An in silico Study of Two Transcription Factors Controlling Diazotrophic Fates of the Azolla Major Cyanobiont Trichormus azollae
Authors: Gunawardana Dilantha 
Published: December 2020

The cyanobiont Trichormus azollae lives symbiotically within fronds of the genus Azolla, and assimilates atmospheric nitrogen upon N-limitation, which earmarks this symbiosis to be a valuable biofertilizer in rice cultivation, among many other benefits that also include carbon sequestration. Therefore, studying the regulation of nitrogen fixation in Trichormus azollae is of great importance and benefit, especially the two topmost rungs of regulation, the NtcA and HetR transcription factors that are able to regulate the expression of myriads of downstream genes. Bioinformatics tools were used to zoom in on the NtcA and HetR transcription factors from Trichormus azollae to elaborate on what makes this particular cyanobiont different from other symbiotic as well as more distinct counterparts, in their commitment to nitrogen fixation. The utility of Azolla plants in tropical agriculture in particular merits the “top down N-regulation” by cyanobiont as a significant niche area of study, to make sense of superior N-fixing capabilities. The Trichormus azollae NtcA sequence was found as a phylogenetic outlier to horizontally infecting cyanobionts, which points to a distinct identity compared to symbiotic counterparts. There were borderline (60%-70%) levels of acceptable bootstrap support for the phylogenetic position of the Azolla cyanobiont’s NtcA protein compared to other cyanobionts. Furthermore, the NtcA global nitrogen regulator in the Azolla cyanobiont has an extra cysteine at position 128, in addition to two other more conspicuous cysteines (positions, 157 and 164). A simulated homology model of the NtcA protein from Trichormus azollae, points to a single unique cysteine (Cysteine-128) as a key residue at the center of a lengthy C-helix, which forms a coiled-coil interface, through likely disulfide bond formation. Three cysteine (Cysteines: 128, 157, 164) architecture is exclusively found in Trichormus azollae and is absent in other cyanobacteria. A separate proline to alanine mutation in position 97—again exclusive to Trichormus azollae—appears to influence the flexibility of effector binding domain (EBD) to 2-oxoglutarate. The Trichormus azollae HetR sequence was found outside of horizontally-infecting cyanobiont sequences that formed a common clade, with the exception of the cyanobiont from the genus Cycas that formed one line of descent with the Trichormus azollae counterpart. Five (out of 6) serines predicted to be phosphorylated in the Trichormus azollae HetR sequence, are conserved in the Nostoc punctiforme counterpart, showcasing that phosphorylation is likley conserved in both vertically-transmitted and horizontally-acquired cyanobionts. A key Serine-127, within a conserved motif TSLTS, although conserved in heterocystous subsection IV and V cyanobacteria, are mutated in subsection III cyanobacteria that form trichomes but are unable to form heterocysts. I conclude that the NtcA protein from Trichormus azollae to be strategically divergent at specific amino acids that gives it an advantage in function as a 2-oxoglutarate-mediated transcription factor. The Trichormus azollae HetR transcription factor appears to possess parallel functionality to horizontally acquired counterparts. Especially Cysteine-128 in the NtcA transcription factor of the Azolla cyanobiont is an interesting proposition for future structure-function studies.