Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid.

Plasmids and stability of symbiotic properties of Rhizobium trifolii.

A conjugal plasmid which encodes both peak nodulation genes and nitrogenase genes, and which is labeled with the transposon Tn5, was transferred to a wild-type Rhizobium trifolii strain to examine the stability and expression of the host range and fixation (Fix+) phenotypes. Transconjugates were isolated which were shown to initially form nitrogen-fixing nodules (Nod+ Fix+) on both clovers and peas. These hybrid strains were then repeatedly passaged through either pea or clover nodules or onto a solid agar medium to determine whether these broadened-host-range characteristics were stably maintained.

Molecular mechanism for loss of nodulation properties of Rhizobium trifolii.

Of 18 Rhizobium trifolii strains tested, 12 showed a high frequency of loss of nodulation ability after incubation in cultures at elevated temperatures. A correlation between loss of nodulation ability and loss of a large plasmid was demonstrated for R. trifolii.

Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation.

A transposon (Tn5)-induced mutant (strain ANU437) of Rhizobium trifolii was isolated in which no water-soluble exopolysaccharide (EPS) could be detected. This mutant was also incapable of forming nitrogen-fixing root nodules on clover plants. Molecular cloning has demonstrated that the Tn5 transposon was responsible for both of these mutant phenotypes and that there is a direct correlation between EPS synthesis in this bacterial strain and its ability to carry out symbiotic nitrogen fixation.

Specific adsorption of bacteria to clover root hairs, related to the presence of the plasmid pWZ2 in cells of Rhizobium trifolii.

Rhizobium trifolii strains containing plasmid pWZ2, the genetic element indispensable for nodule induction, showed a strong specific adsorption of cells to clover roots. Elimination of the plasmid pWZ2 from bacteria caused loss of adsorption capability, and this property could be recoverd by introduction of the plasmid into bacterial cells. The results obtained indicate that the plasmid pWZ2 contains gene(s) controlling the first stage of Rhizobium-legume symbiosis which is a specific adsorption of nodule bacteria to a suitable plant host.