Molecular Characterization ofFischerella uthpalarensis, the first subsection V cyanobiont from a tropicalAzollaspecies containing dual nitrogenases

Abstract

AbstractThere have been theories presented onAzollacyanobionts, known for voracious nitrogen fixation, vertical transmission of cyanobiont and helping transform a greenhouse planet to an icehouse one ~49 Mya. One such theory encapsulates the existence of two cyanobionts, named Major and Minor. We show here the identity of a possible minor cyanobiont ofAzollanamedFischerella uthpalarensis. A likely cyanobiont with straight or curved filaments that were truly branched was isolated. Seven gene fragments, namely 16s rDNA (Forward and Reverse), RNA Polymerase, ITS1 region (Forward and Reverse),nifDand GroEL genes were utilized to identify the isolated cyanobiont. The best match based on BLASTn search tool was found in the RNA Polymerase beta subunit (rpoC) gene fragment, that showed 99.54% identity with 55% coverage toFischerella muscicola. Phylogenetic inferences with therpoCgenetic locus and the GroEL protein sequence suggest a likelyFischerellagenus identity. Furthermore,VnfDGandVnfNfragments too were amplified using PCR and sequenced to demonstrate that this cyanobiont has alternate nitrogenase genes, on top of the molybdenum counterpart, providing an advantage in lifestyle. We encountered a higher level of genomic-level synonymous substitutions, which was not reflected in protein sequences, namelyVnfDGandVnfNgene products, which may be due to codon heterogeneity. We also propose forF. uthpalarensisatypicality in codon usage due to the likely acquisition of the V-nitrogenase operon from a presumed recent horizontal gene transfer (HGT) event. The cyanobiont from this study showcases a higher preference for AT over GC at theVnfDGcomposite locus again hinting at a symbiotic lifestyle.