Redox Homeostasis in Cyanobacteria

Oxygenic photosynthetic organisms utilize high-energy electron transfer chains comprised of redox active intermediates and light harvesting complexes. While oxygen is a necessary byproduct of water oxidation and the source of photosynthetic electrons, its presence is also dangerous because leakage of electrons and excitation energy can interact with molecular oxygen to generate reactive oxygen species: ROS). Elaborate antioxidant networks and redox buffering systems have evolved to protect photosynthetic organisms from the threat of ROS. Glutathione: GSH) is a multifunctional molecule that is involved in core metabolism, detoxification of xenobiotics and in maintenance of cellular redox poise. The ubiquitous nature of glutathione and its importance to cellular metabolism has been observed in many organisms, however the specific roles of glutathione in photosynthetic organisms are not fully understood. To address these questions, we have generated several mutants in the glutathione biosynthesis and degradation pathways in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803), an oxygenic photosynthetic cyanobacterium. We utilized targeted homologous recombination to generate deletion mutants of glutamate-cysteine ligase: GshA) and glutathione synthetase: GshB) in Synechocystis 6803. Our results indicate that GshA activity is essential for growth in cyanobacteria because we were unable to isolate a fully segregated ∆gshA deletion mutant. We did isolate a ∆gshB mutant strain that accumulates the biosynthetic intermediate γ-glutamylcysteine: γ-EC) instead of GSH. In this work, I have characterized the physiology of the ∆gshB mutant following environmental, genetic and redox perturbations. The results presented here also shed light on the dynamic nature of the low-molecular weight thiol pool in cyanobacteria. We quantified the levels of cellular thiols in Synechocystis 6803 during exposure to multiple environmental and redox perturbations and found that conditions promoting increased cellular metabolism and increased ROS production, including during high-light treatment and photomixotrophic growth, lead to higher cellular thiol levels. Furthermore, the intracellular pools of thiols decrease when the cell exhibits reduced metabolic capacity during conditions such as nutrient deprivation and dark incubation. Sulfate limitation results in dramatically decreased cellular thiol contents in a short period of time. We found that the ∆gshB strain is sensitive to sulfate limitation and exhibits delayed recovery upon sulfate repletion, indicating that GSH is important for acclimation to sulfate limiting conditions. To facilitate our understanding of GSH degradation in Synechocystis 6803 during sulfate limitation, we generated a mutant lacking γ-glutamyltranspeptidase: Ggt), an enzyme with GSH degradation activity. However, the ∆ggt mutant still exhibited GSH degradation during sulfate depletion, indicating the presence of an alternative system or mechanism. We did find increased levels of GSH in the growth media of the ∆ggt strain compared to the WT, which suggests a role in GSH uptake or prevention of leakage. Our results demonstrate that GSH is essential for protection from multiple environmental and redox perturbations in cyanobacteria. However, there are many pathways involved in maintenance of redox homeostasis in cyanobacteria. Therefore, we also aimed to determine whether these pathways function cooperatively to ameliorate damage from ROS. Several flavodiiron: Flv) proteins have been identified in Synechocystis 6803 that are involved in reduction of O2 to H2O without the formation of ROS intermediates. However, single ∆flv3 mutants do not exhibit severe growth defects under normal conditions. Therefore, we generated a ∆gshB/∆flv3 mutant to examine whether these systems cooperate to maintain redox homeostasis. Our results show that the ∆gshB/∆flv3 mutant exhibits reduced growth than either of the single mutants when grown on solid media, suggesting a degree of interaction between these pathways in cyanobacteria

Edible cyanobacterial genus <i>Arthrospira</i>: actual state of the art in cultivation methods, genetics, and application in medicine

The cyanobacterial genus Arthrospira appears very conserved and has been divided into five main genetic clusters on the basis of molecular taxonomy markers. Genetic studies of seven Arthrospira strains, including genome sequencing, have enabled a better understanding of those photosynthetic prokaryotes. Even though genetic manipulations have not yet been performed with success, many genomic and proteomic features such as stress adaptation, nitrogen fixation, or biofuel production have been characterized. Many of above-mentioned studies aimed to optimize the cultivation conditions. Factors like the light intensity and quality, the nitrogen source, or different modes of growth (auto-, hetero-, or mixotrophic) have been studied in detail. The scaling-up of the biomass production using photobioreactors, either closed or open, was also investigated to increase the production of useful compounds. The richness of nutrients contained in the genus Arthrospira can be used for promising applications in the biomedical domain. Ingredients such as the calcium spirulan, immulina, C-phycocyanin, and γ-linolenic acid (GLA) show a strong biological activity. Recently, its use in the fight against cancer cells was documented in many publications. The health-promoting action of “Spirulina” has been demonstrated in the case of cardiovascular diseases and age-related conditions. Some compounds also have potent immunomodulatory properties, promoting the growth of beneficial gut microflora, acting as antimicrobial and antiviral. Products derived from Arthrospira were shown to successfully replace biomaterial scaffolds in regenerative medicine. Supplementation with the cyanobacterium also improves the health of livestock and quality of the products of animal origin. They were also used in cosmetic preparations

