Arlete Santos

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.

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N(2)-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles

UNLABELLED The effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110 were evaluated. Our results clearly show that each heavy metal affects the cells in a particular manner, triggering distinctive responses. Concerning chronic exposure, cells were more affected by Cu(2+) followed by Pb(2+), Cd(2+), and Li(+). The presence of metal leads to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes (iTRAQ) allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu(2+)) and a non-essential (Cd(2+)) metal. The majority of the proteins identified and with fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Moreover, our results indicate that during chronic exposure to sub-lethal concentrations of Cu(2+), the cells tune down their metabolic rate to invest energy in the activation of detoxification mechanisms, which eventually result in a remarkable recovery. In contrast, the toxic effects of Cd(2+) are cumulative. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. BIOLOGICAL SIGNIFICANCE This work shows the holistic effects of different heavy metals on the cells of the highly efficient EPS-producer the cyanobacterium Cyanothece sp. CCY 0110. The growth/survival, EPS production, ultrastructure, protein profiles and stress response were evaluated. The knowledge generated by this study will contribute to the implementation of heavy-metal removal systems based on cyanobacteria EPS or their isolated polymers.

Immunolocalization of the uptake hydrogenase in the marine cyanobacterium Lyngbya majuscula CCAP 1446/4 and two Nostoc strains

In N(2)-fixing cyanobacteria, the reduction of N(2) to NH(3) is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N(2)-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula, reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme, the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.

Effect of hydrogen peroxide on catalase gene expression, isoform activities and levels in leaves of potato sprayed with homobrassinolide and ultrastructural changes in mesophyll cells

The effect of hydrogen peroxide (H2O2) on catalase (CAT) isoform activities and amounts and on mRNA levels was studied in leaves from potato plants untreated and treated with homobrassinolide (HBR). Northern blot analysis revealed that 100 mm H2O2 supplied through the leaf petiole for 4 h did not induce CAT expression. In contrast, CAT1 and CAT2 responded differently to longer treatment, as CAT2 transcript levels increased markedly whereas CAT1 transcript levels remained unchanged. Western blot analysis showed disparity between the level of CAT1 transcript and CAT1 amount, which actually decreased after 28 h. CAT2 amount correlated well with transcript accumulation and CAT2 activity as visualised by zymogram analysis. H2O2 modified the relative importance of CAT isoforms. After 4 h, CAT1 was prevalent in untreated and H2O2-treated leaves. After 28 h, CAT2 was prevalent in H2O2-treated leaves; therefore, the quantified increase in total CAT activity in these leaves was due to the rise in CAT2. HBR pre-treatment increased CAT2 basal level not changing the pattern of CAT responses to H2O2, only lowering its amplitude. Even so, ultrastructural studies showed that HBR significantly reduced H2O2 negative effects on cellular sub-structures, allowing better recovery of affected structures and reducing the macroscopic injury symptoms on leaves, thus data point to a HBR protective role.

Unusual symbiotic cyanobacteria association in the genetically diverse intertidal marine sponge Hymeniacidon perlevis (Demospongiae, Halichondrida).

Cyanobacteria represent one of the most common members of the sponge-associated bacterial community and are abundant symbionts of coral reef ecosystems. In this study we used Transmission Electron Microscopy (TEM) and molecular techniques (16S rRNA gene marker) to characterize the spatial distribution of cyanobionts in the widely dispersed marine intertidal sponge Hymeniacidon perlevis along the coast of Portugal (Atlantic Ocean). We described new sponge associated cyanobacterial morphotypes (Xenococcus-like) and we further observed Acaryochloris sp. as a sponge symbiont, previously only reported in association with ascidians. Besides these two unique cyanobacteria, H. perlevis predominantly harbored Synechococcus sp. and uncultured marine cyanobacteria. Our study supports the hypothesis that the community of sponge cyanobionts varies irrespective of the geographical location and is likely influenced by seasonal fluctuations. The observed multiple cyanobacterial association among sponges of the same host species over a large distance may be attributed to horizontal transfer of symbionts. This may explain the absence of a co-evolutionary pattern between the sponge host and its symbionts. Finally, in spite of the short geographic sampling distance covered, we observed an unexpected high intra-specific genetic diversity in H. perlevis using the mitochondrial genes ATP6 (π = 0.00177), COI (π = 0.00241) and intergenic spacer SP1 (π = 0.00277) relative to the levels of genetic variation of marine sponges elsewhere. Our study suggests that genotypic variation among the sponge host H. perlevis and the associated symbiotic cyanobacteria diversity may be larger than previously recognized.

