Abstract
The process by which free-living Rhizobium forms nitrogen-fixing nodules on the roots of leguminous plants involves the induction and repression of a number of bacterial genes. To help identify these genes and to determine their mode of regulation, we have transposed the E. coli lac operon, minus its promoter, randomly around the chromosome of R. japonicum, a soybean nodulating bacterium. This was accomplished using the defective phage, mu-dI (Kan, lac) placed onto a self-transmissible, narrow host-range suicide vector, pGS6. Among the mu-dI insertion mutants examined, auxotrophs were detected at frequencies of about 0.3%, and nodulation and nitrogen fixation defective mutants at about 4%. Many of the symbiotically defective mutants were found to have deletions in their indigenous plasmids. Three mutants showed increased β-galactosidase levels when grown in the presence of soybean or kidney bean root-extracts but not in the presence of root-extracts from other legumes or a non-legume, indicating specific plant control of Rhizobium gene expression.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Jordon, D.C. and Allen, O.N. 1974. Genus II. Rhizobium, p. 262–264. In: Bergey's Manual of Determinative Bacteriology, 8th ed. R. E. Buchanan and N. E. Gibbons (eds.) The Williams and Wilkins Co., Baltimore.
Vincent, J.M. 1974. Root-nodule symbiosis with Rhizobium, p. 266–341. In: Biology of Nitrogen Fixation. Quispel, A. (ed.) North Holland Publishing Co., Amsterdam.
Verma, D.P.S. and Long, S. 1983. The molecular biology of Rhizobium-legume symbiosis, p. 211–245. Int. Rev. Cytol. Suppl. 14, K. Jeon (ed.), Academic Press, New York.
Beynon, J.L., Beringer, J.E. and Johnston, A.W.B. 1980. Plasmids and host-range in Rhizobium leguminosarum and Rhizobium phaseoli. J. Gen. Microbiol. 120: 421–429.
Brewin, N.J., Beringer, J.E. and Johnston, A.W.B. 1980. Plasmid-mediated transfer of host-range specificity between two strains of Rhizobium leguminosarum. J. Gen. Microbiol. 120: 413–420.
Brewin, N.J., Beringer, J.E., Buchanan-Wollaston, A.V., Johnston, A.W.B. and Hirsch, P.R. 1980. Transfer of symbiotic genes with bacteriocinogenic plasmids in Rhizobium leguminosarum. J. Gen. Microbiol. 116: 261–270.
Djordjevic, M.A., Zurkowski, W. and Rolfe, B.G. 1982. Plasmids and stability of symbiotic properties of Rhizobium trifolii. J. Bacteriol. 151: 560–568.
Hombrecher, G., Brewin, N.J. and Johnston, A.W.B. 1981. Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium leguminosarum and R. phaseoli. Mol. Gen. Genet. 182: 133–136.
Hooykaas, P.J.J., Van Brussel, A.A.N., den Dulk-Ras, H., van Slogteren, S. and Schilperoort, R.A. 1981. Sym plasmid of Rhizobium trifolii expressed in different rhizobial species and Agrobacterium. Nature 291: 351–353.
Johnston, A.W.B., Beynon, J.L., Buchanan-Wollaston Setchell, S.M., Hirsch, P.R. and Beringer, J.E. 1978. High frequency transfer of nodulation ability between strains and species of Rhizobium. Nature 276: 634–636.
Nuti, M.P., Lepidi, A.A., Prakash, R.K., Schilperoort, R.A. and Cannon, F.C. 1979. Evidence for nitrogen fixation (nif) genes on indigenous Rhizobium plasmids. Nature 282: 533–535.
Beringer, J.E. 1980. The development of Rhizobium genetics. J. Gen. Microbiol. 116 1–7.
Beringer, J.E., Beynon, J.L., Buchanan-Wollaston, A.V. and Johnston, A.W.B. 1978. Transfer of drug resistance transposon Tn5 to Rhizobium. Nature 276: 633–634.
Berg, D.E. and Berg, C.M. 1983. The prokaryotic transposable element Tn5. Bio/technology 1: 417–433.
Hom, S.S.M., Uratsu, S.L. and Hoang, F. 1984. Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants. J. Bacteriol. 159: 335–340.
Kondorosi, E., Banfalvi, Z. and Kondorosi, A. 1984. Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes. Mol. Gen. Genet. In press.
Kondorosi, E., Banfalvi, Z., Slaska-Kiss, K. and Kondorosi, A. 1983. Analysis of symbiotic nitrogen fixation genes carried by Rhizobium meliloti megaplasmid, p. 259–275. In: UCLA Symposium on molecular and cellular biology; Plant Molecular Biology. R. Goldberg (ed.), AR Liss Inc., New York.
