Photoautotrophic black-colored cyanobacterial soil crust biosynthesizes photoprotective compounds and is capable of using blue, green, and red wavelengths of light for its growth.

Several cyanobacteria can adjust their light-harvesting machinery in response to existing light signals in a process called chromatic acclimation (CA) which permits the utilization of available light resources for photosynthesis. CA involves alteration in the pigment composition of a major light-harvesting complex called phycobilisome (PBS) and allows some cyanobacteria to utilize green light (GL) to drive photosynthesis. However, cyanobacteria, in contrast with eukaryotic algae and higher plants, can not utilize blue light (BL) for photosynthesis due to their dependency on PBS. Here, we studied a black-colored soil crust that was composed of a single cyanobacterium identified and named Oscillatoria sp. Malviya-1 after phenotypic and phylogenetic analyses. The black-colored crust can absorb light from almost all parts of photosynthetically active radiation (400-700 nm) and ultraviolet radiation (280-400 nm) due to the presence of photosynthetic pigments and microbial sunscreens such as chlorophyll ɑ, carotenoids, phycoerythrin, phycocyanin, allophycocyanin, mycosporine-like amino acids, and scytonemin. Unlike other cyanobacteria, Oscillatoria sp. Malviya-1 can grow using GL, BL, and red light (RL) in addition to white light (WL) which was accompanied by the different colors of the mat under different light conditions. The presence of CA and sunscreens compounds can maximize the fitness of soil crust under a dynamic light environment, UVR, and desiccation. Detailed study of Oscillatoria sp. Malviya-1 will provide information on the mechanism of CA in cyanobacterial soil crust and its unique ability to use both GL and BL.

New insights on thioredoxins (Trxs) and glutaredoxins (Grxs) by in silico amino acid sequence, phylogenetic and comparative structural analyses in organisms of three domains of life.

Thioredoxins (Trxs) and Glutaredoxins (Grxs) regulate several cellular processes by controlling the redox state of their target proteins. Trxs and Grxs belong to thioredoxin superfamily and possess characteristic Trx/Grx fold. Several phylogenetic, biochemical and structural studies have contributed to our overall understanding of Trxs and Grxs. However, comparative study of closely related Trxs and Grxs in organisms of all domains of life was missing. Here, we conducted in silico comparative structural analysis combined with amino acid sequence and phylogenetic analyses of 65 Trxs and 88 Grxs from 12 organisms of three domains of life to get insights into evolutionary and structural relationship of two proteins. Outcomes suggested that despite diversity in their amino acids composition in distantly related organisms, both Trxs and Grxs strictly conserved functionally and structurally important residues. Also, position of these residues was highly conserved in all studied Trxs and Grxs. Notably, if any substitution occurred during evolution, preference was given to amino acids having similar chemical properties. Trxs and Grxs were found more different in eukaryotes than prokaryotes due to altered helical conformation. The surface of Trxs was negatively charged, while Grxs surface was positively charged, however, the active site was constituted by uncharged amino acids in both proteins. Also, phylogenetic analysis of Trxs and Grxs in three domains of life supported endosymbiotic origins of chloroplast and mitochondria, and suggested their usefulness in molecular systematics. We also report previously unknown catalytic motifs of two proteins, and discuss in detail about effect of abovementioned parameters on overall structural and functional diversity of Trxs and Grxs.

Flow Cytometry-based Measurement of Reactive Oxygen Species in Cyanobacteria.

Cyanobacteria are Gram-negative oxygen-producing photosynthetic bacteria that are useful in the pharmaceutical and biofuel industries. Monitoring of oxidative stress under fluctuating environmental conditions is important for determining the fitness, survival, and growth of cyanobacteria in the laboratory as well as in large scale cultivation systems. Here, we provide a protocol developed using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria for high-throughput oxidative stress measurement by 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) and flow cytometry (FCM). We also provide details for the optimization of cell number, dye concentration, and FCM parameters for each organism before it can be utilized to quantify reactive oxygen species (ROS). FCM-based method can be used to measure ROS in a large population of cyanobacterial cells in a high-throughput manner. Graphical abstract.

Computational studies on photolyase (Phr) proteins of cyanobacteria.

Photolyases (Phrs) are enzymes that utilize the blue/ultraviolet (UV-A) region of light for repairing UV-induced cyclopyramidine dimers. We studied Phr groups by bioinformatic analyses as well as active-site and structural modeling. Analysis of 238 amino acid sequences from 85 completely sequenced cyanobacterial genomes revealed five classes of Phrs, CPD Gr I, 6-4 Phrs/cryptochrome, Cry-DASH, Fe-S bacteria Phrs, and a group with fewer amino acids (276-385) in length. The distribution of Phr groups in cyanobacteria belonging to the order Synechococcales was found to be influenced by the habitats of the organisms. Class V Phrs are exclusively present in cyanobacteria. Unique motifs and binding sites were reported in groups II and III. The Fe-S protein binding site was only present in group V and the active site residues and putative CPD/6-4PP binding residues are charged amino acids present on the surface of the proteins. The majority of hydrophilic amino acid residues were present on the surface of the Phrs. Sequence analysis confirmed the diverse nature of Phrs, although sequence diversity did not affect the overall three-dimensional structure. Protein-ligand interaction analysis identified novel CPD/6-4PP binding sites on Phrs. This structural information of Phrs can be used for the preparation of efficient Phr-based formulations.