Characterization of an intertidal cyanobacterium that constitutes a separate clade together with thermophilic strains

This work reports the characterization of a unicellular cyanobacterium (LEGE 06123) isolated from an intertidal diazotrophic mat at Luz, a rocky beach in southern Portugal. This organism aggregates to form colonies held together by extracellular polymeric substances (EPS). Cells or cell groups are surrounded by a laminated sheath, and division occurs by binary fission, often in irregular planes. Dispersion is achieved by liberation of daughter cells from the ruptured mother sheath. LEGE 06123 is halotolerant, withstands a considerable range of iron concentrations, and favours growth at moderately high temperatures (35, 40°C). According to the criteria defined in 1998 by Komárek and Anagnostidis, LEGE 06123 could belong to the species Gloeocapsopsis crepidinum (Thuret) Geitler ex Komárek 1993. However, our phylogenetic analyses clearly show that LEGE 06123 is closely related to three cyanobacteria isolated from the Great Yellowstone and Zerka Ma’in hot springs, one of them (5.2 s.c.1) being described as a new genus and a new species – Chroogloeocystis siderophila Brown, Mummey et Cooksey. The polyphasic characterization performed here has exposed the unusual features of LEGE 06123, which along with the three thermophilic strains, forms a distinct clade deserving further investigation.

Description of new genera and species of marine cyanobacteria from the Portuguese Atlantic coast

Aiming at increasing the knowledge on marine cyanobacteria from temperate regions, we previously isolated and characterized 60 strains from the Portuguese foreshore and evaluate their potential to produce secondary metabolites. About 15% of the obtained 16S rRNA gene sequences showed less than 97% similarity to sequences in the databases revealing novel biodiversity. Herein, seven of these strains were extensively characterized and their classification was re-evaluated. The present study led to the proposal of five new taxa, three genera (Geminobacterium, Lusitaniella, and Calenema) and two species (Hyella patelloides and Jaaginema litorale). Geminobacterium atlanticum LEGE 07459 is a chroococcalean that shares morphological characteristics with other unicellular cyanobacterial genera but has a distinct phylogenetic position and particular ultrastructural features. The description of the Pleurocapsales Hyella patelloides LEGE 07179 includes novel molecular data for members of this genus. The filamentous isolates of Lusitaniella coriacea - LEGE 07167, 07157 and 06111 - constitute a very distinct lineage, and seem to be ubiquitous on the Portuguese coast. Jaaginema litorale LEGE 07176 has distinct characteristics compared to their marine counterparts, and our analysis indicates that this genus is polyphyletic. The Synechococcales Calenema singularis possess wider trichomes than Leptolyngbya, and its phylogenetic position reinforces the establishment of this new genus.

In vitro bulb formation of Narcissus asturiensis, a threatened species of the Amaryllidaceae

Summary The culture conditions for in vitro production of bulbs of Narcissus asturiensis were determined. The process was initiated using twin-scales as primary explants that were cultured on a modified MS medium supplemented with IBA (4.9 μM) 1 BA (8.87 μM) or NAA (0.65 μM) 1 BA (26.6 μM). Both media were appropriate for shoot induction and proliferation, although the multiplication rate of leafy shoots was higher on the medium with NAA and BA. After 60 d of culture on both media, tiny bulb-like structures appeared at the base of the leaves; however, the medium with IBA was the most suitable for bulb induction. Further growth of the bulbs was achieved on a similar basal medium with only NAA and with an increased sucrose content (9%). After 90.d of culture on this growth medium, bulbs attained a mean diameter of 8 mm but the production of roots was low. Root induction was performed on four rooting media (MS salt solution with NAA or IBA and half strength MS salt solution with NAA or IBA), the highest number of rooted bulbs was on the medium with reduced inorganic salt concentration plus NAA. Rooted bulbs adequately prepared for ex vitro culture were then transferred to a glasshouse and the success of transplantation is under evaluation.

Induction of somatic embryogenesis in Iris hollandica Hort. cv. 'Bronze Queen'

Summary Twin-scale explants of Iris hollandica cv. ‘Bronze Queen’ produced callus when cultured on a modified Murashige and Skoog medium supplemented with 9.0, 18.1, 27.2 or 36.2 µM 2,4-dichlorophenoxyacetic acid (2,4-D). Induction of embryogenic callus was obtained at 9.0 or 18.1 µM 2,4-D, while higher concentrations of the auxin (27.2 or 36.2 µM) were not effective. Embryo-like structures developed on the surface of embryogenic calli. Somatic embryo maturation was achieved when the bipolar structures produced were cultured on medium without auxin. To improve embryo germination, the developing embryos were transferred to medium supplemented with 0.95 µM abscisic acid (ABA). In this medium, mature embryos developed into normal plantlets. Histological studies confirmed that the structures exhibiting bipolar differentiation were somatic embryos. Regeneration of I. hollandica cv. ‘Bronze Queen’ plants through somatic embryogenesis was thus established.