Characteristics of Plasmids in Rhizobium huakuii

Abstract.

Rhizobium huakuii nodulates Astragalus sinicus, an important green manuring crop in Southern China, which can be used as forage. The plasmid profiles of 154 R. huakuii strains were examined with the Eckhardt procedure. The plasmid number of the strains varied from one to five, and their molecular weights were estimated from 42 to 600 mDa or more. The plasmids were hybridized with probes nodABC and nifHDK. The results showed that there was one plasmid carrying the nod and nif genes in the strains that harbor two or more plasmids, and the molecular weights of the symbiotic plasmids varied from 117 to 251 mDa. Homology was not observed on plasmids in the strains having only one plasmid; presumably the symbiotic genes are on the chromosome. Plasmid curing was carried out with the Bacillus subtilus sacB to generate derivatives of Rhizobium huakuii strain CH203, which harbors three plasmids, pRHa(97MD), pRHb(168MD), and pRHc(251MD). The largest plasmid (pRHc) carried both nodulation and nitrogen fixation genes. When pRHc was cured, the strain lost its symbiotic ability. The other two plasmids were also related to symbiosis. The derivative cured of pRHb did not nodulate on the host plant, had an altered lipopolysaccharide, and grew much more slowly than the parent strain. Curing of the smallest plasmid (pRHa) resulted in delaying the strain nodulation and made it lose nitrogen fixation ability. Curing of each plasmid in strain CH203 reduced its acid tolerance. Complementation of plasmid-cured strains with appropriate plasmids restored their original phenotypes.