A NEW RHIZOBIUM-MELILOTI SYMBIOTIC MUTANT ISOLATED AFTER INTRODUCING FRANKIA DNA-SEQUENCE INTO A NODA=TN5 STRAIN

Our goal was to isolate Frankia genes by complementation of Rhizobium meliloti mutants. By mating a library of Frankia genes in pLAFR3 with a R. meliloti nodA = Tn5 recipient, we isolated a clone that appeared to confer nodulation functions, although the apparently complemented transconjugant (Rm5610 NS6) remained Fix- on alfalfa. Further experiments have shown that the Nod+ Fix- phenotype was due to genomic mutations in R. meliloti and was independent of the Frankia DNA. Rm5610 NS6 had apparently lost Tn5 and simultaneously acquired neomycin resistance. We subsequently isolated a spontaneous neomycin-resistant derivative of R. meliloti 1021 that appeared to be identical to NS6. The nodules elicited by the neomycin-resistant mutants contained infection threads that penetrated the nodule; rhizobia were released from the threads, but they did not differentiate into elongate bacteroids. We examined nodulin gene expression in the ineffective mutant-induced nodules by northern blot and by in situ hybridization analyses and found that transcripts for the nodulins MsENOD2, MsENOD12-1, and leg-hemoglobin were detectable in the same cellular location as in wild type-induced nodules. These results confirm that the expression of these nodulin genes is regulated by signals exclusive of bacteroid differentiation and nitrogen fixation.