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

Abstract

Genomic digests from 25 strains of Bradyrhizobium japonicum, for which the phenotypes have been determined with respect to production of rhizobitoxine, hydrogen uptake (Hup) and composition of extracellular polysaccharide (EPS), were hybridized with probe DNAs of the nifDK and nifE genes of B. japonicum USDA 110. The degree of the estimated base substitution in and around nifDKE clearly divided the strains of B. japonicum into two markedly divergent groups, which were designated as genotype I and II. Moreover, a strict correlation was observed between these genotypes, production of rhizobitoxine and EPS composition. The genotype I strains produced no rhizobitoxine and an EPS composed of glucose, mannose, galactose, 4-Omethyl galactose and galacturonic acid, whereas the genotype II strains produced rhizobitoxine and an EPS composed of rhamnose and 4-O-methyl glucuronic acid. strains were confined exclusively to the genotype I. Hind III digests of genomic DNAs from the 25 strains were hybridized with probe DNA of structural genes for the uptake hydrogenase from B. japonicum. In 23 wild-type strains, strains generated a 5.9-kb band that hybridized to the probe under high-stringency conditions, while Hup strains did not generate the band. These results suggest that the genotypes I and II are two highly divergent evolutionary lines that define a marked division of various phenotypes, such as production of rhizobitoxine, EPS composition and hydrogen uptake.