An in silico Study of Two Transcription Factors Controlling Diazotrophic Fates of the Azolla Major Cyanobiont Trichormus azollae

Abstract

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.

The Microbiome of Suaeda monoica and Dipterygium glaucum From Southern Corniche (Saudi Arabia) Reveals Different Recruitment Patterns of Bacteria and Archaea

“…In terms of Sphaerospermopsis abundance in S. monoica's crust, Cyanobacteria from Sphaerospermopsis BCCUSP55 genus could be related to nitrogen fixation at the soil surface. Sphaerospermopsis genus was previously reported to be closely related to a well-known nitrogen-fixing cyanobiont bacteria known as Nostoc (Gunawardana, 2020). This can be supported by the fact that at the family level, Nostocaceae cyanobacteria were abundant in S. monoica-associated samples, especially S. monoica's crust, while Balneolaceae was abundant in Sr. Another species, Sphaerospermopsis aphanizomenoides, was reported to not only grow faster than other Nostocales families in high temperature but also to be correlated with phytoplankton (Budzyńska et al, 2019).…”

Genetic, Structural, Physicochemical, and Molecular Epidemiological Landscape of Three New Mutations Found in the Spike (S) Protein of Highly Transmissible Sri Lankan Delta Variant Sub‐lineage AY.28: A222V, A701S, and A1078S

“…Serine is the most frequently found mutation and one of the most targeted destinations in relation to amino acids and consequently, is called a mutational hotspot [8]. is is primarily due to the two disjointed types of codons that are found in the genetic code that are encoding for the same amino acid, serene [9]. ey are as follows: UCN (UCA, UCC, UCG, and UCU) and AGY (AGU and AGC), which are farther apart than a single nucleotide substitution, which makes two distant groupings and six unique codons for serine (Figure 1).…”

A Hypothesis on How the Azolla Symbiosis Mitigates Nitrous Oxide Based on In Silico Analyses

“…One key regulator of nitrogen fixation, heterocyst development and function, is the NtcA transcription factor, which is thought to regulate the downstream function of >2000 mostly nitrogen-metabolism-related genes [22]. We searched for the NtcA binding promoter region in the WP_013192178.1 encoding gene to see whether it was under top-down regulation.…”

“…Under nitrogen-rich conditions, 2-oxoglutarate counts are low and NtcA is found in an inactive form [22]. In a recent publication, it was shown that the NtcA transcription factor of T. azollae putatively uses a putative disulfide bond to perform its robust function in modifying gene expression using NtcA responsive promoters [22].…”

“…Under nitrogen-rich conditions, 2-oxoglutarate counts are low and NtcA is found in an inactive form [22]. In a recent publication, it was shown that the NtcA transcription factor of T. azollae putatively uses a putative disulfide bond to perform its robust function in modifying gene expression using NtcA responsive promoters [22]. We propose here that nitrous oxide released from urea can gradually replace or function secondarily to the canonical function of reducing oxygen to water by the WP_013192178.1 enzyme due to extremely low levels of oxygen found inside heterocysts.…”