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. 1995 Nov;61(11):4051–4056. doi: 10.1128/aem.61.11.4051-4056.1995

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti

S O'Flaherty, Y Moenne-Loccoz, B Boesten, P Higgins, D N Dowling, S Condon, F O'Gara
PMCID: PMC1388604  PMID: 16535168

Abstract

The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa. The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(sup-)). Under greenhouse conditions, Thy(sup-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(sup-) rhizobia contained only Thy(sup+) revertants. In soil, Thy(sup-) rhizobia were compromised and failed to nodulate alfalfa. Thy(sup-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(sup-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY. At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602. The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.

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Selected References

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  1. Bosworth A. H., Williams M. K., Albrecht K. A., Kwiatkowski R., Beynon J., Hankinson T. R., Ronson C. W., Cannon F., Wacek T. J., Triplett E. W. Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression. Appl Environ Microbiol. 1994 Oct;60(10):3815–3832. doi: 10.1128/aem.60.10.3815-3832.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Bánfalvi Z., Sakanyan V., Koncz C., Kiss A., Dusha I., Kondorosi A. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet. 1981;184(2):318–325. doi: 10.1007/BF00272925. [DOI] [PubMed] [Google Scholar]
  4. Chen H., Richardson A. E., Gartner E., Djordjevic M. A., Roughley R. J., Rolfe B. G. Construction of an Acid-Tolerant Rhizobium leguminosarum Biovar Trifolii Strain with Enhanced Capacity for Nitrogen Fixation. Appl Environ Microbiol. 1991 Jul;57(7):2005–2011. doi: 10.1128/aem.57.7.2005-2011.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S. S., Barner H. D. STUDIES ON UNBALANCED GROWTH IN ESCHERICHIA COLI. Proc Natl Acad Sci U S A. 1954 Oct;40(10):885–893. doi: 10.1073/pnas.40.10.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fenton A. M., Stephens P. M., Crowley J., O'Callaghan M., O'Gara F. Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl Environ Microbiol. 1992 Dec;58(12):3873–3878. doi: 10.1128/aem.58.12.3873-3878.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hynes M. F., Quandt J., O'Connell M. P., Pühler A. Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene. Gene. 1989 May 15;78(1):111–120. doi: 10.1016/0378-1119(89)90319-3. [DOI] [PubMed] [Google Scholar]
  11. Lambert G. R., Harker A. R., Cantrell M. A., Hanus F. J., Russell S. A., Haugland R. A., Evans H. J. Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes. Appl Environ Microbiol. 1987 Feb;53(2):422–428. doi: 10.1128/aem.53.2.422-428.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. O'Donovan G. A., Neuhard J. Pyrimidine metabolism in microorganisms. Bacteriol Rev. 1970 Sep;34(3):278–343. doi: 10.1128/br.34.3.278-343.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. O'Gara F., Shanmugam K. T. Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4. Biochim Biophys Acta. 1976 Jul 21;437(2):313–321. doi: 10.1016/0304-4165(76)90001-5. [DOI] [PubMed] [Google Scholar]
  14. OKADA T., YANAGISAWA K., RYAN F. J. Elective production of thymine-less mutants. Nature. 1960 Oct 22;188:340–341. doi: 10.1038/188340a0. [DOI] [PubMed] [Google Scholar]
  15. Rafii F., Crawford D. L. Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil. Appl Environ Microbiol. 1988 Jun;54(6):1334–1340. doi: 10.1128/aem.54.6.1334-1340.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ross P., O'Gara F., Condon S. Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis. Appl Environ Microbiol. 1990 Jul;56(7):2156–2163. doi: 10.1128/aem.56.7.2156-2163.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ross P., O'Gara F., Condon S. Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes. Appl Environ Microbiol. 1990 Jul;56(7):2164–2169. doi: 10.1128/aem.56.7.2164-2169.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stephens P. M., O'sullivan M., O'gara F. Effect of Bacteriophage on Colonization of Sugarbeet Roots by Fluorescent Pseudomonas spp. Appl Environ Microbiol. 1987 May;53(5):1164–1167. doi: 10.1128/aem.53.5.1164-1167.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Triplett E. W. Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness. Appl Environ Microbiol. 1990 Jan;56(1):98–103. doi: 10.1128/aem.56.1.98-103.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang Y. P., Birkenhead K., Boesten B., Manian S., O'Gara F. Genetic analysis and regulation of the Rhizobium meliloti genes controlling C4-dicarboxylic acid transport. Gene. 1989 Dec 21;85(1):135–144. doi: 10.1016/0378-1119(89)90473-3. [DOI] [PubMed] [Google Scholar]

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