Joana Martins

Peptide diversity in strains of the cyanobacterium Microcystis aeruginosa isolated from Portuguese water supplies

Strains of the cyanobacterium Microcystis aeruginosa were isolated into pure culture from a variety of lakes, rivers, and reservoirs in Portugal. Samples were tested with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the presence of various peptide groups including aeruginosins, microginins, anabaenopeptins, cyanopeptilins, microcystins, and microviridins and other peptide-like compounds. Binary data, based on the presence and absence of different peptide groups, were analyzed by phylogenetic inference. DNA was also extracted from the samples and tested using a range of primers. Those strains that gave positive results for a Microcystis-specific primer pair were further analyzed for the presence of genes linked to the biosynthesis of microginin and microcystin. The results showed that a wide range of microcystin forms were produced by the strains among which MC-LR, -FR, -RR, -WR, and -YR were the most common. The peptide profiles obtained from the MALDI analysis were assessed using cluster analysis which resulted in the formation of distinct groups or chemotypes.

Cyanobactins from Cyanobacteria: Current Genetic and Chemical State of Knowledge.

Cyanobacteria are considered to be one of the most promising sources of new, natural products. Apart from non-ribosomal peptides and polyketides, ribosomally synthesized and post-translationally modified peptides (RiPPs) are one of the leading groups of bioactive compounds produced by cyanobacteria. Among these, cyanobactins have sparked attention due to their interesting bioactivities and for their potential to be prospective candidates in the development of drugs. It is assumed that the primary source of cyanobactins is cyanobacteria, although these compounds have also been isolated from marine animals such as ascidians, sponges and mollusks. The aim of this review is to update the current knowledge of cyanobactins, recognized as being produced by cyanobacteria, and to emphasize their genetic clusters and chemical structures as well as their bioactivities, ecological roles and biotechnological potential.

Unraveling cyanobacteria ecology in wastewater treatment plants (WWTP).

Cyanobacteria may be important components of wastewater treatment plants’ (WWTP) biological treatment, reaching levels of 100% of the total phytoplankton density in some systems. The occurrence of cyanobacteria and their associated toxins in these systems present a risk to the aquatic environments and to public health, changing drastically the ecology of microbial communities and associated organisms. Many studies reveal that cyanotoxins, namely microcystins may not act as antibacterial compounds but they might have negative impacts on protozoans, inhibiting their growing and respiration rates and leading to changes in cellular morphology, decreasing consequently the treatment efficacy in WWTP. On the other side, flagellates and ciliates may ingest some cyanobacteria species while the formation of colonies by these prokaryotes may be seen as a defense mechanism against predation. Problems regarding the occurrence of cyanobacteria in WWTP are not limited to toxin production. Other cyanobacterial secondary metabolites may act as antibacterial compounds leading to the disruption of bacterial communities that biologically convert organic materials in WWTP being fundamental to the efficacy of the process. Studies reveal that the potential antibacterial capacity differs according to cyanobacteria specie and it seems to be more effective in Gram (+) bacteria. Thus, to understand the effects of cyanobacterial communities in the efficiency of the waste water treatment it will be necessary to unravel the complex interactions between cyanobacterial populations, bacteria, and protozoa in WWTP in situ studies.

DNA profiling of complex bacterial populations: toxic cyanobacterial blooms.

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.

Cyanobacteria and bacteria co-occurrence in a wastewater treatment plant: absence of allelopathic effects.

Fifty-one cyanobacteria strains, belonging to Phormidium autumnale, Planktothrix mougeotii, Limnotrix sp. and Synechocystis sp. species, were isolated during a twelve months period, from a Wastewater Treatment Plant (WWTP) located in the north of Portugal. Isolated strains and environmental samples were analyzed by Polymerase Chain Reaction (PCR) technique to evaluate the presence of the genes linked to the biosynthesis of the cyanotoxins, microcystin (MC), nodularin (NOD) and cylindrospermopsin (CYN). Even though none of the isolated strains contained the genes responsible for these toxins production, the use of PCR in the water samples detected the mcyE gene, in the biological treatment tank, and the result of its sequence revealed 92% of maximum identity with the microcystin gene. ELISA assay confirmed the presence of the toxin with values of 0.60 μg MC-LR eq/l. The effects of extracts of the cyanobacteria strains on bacteria isolated from the WWTP, revealed that none of the tested strains inhibited or reduced the bacterial growth. This may indicate that those prokaryote communities may have co-evolved in a way that success of both cyanobacteria and bacteria was achieved.

N-Terminal Protease Gene Phylogeny Reveals the Potential for Novel Cyanobactin Diversity in Cyanobacteria

Cyanobactins are a recently recognized group of ribosomal cyclic peptides produced by cyanobacteria, which have been studied because of their interesting biological activities. Here, we have used a PCR-based approach to detect the N-terminal protease (A) gene from cyanobactin synthetase gene clusters, in a set of diverse cyanobacteria from our culture collection (Laboratory of Ecotoxicology, Genomics and Evolution (LEGE) CC). Homologues of this gene were found in Microcystis and Rivularia strains, and for the first time in Cuspidothrix, Phormidium and Sphaerospermopsis strains. Phylogenetic relationships inferred from available A-gene sequences, including those obtained in this work, revealed two new groups of phylotypes, harboring Phormidium, Sphaerospermopsis and Rivularia LEGE isolates. Thus, this study shows that, using underexplored cyanobacterial strains, it is still possible to expand the known genetic diversity of genes involved in cyanobactin biosynthesis.

