Phylogenetic analysis of Nostocales (Cyanobacteria) based on two novel molecular markers, implicated in the nitrogenase biosynthesis.

The characterization of cyanobacteria communities remains challenging, as taxonomy of several cyanobacterial genera is still unresolved, especially within Nostocales taxa. Nostocales cyanobacteria are capable of nitrogen fixation; nitrogenase genes are grouped into operons and are located in the same genetic locus. Structural nitrogenase genes (nifH, nifK and nifD) as well as 16S rRNA have been shown to be adequate genetic markers for distinguishing cyanobacterial genera. However, there is no available information regarding the phylogeny of regulatory genes of the nitrogenase cluster. Aiming to provide a more accurate overview of the evolution of nitrogen fixation, this study analyzed for the first time nifE and nifN genes, which regulate the production of nitrogenase, alongside nifH. Specific primers were designed to amplify nifE and nifN genes, previously not available in literature and phylogenetic analysis was carried out in 13 and 14 TAU-MAC culture collection strains, respectively, of ten Nostocales genera along with other sequences retrieved from cyanobacteria genomes. Phylogenetic analysis showed that these genes seem to follow a common evolutionary pattern with nitrogenase structural genes and 16S rRNA. The classification of cyanobacteria based on these molecular markers seems to distinguish Nostocales strains with common morphological, ecological, and physiological characteristics.