Symbiotic and galactose utilization properties of phage RMP64-resistant mutants affecting three complementation groups in Rhizobium meliloti

Abstract

Random Tn5 insertional mutants were induced in Rhizobium meliloti Rmd201, a streptomycin-resistant mutant of AK631 (which is itself a compact colony morphology mutant of the wild-type strain Rm41), and screened for sensitivity to a set of 16 phages. Out of 3000 mutants 240 were found to be phage-resistant. The phage-resistant mutants were separable into six groups on the basis of their sensitivity pattern against test phages. Nodulation tests on alfalfa showed that although all the phage-resistant mutants induced root nodules, 7 mutants out of 12 of a class resistant to phage RMP64 (Sxf⁻) induced atypical nodules that were ineffective in nitrogen fixation (Fix⁻). The aberrant nodules were small, white, contained only a few bacteria and no bacteroids, and phenotypically resembled nodules elicited by already known exoB, exoH, ndvA and ndvB mutants of R. meliloti. Spontaneous mutants selected for resistance to RMP64 also fell into two groups: Fix⁺ and Fix⁻. Genetic complementation tests between the Sxf⁻ mutants defined three genes sxfA, sxfB and sxfC, of which sxfA and sxfB comprise an operon. These also demonstrated that sxfA, sxfB and sxfC must be located on the same replicon. All the Sxf⁻ mutants were Calcofluor-positive, like their parent strains Rmd201 and AK631. Characterization of carbohydrate metabolism of the mutants revealed that while the sxfA (Fix⁻) and sxfB (Fix⁺) mutants utilized galactose as sole carbon source, sxfC (Fix⁻) mutants did not. It has been concluded that sxf A, sxfB and sxfC are new genetic loci and that sxfA and sxfC have roles in nodule invasion and development.