Division of Rhizobitoxine-Producing and Hydrogen-Uptake Positive Strains of Bradyrhizobium japonicum by nifDKE Sequence Divergence

Genomic digests from 25 strains of Bradyrhizobium japonicum,for which the phenotypes have been determined with respect toproduction of rhizobitoxine, hydrogen uptake (Hup) and compositionof extracellular polysaccharide (EPS), were hybridized withprobe DNAs of the nifDK and nifE genes of B. japonicum USDA110. The degree of the estimated base substitution in and aroundnifDKE clearly divided the strains of B. japonicum into twomarkedly divergent groups, which were designated as genotypeI and II. Moreover, a strict correlation was observed betweenthese genotypes, production of rhizobitoxine and EPS composition.The genotype I strains produced no rhizobitoxine and an EPScomposed of glucose, mannose, galactose, 4-O-methyl galactoseand galacturonic acid, whereas the genotype II strains producedrhizobitoxine and an EPS composed of rhamnose and 4-O-methylglucuronic acid. Hup⁺ strains were confined exclusively to thegenotype I. Hind III digests of genomic DNAs from the 25 strainswere hybridized with probe DNA of structural genes for the uptakehydrogenase from B. japonicum. In 23 wild-type strains, Hup⁺strains generated a 5.9-kb band that hybridized to the probeunder high-stringency conditions, while Hup^– strains didnot generate the band. These results suggest that the genotypesI and II are two highly divergent evolutionary lines that definea marked division of various phenotypes, such as productionof rhizobitoxine, EPS composition and hydrogen uptake. (Received July 10, 1989; Accepted November 4, 1989)