Pedro Moradas-Ferreira

Complexity of cyanobacterial exopolysaccharides: composition, structures, inducing factors and putative genes involved in their biosynthesis and assembly

Cyanobacterial extracellular polymeric substances (EPS) are mainly composed of high-molecular-mass heteropolysaccharides, with variable composition and roles according to the microorganism and the environmental conditions. The number of constituents - both saccharidic and nonsaccharidic - and the complexity of structures give rise to speculations on how intricate their biosynthetic pathways could be, and how many genes may be involved in their production. However, little is known regarding the cyanobacterial EPS biosynthetic pathways and regulating factors. This review organizes available information on cyanobacterial EPS, including their composition, function and factors affecting their synthesis, and from the in silico analysis of available cyanobacterial genome sequences, proposes a putative mechanism for their biosynthesis.

The molecular defences against reactive oxygen species in yeast

There is rapidly expanding interest into the protective systems against reactive oxygen species (ROS) in the eukaryotic cell, now that the links between oxidative damage, various disease states, and ageing, are firmly established in higher organisms. Yeast molecular genetics should be able to provide powerful insight into these mechanisms; this potential is now starting to be exploited. A number of primary antioxidant activities and systems of metal‐ion homeostasis or detoxification have now been demonstrated to contribute to oxidative‐stress protection in yeast. Also, evidence is emerging that the oxidative‐stress response of this organism is complex, involving separate transcription‐factor responses to peroxide, superoxide anion and metal ions.

Oxidative stress and signal transduction in Saccharomyces cerevisiae: insights into ageing, apoptosis and diseases.

In yeast, as in higher eukaryotes, reactive oxygen species are produced as normal by-products of cellular metabolism. Under physiological conditions, the cell defence mechanisms are able to avoid molecular damages. This balance is disturbed when yeast cells are exposed to diverse environmental stress conditions, such as the presence of oxidants, heat shock, ethanol and metal ions. The increased production of reactive oxygen species is sensed by the cell, leading to the induction of defence mechanisms - the oxidative stress response. The present review discusses the mechanisms by which reactive oxygen species are sensed and the signalling pathways that are coupled with changes in genomic expression programs. Yeast has been used as an eukaryotic cell system to characterise the molecular mechanisms underlying the oxidative stress response. Furthermore, yeast has been utilised to elucidate the role of oxidative stress in ageing, apoptosis, and diseases, such as familial amyotrophic lateral sclerosis and Friedreichs ataxia.

HYDROGEN PEROXIDE-INDUCED CARBONYLATION OF KEY METABOLIC ENZYMES IN SACCHAROMYCES CEREVISIAE : THE INVOLVEMENT OF THE OXIDATIVE STRESS RESPONSE REGULATORS Yap1 AND Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.

Mitochondrial superoxide dismutase is essential for ethanol tolerance of Saccharomyces cerevisiae in the post-diauxic phase

This work reports the role of both superoxide dismutases-CuZnSOD (encoded by SOD1) and MnSOD (encoded by SOD2)-in the build-up of tolerance to ethanol during growth of Saccharomyces cerevisiae from exponential to post-diauxic phase. Both enzyme activities increase from the exponential phase to the diauxic shift and from the diauxic shift to the post-diauxic phase. The levels of mRNA-SOD1 and mRNA-SOD2 increase from the exponential phase to the diauxic shift; however, during the post-diauxic phase mRNA-SOD1 levels decrease while mRNA-SOD2 levels remain unchanged. These data indicate the existence of two regulatory mechanisms involved in the induction of SOD activity during growth: synthesis de novo of the proteins (until the diauxic shift), and post-transcriptional or post-translational regulation (during the post-diauxic phase). Ethanol does not alter the activities of either enzyme in cells from the diauxic shift or post-diauxic-phases, although the respective mRNA levels decrease in post-diauxic-phase cells treated with ethanol (14% or 20%). Results of experiments with sod1 and sod2 mutants show that MnSOD, but not CuZnSOD, is essential for ethanol tolerance of diauxic-shift and post-diauxic-phase cells. Evidence that ethanol toxicity is correlated with the production of reactive oxygen species in the mitochondria is obtained from results with respiration-deficient mutants. In these cells, the induction of superoxide dismutase activity by ethanol is low; also, the respiratory deficiency restores the capacity of sod2 cells to acquire ethanol tolerance.

