsch PR (2001) Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia. Antonie van Leeuwenhoek 79: 189–197.

    Article  PubMed  CAS  Google Scholar 

  • Palmer KM, Turner SL, Young JPW (2000) Sequence diversity of the plasmid replication gene repC in the Rhizobiaceae. Plasmid 44: 209–219.

    Article  PubMed  CAS  Google Scholar 

  • Rigottier-Gois L, Turner SL, Young JPW & Amarger N (1998) Distribution of repC plasmid-replication sequences among plasmids and isolates of Rhizobium leguminosarum bv. viciae from field populations. Microbiol. UK 144: 771–780.

    CAS  Google Scholar 

  • Schneiker S, Keller M, Dröge M, Lanka E, Pühler A & Selbitshka W (2001) The genetic organisation and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa. Nucl. Acids Res. 29: 5169–5181.

    Article  PubMed  CAS  Google Scholar 

  • Schwieger F & Tebbe CC (2000) Effect of field inoculation with Sinorhizobium meliloti L33 on the composition of bacterial communities in rhizospheres of a target plant (Medicago sativa) and a non-target plant (Chenopodium album) - Linking of 16S rRNA gene-based single-strand conformation polymorphism community profiles to the diversity of cultivated bacteria. Appl. Environ. Microbiol. 66: 3556–3565.

    Article  PubMed  CAS  Google Scholar 

  • Selbitschka W, Jording D, Nieman S, Schmidt R, Puhler A, Mendum T & Hirsch P (1995) Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment. FEMS Microbiol. Lett. 128: 255–263.

    Article  PubMed  CAS  Google Scholar 

  • Sørensen AB, Duch M & Pedersen FS (1999) Isolation of unknown flanking DNA by a simple two-step polymerase chain reaction method. DYNALogue 3: 2–3.

    Google Scholar 

  • Turner SL, Rigottier-Gois L, Power RS, Amarger N & Young JPW (1996) Diversity of repC plasmid replication sequences in Rhizobium leguminosarum. Microbiol. UK 142: 1705–1713.

    Article  CAS  Google Scholar 

  • Van Elsas JD, Fry J, Hirsch P & Molin S (2000) Ecology of plasmid transfer and spread. In: Thomas CM (Ed) The Horizontal Gene Pool (pp 175–206). Harwood Academic Publishers, Amsterdam, The Netherlands.

    Google Scholar 

  • Villadas PJ, Burgos P, Rodríguez-Navarro DN, Temprano F & Toro N (1998) Characterization of rhizobia homologues of Sinorhizobium meliloti insertion sequences ISRm3 and ISRm4. FEMS Microbiol. Ecol. 25: 341–348.

    Article  CAS  Google Scholar 

  • Wilson M & Lindow SE (1993) Release of recombinant microorganisms. Ann. Rev. Microbiol. 47: 913–944.

    Article  CAS  Google Scholar 

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Correspondence to Penny R Hirsch.

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Clark, I.M., Mendum, T.A. & Hirsch, P.R. The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia in soil and crop rhizospheres, and implications for gene flow. Antonie Van Leeuwenhoek 81, 607–616 (2002). https://doi.org/10.1023/A:1020574009445

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