Characterization of a nitrogen-fixation (nif) gene cluster from Anabaena azollae 1a shows that closely related cyanobacteria have highly variable but structured intergenic regions

The exact identity of cyanobacteria that have been cultured from symbiotic associations with the water fern Azolla spp., whether they are required in the symbiotic process, and their relationship to the symbiotic species, is a matter of some debate. We have characterized a 6 kb region containing the nifB operon and the nifH gene from cyanobacterium Anabaena azollae 1a, a putative symbiont of Azolla caroliniana. Five complete open reading frames have been sequenced. All are very highly conserved when compared with the corresponding regions of Anabaena sp. PCC 7120, with 93% to 97% identity at the nucleotide level and 93% to almost 100% at the amino-acid level. The intergenic regions, however, are not highly conserved (53-89% identity) when compared to the corresponding regions of Anabaena 7120: the A. azollae genome contains both more copies and more types of short tandemly repeated repetitive sequences than Anabaena 7120. The startpoints of transcription for both the nifB and nifH operons were mapped and found to be the same as those in Anabaena 7120. It was not possible to discern an improved consensus nif promoter sequence, but it was possible to define the likely extent of the promoter to within 40 bases upstream of the transcription start-point.