Cyanobacterial Diversity in Microbial Mats from the Hypersaline Lagoon System of Araruama, Brazil: An In-depth Polyphasic Study

Microbial mats are complex, micro-scale ecosystems that can be found in a wide range of environments. In the top layer of photosynthetic mats from hypersaline environments, a large diversity of cyanobacteria typically predominates. With the aim of strengthening the knowledge on the cyanobacterial diversity present in the coastal lagoon system of Araruama (state of Rio de Janeiro, Brazil), we have characterized three mat samples by means of a polyphasic approach. We have used morphological and molecular data obtained by culture-dependent and -independent methods. Moreover, we have compared different classification methodologies and discussed the outcomes, challenges, and pitfalls of these methods. Overall, we show that Araruamas lagoons harbor a high cyanobacterial diversity. Thirty-six unique morphospecies could be differentiated, which increases by more than 15% the number of morphospecies and genera already reported for the entire Araruama system. Morphology-based data were compared with the 16S rRNA gene phylogeny derived from isolate sequences and environmental sequences obtained by PCR-DGGE and pyrosequencing. Most of the 48 phylotypes could be associated with the observed morphospecies at the order level. More than one third of the sequences demonstrated to be closely affiliated (best BLAST hit results of ≥99%) with cyanobacteria from ecologically similar habitats. Some sequences had no close relatives in the public databases, including one from an isolate, being placed as “loner” sequences within different orders. This hints at hidden cyanobacterial diversity in the mats of the Araruama system, while reinforcing the relevance of using complementary approaches to study cyanobacterial diversity.

Cyanobacterial diversity held in microbial biological resource centers as a biotechnological asset: the case study of the newly established LEGE culture collection

Cyanobacteria are a well-known source of bioproducts which renders culturable strains a valuable resource for biotechnology purposes. We describe here the establishment of a cyanobacterial culture collection (CC) and present the first version of the strain catalog and its online database (http://lege.ciimar.up.pt/). The LEGE CC holds 386 strains, mainly collected in coastal (48%), estuarine (11%), and fresh (34%) water bodies, for the most part from Portugal (84%). By following the most recent taxonomic classification, LEGE CC strains were classified into at least 46 genera from six orders (41% belong to the Synechococcales), several of them are unique among the phylogenetic diversity of the cyanobacteria. For all strains, primary data were obtained and secondary data were surveyed and reviewed, which can be reached through the strain sheets either in the catalog or in the online database. An overview on the notable biodiversity of LEGE CC strains is showcased, including a searchable phylogenetic tree and images for all strains. With this work, 80% of the LEGE CC strains have now their 16S rRNA gene sequences deposited in GenBank. Also, based in primary data, it is demonstrated that several LEGE CC strains are a promising source of extracellular polymeric substances (EPS). Through a review of previously published data, it is exposed that LEGE CC strains have the potential or actual capacity to produce a variety of biotechnologically interesting compounds, including common cyanotoxins or unprecedented bioactive molecules. Phylogenetic diversity of LEGE CC strains does not entirely reflect chemodiversity. Further bioprospecting should, therefore, account for strain specificity of the valuable cyanobacterial holdings of LEGE CC.

Modulation of hepatic glutathione transferases isoenzymes in three bivalve species exposed to purified microcystin-LR and Microcystis extracts

ABSTRACT This study compares the role of hepatic cytosolic glutathione transferases (cGST) isoforms of three different bivalve species to a Microcystis aeruginosa extract and purified MC‐LR exposure (both at 150 &mgr;g MC‐LR L−1) for 24 h. Characterization and alterations of the cytosolic GST activities in Mytilus galloprovincialis, Ruditapes philippinarum and Corbicula fluminea were measured using four class‐specific substrates and changes in individual GST isoforms expression were achieved by a subsequent two‐dimensional electrophoresis analysis. Evaluation of cGST activity basal levels using the four class‐specific substrates denoted quantitative differences between the three bivalves. Purified MC‐LR did not induce any significant response from bivalves. On the other hand, cell extracts caused significant alterations according to bivalves and substrates. Among the three bivalves, only R. philippinarum showed a significant induction of cGST activity using generic 1‐chloro‐2,4‐dinitrobenzene (CDNB) substrate. However, no significant alterations were detected in these clams by cell extracts using the other specific substrates. In contrast, C. fluminea revealed significant induction of cGST activity when using 3,4‐dichloronitrobenzene (DCNB) and ethacrynic acid (EA). In M. galloprovincialis, cell extracts promoted a significant decrease of cGST activity when using EA substrate. Altered protein expression was quantitatively detected upon exposure to cell extracts for one spot in R. philippinarum and another for C. fluminea, both upregulated (2.0 and 8.5‐fold, respectively) and identified as a sigma1‐class GST in the case of the first. The results showed that the three bivalves presented specific adaptive biotransformation responses to MCs and other cyanobacteria compounds supported by the modulation of distinct cGST classes. HIGHLIGHTSHepatic cGST activity basal levels denoted quantitative differences between bivalves.Purified MC‐LR did not induce responses on bivalves hepatic cGST system.M. extracts caused different induction of hepatic cGST in C. fluminea and R. philippinarum.M. galloprovincialis hepatic cGST system was less affected by the cell extracts.

Towards Automatic Calibration of Dotblot Images.

This paper addresses the issue of calibration (or normalization) of macroarray (dotblot) images. It proposes 3 parameters for the evaluation of the impact on the recorded markers of under- and over-exposure during the experimental acquisition of dotblot images – volume (V), saturation (S) and apparent radius (R). These parameters were evaluated using 101 dotblot images obtained from 16 different experiments, with 404 control markers in total. A procedure to simulate the changes on markers by increasing and decreasing exposure times is also presented. This can be the basis of a normalization procedure for dot blot images, which would be an important improvement in the current laboratory image acquisition protocol, reducing the subjectivity both at the acquisition level and at the subsequent image analysis stage.