Meade, H.M., Long, S.R., Ruvkun, G.B., Brown, S.E. and Ausubel, F.M. 1983. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J. Bacteriol. 149: 114–122.
Rolfe, B.G., Djordjevic, M., Scott, K.F., Hughes, J.E., Badenoch-Jones, J., Gresshoff, P.M., Cen, Y., Dudman, W.F., Zurkowski, W. and Schine, J. 1981. Analysis of nodule forming ability of fast-growing Rhizobium strains, p. 142–145. In: Current Perspectives in Nitrogen Fixation. A, H. Gibson, and W. E. Newton (eds.), Australian Academy of Sciences, Canberra.
Rostas, K., Sista, P.R., Stanley, J. and Verma, D.P.S. 1984. Transposon mutagenesis of Rhizobium japonicum. Mol. Gen. Genet. In press.
Selvaraj, G. and Iyer, V.N. 1983. Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J. Bacteriol. 156: 1292–1300.
Simon, R., Priefer, U. and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/technology 1: 784–791.
Dixon, R., Eady, R.R., Espin, G., Hill, S., Iaccarino, M., Kahn, D. and Merrick, M. 1980. Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature 286: 128–132.
MacNeil, D., Zhu, J. and Brill, W.J. 1981. Regulation of nitrogen fixation in Klebsiella pneumoniae: isolation and characterization of strains with nif-lac fusions. J. Bacteriol. 145: 348–351.
Sundaresen, V., Jones, J.D.G., Ow, D.W. and Ausubel, F.M. 1983. Conservation of nitrogenase promoters from Rhizobium meliloti and Klebsiella pneumoniae. Nature 301: 728–731.
Sundaresan, V., Ow, D.W. and Ausubel, F.M. 1983. Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins. Proc. Natl. Acad. Sci. USA 80: 4030–4034.
Szeto, W.W., Zimmerman, J.L., Sundaresan, V. and Ausubel, F.M. 1984. A Rhizobium meliloti symbiotic regulatory gene. Cell 36: 1035–1043.
Kahn, M.L. and Timblin, C.R. 1984. Gene fusion vehicles for the analysis of gene expression in Rhizobium meliloti. J. Bacteriol. 158: 1070–1077.
Keyser, H.H., Bohlool, B.B., Hu, T.S. and Weber, D.F. 1982. Fast-growing rhizobia isolated from root nodules of soybean. Science 215: 1631–1632.
Sadowsky, M.J., Keyser, H.H. and Bohlool, B.B. 1983. Biochemical characterization of fast- and slow-growing rhizobia that nodulate soybeans. Int. J. Syst. Bacteriol. 33: 716–722.
Masterson, R.V., Russell, P.R. and Atherly, A.G. 1982. Nitrogen Fixation (nif) genes and large plasmids of Rhizobium japonicum. J. Bacteriol. 152: 928–931.
Sadowsky, M.J. and Bohlool, B.B. 1983. Possible involvement of a megaplasmid in nodulation of soybeans by fast growing rhizobia from China. Appl. Environ. Microbiol. 46: 906–911.
Castilho, B.A., Olfson, P. and Casadaban, M.J. 1984. Plasmid insertion mutagenesis and lac fusion with mini-bacteriophage transposon. J. Bacteriol. 158: 488–495.
Casadaban, M.J. and Cohen, S.N. 1979 Lactose genes fused to exogenous promoters in one step using a mu-lac bacteriophage. Proc. Natl. Acad. Sci. USA 76: 4530–4533.
Wanner, B.W. and McSharry, R. 1982. Phosphate-controlled gene expression in Escherichia coli K12 using mu-dI directed lacZ fusions. J. Mol. Biol. 158: 347–363.
Hughes, K.T. and Roth, J.R. 1984. Conditionally transposition-defective derivative of mu-dI (Amp, lac). J. Bacteriol. 159: 130–137.
Lee, J.H., Heffernan, L. and Wilcox, G. 1980. Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage mu-d (Apr, lac). J. Bacteriol. 143: 1325–1331.
Rosenfeld, S.A. and Brenchley, J.E. 1980. Bacteriophage P1 as a vehicle for mu mutagenesis of Salmonella typhimurium. J. Bacteriol. 144: 848–851.
Jayasawal, R.K., Bressan, R.A. and Handa, A.K. 1984. Mutagenesis of Erwinia cartovora subsp. cartovora with bacteriophage mu-dI (Apr, lac, cts62): construction of his-lac gene fusions. J. Bacteriol. 158: 764–766.
Casadaban, M.J. and Chou, J. 1984. In vivo formation of gene fusions encoding hybrid B-galactosidase proteins in one step with transposable Mu-lac transducing phage. Proc. Natl. Acad. Sci. USA 81: 535–539.