Antioxidant Effects of Selenium upon Exposure to Zinc and World Trade Center Particulate Matter in Prokaryotic and Eukaryotic Systems

The antitoxic effect of selenium on zinc has been evaluated in different levels of living organisms. The growth was observed under various concentrations of zinc chloride as well as zinc chloride (ZnCl2)/Selenium dioxide (SeO2) using direct count and turbidity method while morphology and DNA is studied by performing DAPI stain. In prokaryotic Synechococcus sp. IU 625 (SIU 625), the growth was very similar to the control at the concentrations of lOmg/L, but reduced at 25mg/L while 50mg/L ZnCl2 was lethal dosage and hence inhibited the growth completely. Morphological study indicated that the cells become longer and colorless at high concentration. Y and V shape (curved cells and cells with ectopic poles) DAPI stained DNA and some fragmentations were observed in 25mg/L and 50mg/L ZnCl2 treated SIU 625 at 24hours and day 3. SeO2 is able to reduce the availability of ZnCl2 thus reduces the growth at lOmg/L ZnCl2 which is the concentration that enhance the growth of SIU 625. At higher concentration of ZnCl2 (50mg/L), SeO2 is able to reduce the toxicity of ZnCl2 and the long filament shape was observed at day 3 in SIU 625.The eukaryotic model, Chlamydomonas reinhardtii (C. reinhardtii) was very resistant to ZnCl2 treatment. Lower concentrations (l0mg/L and 25mg/L) did not affect the growth of the cells. At the 50mg/L of ZnCl2, the inhibition of growth was observed suggesting that C. reinhardtii might have stronger heavy metal tolerant mechanism compare to SIU 625 which helps them to combat stress caused by zinc. As the concentration of ZnCl2 increase, some cells appear light green and the dead cells appear dark brown at 12 and 24 hours under higher concentration (25 and 50mg/L ZnCl2). At the concentration of lOmg/L and 25mg/L of ZnCl2 in combination with lmg/L of SeO2, better growth was observed. At higher concentration (50mg/L), no significant effect of SeO2 on the toxicity of ZnCl2 was observed. DNA demargination was also observed in DAPI stained DNA under ZnCl2 and ZnCl2/SeO2 treatment. In mammalian cells, SeO2 increased viability in CHO cells treated with 25mg/L SeO2 significantly (about 25.25%) and with 50mg/L ZnCl2 slightly (about 12.26%). SeO2 increased viability in MRC-5 cells treated with 25mg/L significantly (about 37.85%) and with 50mg/L ZnCl2 about 12.27%. SeO2 (0.125mg/L) with WTC dust at 1.25, 12.5, and 125mg/L increased viability about 3.59%, 26.16% and 67.76% respectively in CHO cells, but not in MRC-5. Apotox Glo Triplex assay result was inconclusive in mammalian cells. SIU 625 was used as a model to do bioinformatics and proteomic study. A membrane topology prediction indicated that four (st, groEL, hmtA and ShmtA) out of five genes (st, groEL, ct, hmtA and ShmtA) selected for proteomics study were membrane proteins. Identification of these genes and expression study using qPCR based assay suggested that three genes (st, groEL, ct) had similar expression pattern and showed increased expression immediately after heavy metal exposure (10 and 25mg/L of ZnCl2) while hmtA and ShmtA did not express immediately. Both hmtA and ShmtA contained similar cysteine rich domain indicating its high affinity to be able to bind heavy metal, but only expressed significantly under lower concentration of ZnCl2 (l0mg/L)

Characterization of the prokaryotic community associated with the giant barrel sponge, Xestospongia muta across the Caribbean

