Speciation in the genus rhizobium as demonstrated by DNA antibody techniques, serology and DNA homology

Abstract

The results obtained from precipitin tests with antibodies to DNA prepared by the phenol-chloroform (P.C.) method from Rhizobium meliloti 2748 and Rhizobium trifolii 2668 agreed with those obtained from agglutination tests with the antibodies to R. meliloti 2748 and R. trifolii 2668. On exposure to deoxyribonuclease, denatured DNA prepared by P.C. method from homologous strains did not show loss of reactivity to antibodies to DNA 2748 or DNA 2668. It was concluded that these antibodies were not against DNA but were due to contaminants, possibly polysaccharides, that were present in the DNA preparation.

Highly purified DNA which was substantially free from protein, RNA and polysaccharides was prepared by a combination of phenol chloroform and hydroxyapatite-urea (P.C./HA) methods. The combined methods gave a low yield. It was found that antibody to DNA from Rhizobium meliloti 2748 prepared by P.C./HA methods and antibody to calf thymus DNA purified using the hydroxyapatite-urea method reacted with heat denatured DNA from calf thymus, Rhizobium spp and Escherichia coli using agar diffusion. However, they did not react with native DNA purified by stepwise elution chromatography on a hydroxyapatite column. Anti TINA antibodies were shown to be immunoglobulin M class and in every respect similar to the established reports concerning antibodies produced in response to methylated bovine serum albumin-DNA complex immunization.

Using the fluorescent antibody (FA) technique and enzyme-linked immunosorbent assay (ELISA), antibody to R. trifolii 2668 was found to be species and strain specific. Cross reactions among strains of R. meliloti were encountered against the FA and enzyme linked antibody of that bacterium. The ELISA was 20 times more sensitive than the FA method. Although both techniques were easy to perform, the obvious advantage of ELISA was that a fluorescence microscope was not necessary since estimation of the results could be made visually.

The transmission of streptomycin resistance to sensitive cultures of Rhizobium spp by means of DNA isolated from R. meliloti 2748 and R. meliloti 2755 that were resistant to high concentration (1,000 µg ml⁻¹) of streptomycin was possible only among strains of R. meliloti. Transformation frequency was calculated as the number of transformants growing on streptomycin agar divided by the total number of cells plated on agar without streptomycin. Genetically homologous DNA had a better chance of bringing about transformation than heterologous DNA. The transformable strains of R. meliloti were indistinguishable serologically.

DNA homologies found for the strains of Rhizobium species investigated, correlated well with the serological relationships. Strains that could not be distinguished by serological methods gave high % degree of binding (D) whereas strains that could be separated by FA or ELISA gave low or moderate % D. A significant degree of genetic relatedness among the R. meliloti strains tested was disclosed by the DNA hybridization technique.

The use of serological methods described here in taxonomic studies of Rhizobium offers considerable scope for improving the present classification of this genus to indicate more clearly the relationships between species.