Cozzarelli, N.R., Kelly, R.B. and Kornberg, A. 1968. A minute circular DNA from E. coli 15. Proc. Natl. Acad. Sci. USA 60: 992–999.
Konarska-Kozlowska, M. and Iyer, V.N. 1981. Physical and genetic organization of the IncN-group plasmid pCU1. Gene 14: 195–204.
Leemans, J., Inze, D., Villarroel, R., Engler, G., Harnalsteens, J.P., DeBlock, M. and Van Montagu, M. 1981. Plasmid mobilization as a tool for in vivo genetic engineering, p. 401–409. In Molecular biology, pathogenicity, and ecology of bacterial plasmids. S. B. Levy, R. C. Clowes, and E. L. Koenig (eds.) Plenum Press, New York.
Ruvkun, G.B., Long, S.R., Meade, H.M., van den Bos, R.C. and Ausubel, F.M. 1982. ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen-fixation genes. J. Mol. Appl. Genet. 1: 405–418.
Legocki, R. 1981. Identification of “nodule-specific” plant proteins (nodulins) from soybean root nodules. Ph.D. Thesis., Department of Biology, McGill University, Montreal, Quebec, Canada.
Allen, O.N. and Allen, E.K. 1981. The leguminosae. A source book of characteristics, uses and nodulation: University of Wisconsin Press, Madison.
Vincent, J.M. 1970. A manual for the practical study of root-nodule bacteria. IBP Handbook 15. Blackwell Scientific Publications, Oxford.
Faelen, M., Huisman, O. and Toussaint, A. 1978. Involvement of phage Mu-1 early functions in Mu-mediated chromosomal rearrangements. Nature 271: 580–582.
Nano, F.E. and Kaplan, S. 1984. Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides. J. Bacteriol. 158: 1094–1103.
Noel, K.D., Sanchez, A., Fernandez, L., Leemans, T. and Cevallos, M.A. 1984. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions. J. Bacteriol. 158: 148–155.
Hirsch, P.R., Van Montagu, M., Johnston, A.W.B., Brewin, N.J. and Schell, J. 1980. Physical identification of bacteriocinogenic, nodulation, and other plasmids in Rhizobium leguminosarum. J. Gen. Microbiol. 120: 403–412.
Beringer, J.E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84: 188–198.
Miller, J.H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Buchanan-Wollaston, A.V., Beringer, J.E., Brewin, N.J., Hirsch, P.R. and Johnston, A.W.B. 1980. Isolation of symbiotically defective mutants of Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid. Mol. Gen. Genet. 178: 185–190.
Haugland, R. and Verma, D.P.S. 1981. Interspecific plasmid and genomic DNA homologies and localization of nif genes in effective and ineffective strains of Rhizobium japonicum. J. Mol. Appl. Genet. 1: 205–217.
Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. In Molecular cloning a laboratory manual, p. 109. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Southern, E. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503.
Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.
Casadaban, M.J., Chou, J. and Cohen, S.N. 1980. In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J. Bacteriol. 143: 971–980.
Leonard, L.T. 1943. A simple assembly for use in the testing of cultures of rhizobia. J. Bacteriol. 45: 523–527.
Kosslak, R.M. and Bohlool, B.B. 1984. Suppression of nodule development of one side of a split root system of soybeans caused by prior inoculation of the other side. Plant Physiol. 75: 125–130.
Hoagland, D.R. and Arnon, D.I. 1938. The water culture method for growing plants without soil. California A9, Exp. Std. Circular ♯347.
Hardy, R.W.F., Holsten, R.D., Jackson, E.K. and Burns, R.C. 1968. The C2H2-C2H4 assay for nitrogen fixation; laboratory and field evaluation. Plant Physiol. 43: 1185–1204.
Bolivar, F. and Backman, K. 1979. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 68: 243.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Olson, E., Sadowsky, M. & Verma, D. Identification of Genes Involved in the Rhizobium-Legume Symbiosis by Mu-dI (Kan, lac)-Generated Transcription Fusions. Nat Biotechnol 3, 143–149 (1985). https://doi.org/10.1038/nbt0285-143
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0285-143
This article is cited by
-
Effect of divalent cations on succinate transport in Rhizobium tropici, R. leguminosarum bv phaseoli and R. loti
World Journal of Microbiology & Biotechnology (1994)
-
A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins
Plant Molecular Biology (1987)
-
Galactose metabolism inErwinia carotovora Subsp.carotovora
Current Microbiology (1986)
-
Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum
Archives of Microbiology (1986)
-
The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191
Plant Molecular Biology (1986)