Date of Award
Doctor of Philosophy
The research presented in this dissertation represents an initial investigation into the genetics of Rhizobium japonicum, the nitrogen-fixing bacterium which lives in symbiotic association with soybean plants. Genetic studies with any organism requires that methods be available for exchanging genetic information between strains. Because normal methods of gene transfer (conjugation, transduction, and transformation) work poorly or not at all with R. japonicum, a new method of transformation was developed. For the introduction of foreign plasmid DNA, R. japonicum spheroplasts were prepared by culturing cells in the presence of glycine followed by treatment with lysozyme. The speroplasts were examined by scanning electron microscopy and found to be morphologically similar to the bacteroid forms found in soybean root nodules, suggesting that the mechanisms of bacteroid and spheroplast formation may be similar. The P-group resistance plasmids RP1 and RP4, and the Agrobacterium tumefaciens tumor inducing (Ti) plasmid were introduced into R. japonicum by polyethelene-glycol induced transformation of spheroplasts. After cell wall regeneration, transformants were recovered by selecting for plasmid determinants. Plant nodulation, nitrogen-fixation, serological, and bacterial genetics studies revealed that the transformants were derived from the parental strains and possessed the introduced plasmid genetic markers. Agarose gel electrophoresis, restriction enzyme analysis, and DNA hybridization studies showed that many of the transformant strains had undergone extensive genome rearrangements, possibly as a result of the introduction of foreign DNA. In the RP1 transformants, chromosomal DNA was found to have transposed into a large indigenous plasmid of R. japonicum, producing a new, larger plasmid. The introduced R-plasmid DNA was found to be chromosomally integrated. R. japonicum cells transformed with the A. tumefaciens Ti plasmid were found to have undergone similar genome rearrangements, with a section of chromosomal DNA becoming excised and forming a second, chromosomally-derived plasmid. A small section of Ti plasmid DNA, between 20 and 30 Megadaltons in size and including octopine catabolism genes, was found to be chromosomally integrated in the R. japonicum Ti plasmid transformants. Apparently, a similar section of chromosomal DNA was involved in all of the genomic rearrangements observed in the R. japonicum RP1 and Ti plasmid transformant strains.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
James Owen Berry
Berry, James Owen, "Gene transfer and genome rearrangements in Rhizobium japonicum " (1982). Retrospective Theses and Dissertations. 8332.