Summary
The slow-growing soybean symbiont, Rhizobium japonicum, has not readily been accessible so far to classical mutational analysis of genes responsible for symbiotic nitrogen fixation. We have overcome part of this problem by the successful application of a site-directed mutagenesis technique to this organism. The following steps are involved: (i) local Tn5 mutagenesis, in E. coli, of cloned R. japonicum DNA (e.g. the nifDK operon); (ii) conjugational transfer of the mutated DNA into R. japonicum using vectors which are unable to replicate there; (iii) selection of R. japonicum exconjugants which have exchanged their wild-type genomic DNA region for the Tn5-containing fragment by homologous recombination. While using this technique it appeared mandatory to distinguish double-crossover-events (true replacements) from single-crossover events (replicon fusions or cointegrations). Only the true replacement mutants were genetically stable; their phenotypes were determined with respect to nodulation (Nod) and nitrogen fixation (Fix) by plant infection tests. Tn5 mutations within nifD and nifK caused a Nod+ Fix- phenotype, whereas mutants with insertions in the immediate vicinity on either side of nifDK were found to be Nod+ Fix+, suggesting that genes flanking nifDK may not be involved in the nitrogen fixing symbiosis. Nodule reisolates were found to carry Tn5 at their original locations.
Similar content being viewed by others
References
Cen Y, Bender GL, Trinick MJ, Morrison NA, Scott KF, Gresshoff PM, Shine J, Rolfe BG (1982) Transposon mutagenesis in rhizobia which can nodulate both legumes and the nonlegume Parasponia. Appl Environ Microbiol 43:233–236
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Chinault AC, Carbon J (1979) Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the LEU2 gene on yeast chromosome III. Gene 5:111–126
Clewell DB, Helinski DR (1969) Supercoiled DNA protein complexes in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1166
Corbin D, Barran L, Ditta G (1983) Organization and expression of Rhizobium meliloti nitrogen fixation genes. Proc Natl Acad Sci USA 80:3005–3009
Davis RW, Botstein D, Roth JR (1980) Advanced bacterial genetics. Cold Spring Harbor Laboratory. cold Spring Harbor, New York
Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Doctor F, Modi VV (1976) Genetic mapping of leucine and isoleucine-valine loci in Rhizobium japonicum. J Bacteriol 126:997–998
Fuhrmann M, Hennecke H (1982) Coding properties of cloned nitrogenase structural genes from Rhizobium japonicum. Mol Gen Genet 187:417–425
Grunstein M, Hogness DS (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965
Grunstein M, Wallis J (1979) Colony hybridization. Methods Enzymol 68:379–388
Haugland R, Verma DPS (1981) Interspecific plasmid and genomic DNA sequence homologies and localization of nif genes in effective and ineffective strains of Rhizobium japonicum. J Mol Appl Genet 1:205–217
Hennecke H (1981) Recombinant plasmids carrying nitrogen fixation genes from Rhizobium japonicum. Nature 291:354–355
Hennecke H, Günther I, Binder F (1982) A novel cloning vector for the direct selection of recombinant DNA in E. coli. Gene 19:231–234
Hörcher R, Wilcockson J, Werner D (1980) Screening for mutants of Rhizobium japonicum with defects in nitrogen fixing ability. Z Naturforsch 35c:729–732
Hu N, Messing J (1982) The making of strand-specific M13 probes. Gene 17:271–277
Jorgensen RA, Rothstein SJ, Reznikoff WS (1979) A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet 177:65–72
Kaluza K, Fuhrmann M, Hahn M, Regensburger B, Hennecke H (1983) In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated. J Bacteriol 155:915–918
Kennedy C, Dreyfus B, Brockwell J (1981) Transfer, maintenace and expression of P plasmids in strains of cowpea rhizobia. J Gen Microbiol 125:233–240
Klein RD, Selsing E, Wells RD (1980) A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91
Kuykendall LD (1979) Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum. Appl Environ Microbiol 37:862–866
Maier RJ, Brill WJ (1976) Ineffective and non-nodulating mutants strains of Rhizobium japonicum. J Bacteriol 127:763–769
Maniatis T, Jeffrey A, Kleid DG (1975) Nucleotide sequence of the rightward operator of phage λ. Proc Natl Acad Sci USA 72:1184–1188
Masterson RV, Russell PR, Atherly AG (1982) Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum. J Bacteriol 152:928–931
Pilacinski WP, Schmidt EL (1981) Plasmid transfer within and between serologically distinct strains of Rhizobium japonicum, using antibiotic resistance mutants and auxotrophs. J Bacteriol 145:1025–1030
Regensburger B, Hennecke H (1983) RNA polymerase from Rhizobium japonicum. Arch Microbiol 135:103–109
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in procaryotes. Nature 289:85–88
Ruvkun GB, Sundaresan V, Ausubel FM (1982) Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell 29:551–559
Simon R, Priefer U, Pühler A (1983) Vector plasmids for in vivo and in vitro manipulations of gram-negative bacteria. In: Pühler A (ed) Molecular genetics of the bacteria-plant interaction. Springer, Berlin Heidelberg New York, in press
Southern EM (1975) Detection of specific sequence among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Stacey G, Paau AS, Noel KD, Maier RJ, Silver LE, Brill WJ (1982) Mutants of Rhizobium japonicum defective in nodulation. Arch Microbiol 132:219–224
Verma DPS, Long S (1983) The molecular biology of Rhizobium-legume symbiosis. Int Rev Cytol, Suppl 14:211–245
Vincent JM (1970) A manual for the practical study of the root-nodule bacteria. Blackwell scientific Publications, Oxford
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Hahn, M., Hennecke, H. Localized mutagenesis in Rhizobium japonicum . Molec. Gen. Genet. 193, 46–52 (1984). https://doi.org/10.1007/BF00327412
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00327412