Sulfur is in the Air: Cyanolichen Marriages and Pollution.

Cyanolichens are symbiotic organisms involving cyanobacteria and fungi (bipartite) or with the addition of an algal partner (tripartite). Cyanolichens are known for their heightened susceptibility to environmental pollution. We focus here on the impacts on cyanolichens due to rising air pollution; we are especially interested in the role of sulfur dioxide on cyanolichen biology. Cyanolichens due to air pollution including sulfur dioxide exposure, show symptomatic changes including degradation of chlorophyll, lipid membrane peroxidation, decrease in ATP production, changes in respiration rate, and alteration of endogenous auxins and ethylene production, although symptoms are known to vary with species and genotype. Sulfur dioxide has been shown to be damaging to photosynthesis but is relatively benign on nitrogen fixation which proposes as a hypothesis that the algal partner may be more in harm's way than the cyanobiont. In fact, the Nostoc cyanobiont of sulfur dioxide-susceptible Lobaria pulmonaria carries a magnified set of sulfur (alkane sulfonate) metabolism genes capable of alkane sulfonate transport and assimilation, which were only unraveled by genome sequencing, a technology unavailable in the 1950-2000 epoch, where most physiology- based studies were performed. There is worldwide a growing corpus of evidence that sulfur has an important role to play in biological symbioses including rhizobia-legumes, mycorrhizae-roots and cyanobacteria-host plants. Furthermore, the fungal and algal partners of L. pulmonaria appear not to have the sulfonate transporter genes again providing the roles of ambient-sulfur (alkanesulfonate metabolism etc.) mediated functions primarily to the cyanobacterial partner. In conclusion, we have addressed here the role of the atmospheric pollutant sulfur dioxide to tripartite cyanolichen viability and suggest that the weaker link is likely to be the photosynthetic algal (chlorophyte) partner and not the nitrogen-fixing cyanobiont.

Cyanotoxins uptake and accumulation in crops: Phytotoxicity and implications on human health.

The invasive nature of cyanotoxin-producing cyanobacteria and the adverse effects concerning their toxic impacts have gained heightened scientific attention of late. The persistence of cyanotoxins in irrigation water leads to bioaccumulation in plants, the development of phytotoxic effects, and the threat of groundwater contamination. The accumulation of cyanotoxins in plants is caused by several factors leading to severe toxic effects, including reduced plant growth and seed germination, enhanced oxidative stress, lowered rate of mineral uptake, decreased photosynthetic efficiency, and loss of chlorophyll content. The uptake and accumulation of cyanotoxins in plants can be concentration-dependent, as reported in a myriad of studies. Even though several studies have reported phytotoxic effects of cyanotoxin contamination, field-related studies reporting phytotoxic effects are particularly inadequate. Paradoxically, at realistic conditions, some plants are reported to be tolerant of cyanotoxins. Furthermore, the breadth of adverse impacts of cyanotoxins on human health is significant. Cyanotoxins cause major health effects including cancer, oxidative stress, organelle dysfunction, DNA damage, and enzyme inhibition. This review intends to present compelling arguments on microcystins (MCs), cylindrospermopsins (CYN), ß-N-methylamino-L-alanine (BMAA), and anatoxin-a (ANTX-a), their uptake and accumulation in crop plants, phytotoxic effects on plants, and potential health implications to humans. The accumulation of cyanotoxins implants cultivated as food crops, resulting in phytotoxic effects and adverse impacts on human health are serious issues that require scientific inputs to be addressed.

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

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.

An Exploration of Common Greenhouse Gas Emissions by the Cyanobiont of the Azolla-Nostoc Symbiosis and Clues as to Nod Factors in Cyanobacteria.

Azolla is a genus of aquatic ferns that engages in a unique symbiosis with a cyanobiont that is resistant to cultivation. Azolla spp. are earmarked as a possible candidate to mitigate greenhouse gases, in particular, carbon dioxide. That opinion is underlined here in this paper to show the broader impact of Azolla spp. on greenhouse gas mitigation by revealing the enzyme catalogue in the Nostoc cyanobiont to be a poor contributor to climate change. First, regarding carbon assimilation, it was inferred that the carboxylation activity of the Rubisco enzyme of Azolla plants is able to quench carbon dioxide on par with other C3 plants and fellow aquatic free-floating macrophytes, with the cyanobiont contributing on average ~18% of the carboxylation load. Additionally, the author demonstrates here, using bioinformatics and past literature, that the Nostoc cyanobiont of Azolla does not contain nitric oxide reductase, a key enzyme that emanates nitrous oxide. In fact, all Nostoc species, both symbiotic and nonsymbiotic, are deficient in nitric oxide reductases. Furthermore, the Azolla cyanobiont is negative for methanogenic enzymes that use coenzyme conjugates to emit methane. With the absence of nitrous oxide and methane release, and the potential ability to convert ambient nitrous oxide into nitrogen gas, it is safe to say that the Azolla cyanobiont has a myriad of features that are poor contributors to climate change, which on top of carbon dioxide quenching by the Calvin cycle in Azolla plants, makes it an efficient holistic candidate to be developed as a force for climate change mitigation, especially in irrigated urea-fed rice fields. The author also shows that Nostoc cyanobionts are theoretically capable of Nod factor synthesis, similar to Rhizobia and some Frankia species, which is a new horizon to explore in the future.

PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m(7)GpppG.

Fluorescent mRNA molecules offer a wide range of applications for studying capping/decapping reactions, translation, and other biophysical studies. Furthermore, fluorescent tags prove invaluable for tracking RNA molecules in cells. Here, we describe an efficient synthesis of a fluorescent cap analog, anthranioyl-GTP, its purification, and in vitro cap labeling of transcribed mRNA catalyzed by the recombinant vaccinia capping enzyme to produce anthranioyl-m(7)GpppG-capped RNA.

Efficient preparation and properties of mRNAs containing a fluorescent cap analog: Anthraniloyl-m(7)GpppG.

A method has been developed for synthesising fluorescently labeled capped mRNA. The method incorporates a single fluorescent molecule as part of the 5'-mRNA or oligonucleotide cap site. The fluorescent molecule, Ant-m(7)GTP is specifically incorporated into the cap site to yield Ant-m(7)GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m(7)G derivative, which has been previously shown to interact with the eukaryotic cap binding protein eIF4E, is shown in this paper to be a substrate for the Vaccinia capping enzyme and the DCP2 decapping enzyme from Arabidopsis. Further, the Ant-m(7)GTP-capped RNA is readily translated. This Ant-m(7)GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2).

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

Structure and substrate-binding mechanism of human Ap4A hydrolase.

Asymmetric diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.