Summary
We report here successful mutagenesis with Transposon Tn5 of three slow-growing strains of Rhizobium japonicum USDA 122, 61A76, USDA 74 and one fast-growing strain, USDA 191. Strains were chosen as representatives of different DNA homology and serogroups of this divergent species, which effectively nodulate North American soybean cultivars. The source of Tn5 was the suicide plasmid pGS9, which possesses broad host range N-type transfer genes in a narrow host range p15A replicon. The selection of Tn5 mutants was facilitated by the expression of the Tn5 encoded streptomycin gene in R. japonicum. Kanamycin and streptomycin resistant colonies appeared from interspecific crosses with E. coli at optimal frequencies of 10-6 for R. japonicum USDA 61A76 and USDA 191 and 5x10-7 for R. japonicum USDA 122 and USDA 74. Altogether, 6550 Tn5 mutants were isolated in USDA 122 and 61A76, and a small number from USDA 74 and USDA 191. Colony hybridization showed that all tested mutants of 61A76 and USDA 122 contained Tn5. Physical analysis of total DNAs from representative numbers of USDA 122, 61A76 and USDA 191 mutants revealed that each of them carried one copy of the transposon integrated randomly in the genome. This was also true for most USDA 74 mutants. Screening of mutants for auxotrophy showed frequencies of 0.2% for USDA 122 and 0.08% for 61A76. Several symbiotically defective mutants were identified on plants, Glycine soja and G. max.
Similar content being viewed by others
References
Berry JO, Atherly AG (1984) Induced plasmid-genome rearrangements in Rhizobium japonicum. J Bacteriol 157:218–224
Beringer JE, Benyon JL, Buchanan-Wollaston AV, Johnston AWB (1978) Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature 276:633–634
Bishop PE, Guevara JG, Engelke FA, Evans HF (1976) Relation between glutamine synthetase and nitrogenase activities in the symbiotic association between Rhizobium japonicum and Glycine max. Plant Physiol 57:542–546
Chang ACY, Cohen SU (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from p15A cryptic mini-plasmid. J Bacteriol 134:1141–1156
Fuhrmann M, Hennecke H (1982) Coding properties of cloned nitrogenase structural genes from Rhizobium japonicum. Mol Gen Genet 187:419–425
Hahn M, Hennecke H (1984) Localized mutagenesis in Rhizobium japonicum. Mol Gen Genet 193:46–52
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
Holliday R (1956) A new method for the identification of biochemical mutants of micro-organisms. Nature (London) 178:987
Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 14:193–197
Keyser HH, Bohlool BB, Hu TS, Weber DF (1982) Fast-growing rhizobia isolated from root nodules of soybeans. Science 215:1631–1632
Kosslak R, Bohlool BB (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: in press
Ludwig AL (1984) Rhizobium free-living nitrogen fixation occurs in specialized non-growing cells. Proc Natl Acad Sci USA 81:1566–1569
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning — A laboratory manual. pp 80–85. Cold Spring Harbor Laboratories NY
Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122
Meyer MC, Pueppke SG (1980) Differentiation of Rhizobium japonicum strain derivatives by antibiotic sensitivity patterns, lectin binding and utilization of biochemicals. Can J Microbiol 26:600–612
Putnoky P, Kiss GB, Ott I, Kondorosi A (1983) Tn5 carries a streptomycin resistance determinant downstream from the kanamycin-resistance gene. Mol Gen Genet 191:288–294
Ruvkun GB, Long SR, Meade HM, van den Bos RC, Ausubel FM (1982) ISRml: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes. Mol Appl Genet 1:405–418
Selvaraj G, Iyer VN (1983) Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J Bacteriol 158:580–589
Shanabruck WG, Walker BC (1980) Localization of the plasmid (pKM101) gene(s) involved in recA+, leuA+-dependent mutagenesis. Mol Gen Genet 179:289–297
Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791
Sutton BCS, Stanley J, Zelechowska MG, Verma DPS (1984) Isolation and expression of cloned DNA from Rhizobium japonicum encoding an early soybean nodulation function. J Bacteriol 158:920–927
Werner D, Wilcockson I, Zimmermann E (1975) Adsorption and selection of Rhizobia with ion-exchange papers. Arch Microbiol 105:27–32
Yelton MM, Yang SS, Edie SA, Lim ST (1983) Characterization of an effective, salt-tolerant fast-growing strain of Rhizobium japonicum. J Gen Microbiol 129:1537–1547
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
Rostas, K., Sista, P.R., Stanley, J. et al. Transposon mutagenesis of Rhizobium japonicum . Mol Gen Genet 197, 230–235 (1984). https://doi.org/10.1007/BF00330968
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330968