Indigenous plasmids in Anabaena azollae: Their taxonomic distribution and existence of regions of homology with symbiotic genes of Rhizobium

The method of horizontal agarose gel electrophoresis was used to demonstrate the presence of indigenous plasmid DNAs in different isolates of the symbiotic cyanobacterium Anabaena azollae. All isolates extracted from seven distinct species of the host fern Azolla were found to possess one to three cryptic plasmids ranging in sizes from 35 to 100 MDa (million daltons). Anabaenas isolated from Azolla caroliniana, Az. nilotica, and Az. pinnata species contained a single plasmid band of molecular mass approximately 60 MDa, whereas other endosymbiotic cyanobacteria extracted from Azolla filiculoides, Az. rubra, Az. mexicana, and Az. microphylla were shown to possess two or three covalently closed circular (CCC) DNAs. Cloned DNA fragments derived from the plasmid sequences of two different An. azollae isolates were used as hybridization probes. Hybridization data indicated that these symbiotic cyanobacteria possess different but related plasmid species and that it is possible to construct specific plasmid DNA probes capable of distinguishing among several strains of the symbiotic anabaenas. Several heterologous DNA probes, including Rhizobium symbiotic genes, were used to seek homologous sequences on the An. azollae plasmids. DNA sequences homologous to the nod box and nodMN genes were present on the Anabaena plasmids. Moreover, homology of a key Rhizobium exopolysaccharide (exoY) gene to the An. azollae CCC DNAs was detected. In addition, the introduction of the An. azollae plasmid clone into Rhizobium Exo^- mutant (exoY) resulted in the Exo⁺ transconjugants. Those findings suggest that some of the An. azollae plasmids may play a role in symbiotic interactions with Azolla fern.