Physical and genetic characterization of Rhizobium meliloti symbiotic mutants

A set of 19 symbiotic mutants of Rhizobium meliloti obtained by a Tn5 "suicide plasmid" mutagenesis procedure was characterized genetically and physically. As part of this characterization, we showed that R. meliloti strain 1021, like other R. meliloti strains, contains a very large indigenous plasmid (greater than 300 Md) that carries the structural genes for nitrogenase (nifHDK genes). Among the 19 symbiotic mutations studied, at least six were shown to reside on the megaplasmid. By a "walking procedure" we obtained from a cosmid clone bank a set of overlapping cosmids that contained megaplasmid sequences contiguous to nifHDK. A 90 kb region of contiguous DNA from these cosmids was used to probe the mutant strains for rearrangements within this region. The same six mutations that were located on the megaplasmid mapped within the 90 kb region examined, which included the structural genes for nitrogenase (nifHDK). A majority of the mutations characterized in this study could not be correlated with a bona fide Tn5 insertion into a symbiotic gene.