Sponges have long been known to be ecologically important members of the benthic fauna on coral reefs. Recently, it has been shown that sponges, and their symbiotic microbes, are also important contributors to the nitrogen biogeochemistry of coral reefs. Here, I investigate the ecology and physiology of the microbial community associated the ecologically dominant sponge, Xestospongia muta. A natural experiment was conducted with X. muta form three different locations (Florida Keys, USA; Lee Stocking Island, Bahamas, and Little Cayman, Cayman Islands) to compare nitrogen cycling and prokaryotic community composition. The dissolved inorganic nitrogen (DIN) fluxes of sponges were studied using nutrient analysis, stable isotope ratios, and isotope tracer experiments. Results showed that the fluxes of DIN were variable between locations but clearly showed that X. muta can be either a source or sink of DIN. Stable isotope values of sponge and symbiotic bacterial fractions indicate that the prokaryotic community is capable of taking up both NH4+ and NO3 --, and there is potential for translocation of labeled N from the symbiotic bacteria to the host. The prokaryotic community composition of X. muta, and the variability of this community across the Caribbean were quantified using 454 pyrosequencing of the 16S rRNA gene. Phlyogenetic analysis showed differences between the sponge prokaryotic community and the surrounding bacterioplankton. Additionally, both symbiont and bacterioplankton populations were different between locations. In addition to the recovery of many sequences from bacterial phyla commonly found in sponges, a diverse archaeal community was also recovered from X. muta including sequences representing the phyla Euryarchaeota and Thaumarchaeota. Transcriptomic analysis for X. muta and its symbionts revealed a similar prokaryotic community composition to the metagenetic analyses indicating an active and diverse symbiotic community. Additionally, gene specific analyses combined with preliminary metatranscriptome data indicate the presence of genes involved in nitrogen cycling including nifH (nitrogen fixation), amoA (ammonia oxidation), norB (denitrification), and nirK (denitrification). Nitrogen cycling in X. muta appears to be more complex than previous studies have shown. These results have important ecological implications for the understanding of host-microbe associations, and provide a foundation for future studies addressing the functional roles these symbiotic prokaryotes have in the biology of the host sponge and the nutrient biogeochemistry of coral reefs

Microbial Secondary Metabolites and Biotechnology

Many research teams are working to demonstrate that microorganisms can be our daily partners, due to the great diversity of biochemical transformations and molecules they are able to produce. This Special Issue highlights several facets of the production of microbial metabolites of interest. From the discovery of new strains or new bioactive molecules issued from novel environments, to the increase in their synthesis by traditional or innovative methods, different levels of biotechnological processes are addressed. Combining the new dimensions of "Omics" sciences, such as genomics, transcriptomics or metabolomics, microbial biotechnologies are opening up incredible opportunities for discovering and improving microorganisms and their production

Zinc regulation in an open ocean cyanobacterium

Cyanobacteria of the genera Synechococcus and Prochlorococcus are key players in marine CO2 fixation contributing ~25% of the global total. Since oceanic zinc concentrations may directly affect the CO2 fixation process via the role of carbonic anhydrase in cyanobacterial photosynthesis, understanding cyanobacterial zinc metabolism is of critical importance. The current study used the model open ocean cyanobacterium Synechococcus sp. WH8102. In this species zinc uptake is thought to be regulated by a predicted zinc uptake regulator (SynZur) which controls expression of at least four genes thought to be involved in zinc homeostasis: znuA, znuB, znuC and bmtA (encoding a predicted high affinity zinc uptake transporter ZnuABC and a metallothionein SynBmtA). These components and Zur are largely uncharacterized. SynZur was over-expressed recombinantly in E. coli, purified, crystallized and its X-ray crystal structure resolved. This is the first member of the Fur family of proteins from cyanobacteria to have a known structure. Two zinc binding sites were identified: a conserved structural site 1 (Cys4), and a novel sensory site (His2AspCys). Both zinc binding sites reside within the dimerization domain. ESI-MS detected a decrease in the abundance of the SynZur dimer in the absence of zinc at the sensory site, hinting at a potentially novel zinc sensing mechanism that might be typical of cyanobacterial Zur proteins. The affinity of the sensory site for zinc was KD = 8.27×10-13 M. SynZur with two zinc ions per monomer bound also showed selective binding to pznuABC with a KD ~5 nM per dimer or ~10 nM per monomer. ESI-MS revealed that Cd2+ can replace Zn2+ in the structural site suggesting that it is not kinetically inert. A Synechococcus sp. WH8102 zur mutant was also constructed and characterised. Growth rate comparisons showed that the mutant has a lower tolerance to high zinc levels compared to the WT, whilst ICP-MS analysis revealed different accumulation behaviour for trace metals and most significantly for zinc. This is consistent with SynZur repressing a predicted znuABC system. RNA-seq analysis of the WT and zur mutant facilitated experimental identification of new members of the Synechococcus sp. WH8102 Zur regulon. Phyre2 structural prediction suggested that two of the newly identified genes (SYNW0972 and SYNW0973) encode components of a novel outer membrane zinc uptake system. Significantly, these two genes are conserved in other marine cyanobacteria. Furthermore, the RNA-seq data (confirmed using RT-qPCR) showed that SynZur can activate a metallothionein (SYNW0359, SynBmtA) in Synechococcus sp. WH8102 previously thought to be repressed by SynZur. SynZur binds to the operator-promoter region of SynBmtA (pbmtA) in a 4:1 stoichiometry, with an affinity of 26.7 nM per monomer. The SynBmtA protein was over-expressed and purified with four zinc ions bound. SynBmtA was de-metallated and its affinity to Zn2+ was determined (KD = 1.13×10-14 M). ESI-MS showed that apo-SynBmtA is able to remove zinc from metallated SynZurZn2 in agreement with the order of zinc affinities for SynZur and SynBmtA. Finally, SynBmtA was analysed by 1H,15N-NOESY-HSQC and 1H,15N-TOCSY-HSQC NMR spectroscopy. The spectra were partially assigned and the assignment indicated that the characteristic zinc-finger fold of bacterial MTs is indeed present in SynBmtA, but that the C-terminus is likely to differ significantly from that of SmtA from the Synechococcus elongatus strain PCC 7942