Roadmap to sustainable carbon-neutral energy and environment: can we cross the barrier of biomass productivity?

The total number of inhabitants on the Earth is estimated to cross a record number of 9 × 103 million by 2050 that present a unique challenge to provide energy and clean environment to every individual. The growth in population results in a change of land use, and greenhouse gas emission due to increased industrialization and transportation. Energy consumption affects the quality of the environment by adding carbon dioxide and other pollutants to the atmosphere. This leads to oceanic acidification and other environmental fluctuations due to global climate change. Concurrently, speedy utilization of known conventional fuel reservoirs causes a challenge to a sustainable supply of energy. Therefore, an alternate energy resource is required that can maintain the sustainability of energy and environment. Among different alternatives, energy production from high carbon dioxide capturing photosynthetic aquatic microbes is an emerging technology to clean environment and produce carbon-neutral energy from their hydrocarbon-rich biomass. However, economical challenges due to low biomass production still prevent the commercialization of bioenergy. In this work, we review the impact of fossil fuels burning, which is predominantly used to fulfill global energy demand, on the quality of the environment. We also assess the status of biofuel production and utilization and discuss its potential to clean the environment. The complications associated with biofuel manufacturing using photosynthetic microorganisms are discussed and directed evolution for targeted phenotypes and targeted delivery of nutrients are proposed as potential strategies to increase the biomass production.

Oxidative stress measurement in different morphological forms of wild-type and mutant cyanobacterial strains: Overcoming the limitation of fluorescence microscope-based method.

Monitoring of oxidative stress caused by a wide range of reactive oxygen species (ROS) is essential to have an idea about the fitness and growth of photosynthetic organisms. The imaging-based oxidative stress measurement in cyanobacteria using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye has the limitation of small sample size as the only selected number of cells are analyzed to measure the ROS levels. Here, we developed a method for oxidative stress measurement by DCFH-DA and flow cytometer (FCM) using unicellular Synechococcus elongatus PCC 7942 and filamentous Fremyella diplosiphon BK14 cyanobacteria. F. diplosiphon BK14 inherently possess high levels of ROS and showed higher sensitivity to hydrogen peroxide treatment in comparison to S. elongatus PCC 7942. We successfully measured oxidative stress in glutaredoxin lacking strain (Δgrx3) of S. elongatus PCC 7942, and wild-type Synechocystis sp. PCC 6803 using FCM based method. Importantly, ROS were not detected in these two strains of cyanobacteria by fluorescence microscope-based method due to their small spherical morphology. Δgrx3 strain showed high ROS levels in comparison to its wild-type strain. Treatment of abiotic factors such as high PAR in wild-type and Δgrx3 strains of S. elongatus PCC 7942, low PAR or low PAR + UVR in wild-type S. elongatus PCC 7942, and high PAR or high PAR + NaCl in Synechocystis sp. PCC 6803 increased oxidative stress. In summary, the FCM based method can measure ROS levels produced due to physiological conditions associated with genetic changes or abiotic stress in a large population of cells regardless of their morphology. Therefore, the present study shows the usefulness of the method in monitoring the health of organisms in a large scale cultivation system.

Phylogenetic distribution and structural analyses of cyanobacterial glutaredoxins (Grxs).

Glutaredoxins (Grxs), the oxidoreductase proteins, are involved in several cellular processes, including maintenance of cellular redox potential and iron-sulfur homeostasis. The analysis of 503 amino acid sequences from 167 cyanobacterial species led to the identification of four classes of cyanobacterial Grxs, i.e., class I, II, V, and VI Grxs. Class III and IV Grxs were absent in cyanobacteria. Class I and II Grxs are single module oxidoreductase while class V and VI Grxs are multimodular proteins having additional modules at their C-terminal and N-terminal end, respectively. Furthermore, class VI Grxs were exclusively present in marine cyanobacteria. We also report the identification of class VI Grxs with two novel active site motif compositions. Detailed phylogenetic analysis of all four classes of Grxs revealed the presence of several subgroups within each class of Grx having variable dithiol and/or monothiol catalytic active site motif and putative glutathione binding sites. However, class II Grxs possess CGFS-type highly conserved monothiol catalytic active site motif. Sequence analysis confirmed the highly diverse nature of Grx proteins in terms of their amino acid composition; though, sequence diversity does not affect the overall 3D structure of cyanobacterial Grxs. The active site residues and putative GSH binding residues are uncharged amino acids which are present on the surface of the protein. Additionally, the presence of hydrophilic residues at the surface of Grxs confirms their solubility. Protein-ligand interaction analysis identified novel glutathione binding sites on Grxs. Regulation of Grxs encoding genes expression by light quality and quantity as well as salinity suggests their role in determining the fitness of organisms under abiotic factors.