Derivation and molecular characterization of symbiotically deficient mutants of Rhizobium meliloti

A collection of symbiotically defective mutants of Rhizobium meliloti JJ1c10 was derived by Tn5 mutagenesis using the suicide vector pGS9. They include two Nod− and about 250 Fix− mutants. The mutants were found to be heterogenous in acetylene reduction activity and in the morphology and ultrastructure of the nodules which they induced. Over 90% were found to contain bona fide Tn5 insertions in a variety of DNA restriction fragments. When Tn5-carrying DNA segments cloned from 24 of the mutants were introduced into the equivalent location in the genome of the wild-type strain by recombination-mediated replacement, only eight produced a symbiotically defective phenotype similar to that of the original mutant. This result indicated that many of the symbiosis mutations were not directly caused by Tn5 insertion. DNA segments apparently containing mutated fix genes but not containing Tn5 were found in eight mutants by identifying cosmids carrying wild-type DNA which complemented their symbiosis defects. Probing of the DNA of these mutants with their complementing cosmids revealed no detectable physical alteration of the homologous DNA. A segment of DNA including the hsn and nifHDK genes was favoured for these non-Tn5 mutations. Three regions of the genome in which Tn5 caused fix mutations were identified. One of these was the known megaplasmid nod-nif region. The other two regions, designated fix-e5 and fix-h21, were found to be chromosomal. Mutants in one of these chromosomal regions fluoresced more intensely on calcofluor plates than the wild type.