Diversity of regulatory mechanisms in the C/N metabolism of the marine cyanobacteria Prochlorococcus and synechococcus

Marine picocyanobacteria are the most abundant photosynthetic organisms on Earth, with only two genera, Prochlorococcus (Johnson et al., 2006, Olson et al., 1990, Partensky et al., 1999) and Synechococcus (Scanlan, 2003, Scanlan & West, 2002) numerically dominating most oceanic waters. During this research project our main goal was to study the diversity of the regulatory mechanisms in the C/N metabolism of these cyanobacteria. Recent advances in the knowledge of nitrogen metabolism of Prochlorococcus have shown that it has fine regulatory systems to optimize nitrogen assimilation (Rocap et al., 2003, García-Fernández et al., 2004, Lindell et al., 2002). Thus, we have studied the role of 2-oxoglutarate in the control of the C/N balance in order to check whether there exist differences with respect to other model cyanobacteria and among strains of Prochlorococcus. The comparative study performed show that 2-oxoglutarate is the molecule responsible in Prochlorococcus to control the balance between carbon and nitrogen metabolism and there are differences among strains in sensing this metabolite. These results could be an explanation for its adaptation to different ecological niches in the ocean. Besides, we wanted to know how Synechococcus is able to successfully coexist with Prochlorococcus. For that, the hypothesis was that Synechococcus could be more efficient at the utilization of low concentration of nitrate. The results showed that when concentrations of nitrate in the range of nanomolar are present, the genes related with the assimilation of that source are up-regulated in Synechococcus WH7803. Therefore, these facts suggest that the machinery is working at transcriptional level in order to uptake the nitrate. This could be an evolutionary advantage against Prochlorococcus in the real field.Las picocianobacterias marinas son los organismos fotosintéticos más abundantes en la Tierra, con sólo dos géneros, Prochlorococcus (Johnson et al., 2006, Olson et al., 1990, Partensky et al., 1999) y Synechococcus (Scanlan, 2003, Scanlan & West, 2002) dominando la mayor parte de los océanos. El principal objetivo durante este proyecto de investigación ha sido estudiar la diversidad de mecanismos regulatorios del metabolismo del C/N en estas cianobacterias. Recientes avances en la regulación del metabolismo del nitrógeno en Prochlorococcus muestran que tienen una fina regulación para optimizar la asimilación del nitrógeno (Rocap et al., 2003, García-Fernández & Diez, 2004, Lindell et al., 2002). Por lo tanto, hemos estudiado el papel del 2-oxoglutarato en el control del balance C/N con el fin de comprobar si existen diferencias con respecto a otras cianobacterias modelos, e incluso si estas diferencias se encuentran entre estirpes. Lo resultados obtenidos del estudio comparativo mostraron que el 2-oxoglutarato es la molécula responsable del control del balance C/N en Prochlorococcus y que existen diferencias entre estirpes en la detección de este metabolito. Esto pueden ser una explicación de la adaptación a diferentes nichos ecológicos en el océano. Además, nosotros queríamos responder a la pregunta de cómo Synechococcus es capaz de coexistir con éxito con Prochlorococcus. Para ello, la hipótesis era que Synechococcus puede ser más eficaz en la utilización de concentraciones bajas de nitrato. Los resultados mostraron que cuando hay concentraciones de nitrato en el rango de nanomolar, se sobreexpresan los genes relacionados con la asimilación de esta fuente en Synechococcus WH7803. Estos hechos sugieren que hay una regulación a nivel transcripcional con el objetivo de absorber el nitrato a concentraciones bajas. Esto podría ser una ventaja evolutiva respecto a Prochlorococcus en los ecosistemas donde conviven