Biochemical and genetical aspects of the taxonomy of Rhizobium japonicum

Summary The major distinguishing characteristics of fifty diverse strains ofR. japonicum were surveyed. Nodulation onGlycine max, slow growth rate in yeast extract-mannitol broth, alkaline reaction with no serum zone in litmus milk, and lack of an acid reaction in either rhamnose or xylose media rather consistently separatedR. japonicum from other rhizobium species. The degree of relatedness between strains ofR. japonicum was measured using antibiotic resistance, immunological reactions and DNA homology. Generally the strains were resistant to erythromycin, polymyxin B, chloramphenicol, and sensitive to novobiocin. This response pattern differred from control strains consisting ofAgrobacterium tumefaciens, R. meliloti, andR. phaseoli. Response to six other antibiotics was quite random. Serological data show that none of 38 strains shared all somatic antigens. There was a relatively homogeneous group consisting of about ? of the strains tested. Two other, smaller diffuse clusters of strains shared some common antigens, while about one-half of the strains tested reacted only with their own antisera or perhaps reacted with antiserum of one other strain. Comparisons of the theoretical DNA homology between 26 strains showed three statistically significant but overlapping clusters. However, the degree of homology between strains at the extremes of these clusters is only about 70 per cent. Antibiotic response, serological reactions, and DNA homology did not correlate well but demonstrate considerable heterogeneity within this species. Lipid analysis of 5 strains showed the major lipids present to be phospholipids including large amounts of phosphatidylcholine and phosphatidylethanolamine. Such large levels of phosphatidylcholine have previously been found only in the pseudomonads although smaller amounts have been reported in Agrobacterium. Several workers have devised taxonomic schemes based on lipid composition and have thus linked Agrobacterium to the Pseudomonadales. The findings reported here tend to linkR. japonicum to both Agrobacterium and the Pseudomonadales. Glucose and gluconate catabolism were investigated both by the radio-respirometric method and by assaying for key enzymes of the major energy-producing pathways. The results show glucose is metabolized solely by the Entner-Doudoroff Pathway in the one strain studied. The Embden Meyerhoff Parnas and pentose-phosphate pathways were absent. While an inducible Entner-Doudoroff Pathway has been found in many gram-negative Eubacteriales, glucose metabolism solely by this pathway has been shown in the Pseudomonadales, linkingR. japonicum to this order. Gluconate catabolism occurred via the Entner-Doudoroff Pathway but an additional pathway was participating. Evidence was found that this pathway is the ketogluconate pathway previously reported only in the Pseudomonadales. Amino acid analysis patterns and distribution of label from radioactive aspartic acid indicate that glutamate is synthesized in the one strain studied, partly via a pathway previously reported inAcetobacter suboxydans. These investigations indicate thatR. japonicum is a recognizable species, albeit an extremely heterogeneous one. Metabolic data suggest a relatively close genetic relationship between this species and the Pseudomonadales. There is evidence thatR. japonicum may be more closely related to Agrobacterium than to the ‘fast growing’ species of Rhizobium, but because the data indicate thatR. japonicum is an authentic species, combination of these organisms and Agrobacterium is not presently warranted.