Mnsod overexpression extends the yeast chronological (G0) life span but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells

Studies in Drosophila and Caenorhabditis elegans have shown increased longevity with the increased free radical scavenging that accompanies overexpression of oxidant-scavenging enzymes. This study used yeast, another model for aging research, to probe the effects of overexpressing the major activity protecting against superoxide generated by the mitochondrial respiratory chain. Manganese superoxide dismutase (MnSOD) overexpression increased chronological life span (optimized survival of stationary (G(0)) yeast over time), showing this is a survival ultimately limited by oxidative stress. In contrast, the same overexpression dramatically reduced the replicative life span of dividing cells (the number of daughter buds produced by each newly born mother cell). This reduction in the generational life span by MnSOD overexpression was greater than that generated by loss of the major redox-responsive regulator of the yeast replicative life span, NAD+-dependent Sir2p histone deacetylase. It was also independent of the latter activity. Expression of a mitochondrially targeted green fluorescent protein in the MnSOD overexpressor revealed that the old mother cells of this overexpressor, which had divided for a few generations, were defective in segregation of the mitochondrion from the mother to daughter. Mitochondrial defects are, therefore, the probable reason that MnSOD overexpression shortens replicative life span.

SELECTIVE ADVANTAGES CREATED BY CODON AMBIGUITY ALLOWED FOR THE EVOLUTION OF AN ALTERNATIVE GENETIC CODE IN CANDIDA SPP.

Several species of the genus Candida decode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions ‘why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?’ To address these two questions, we have attempted to reconstruct the early stages of Candida albicans CUG reassignment in the closely related yeast Saccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity in S. cerevisiae induces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic of C. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.

Adaptive response of the yeast Saccharomyces cerevisiae to reactive oxygen species: defences, damage and death

Abstract The yeast Saccharomyces cerevisiae has been extensively utilised to address the mechanisms underlying the oxidative stress response. The antioxidant defences can be induced either by respiratory growth or in the presence of pro-oxidants. The cell response involves the transcriptional control of genes by protein regulators that have been recently identified and post-translational activation of pre-existing defences. The current state of the art regarding the induction of antioxidant defences during respiratory growth and by exposure to hydrogen peroxide is reviewed.

Using extracellular polymeric substances (EPS)- producing cyanobacteria for the bioremediation of heavy metals: do cations compete for the EPS functional groups and also accumulate inside the cell?

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly of polysaccharidic nature. These EPS can remain associated to the cell surface as sheaths, capsules and/or slimes, or be liberated into the surrounding environment as released polysaccharides (RPS). The ability of EPS-producing cyanobacteria to remove heavy metals from aqueous solutions has been widely reported in the literature, focusing mainly on the biotechnological potential. However, the knowledge of the effects of the metals in the cells survival/growth is still scarce, particularly when they are simultaneously exposed to more than one metal. This work evaluated the effects of different concentrations of Cu(2+) and/or Pb(2+) in the growth/survival of Gloeothece sp. PCC 6909 and its sheathless mutant Gloeothece sp. CCY 9612. The results obtained clearly showed that both phenotypes are more severely affected by Cu(2+) than Pb(2+), and that the mutant is more sensitive to the former metal than the wild-type. Evident ultrastructural changes were also observed in the wild-type and mutant cells exposed to high levels (10 mg l(-1)) of Cu(2+). Moreover, in bi-metal systems, Pb(2+) was preferentially removed compared with Cu(2+), being the RPS of the mutant that is the most efficient polysaccharide fraction in metal removal. In these systems, the simultaneous presence of Cu(2+) and Pb(2+) caused a mutual inhibition in the adsorption of each metal.

Selection of Suitable Reference Genes for RT-qPCR Analyses in Cyanobacteria

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works.