Publications by authors named "Raquel Castelo-Branco"

12 Publications

  • Page 1 of 1

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria.

BMC Genomics 2021 Aug 31;22(1):633. Epub 2021 Aug 31.

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Matosinhos, Portugal.

Background: Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC - the first predicted dimetal-carboxylate halogenase to be characterized - was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes.

Results: In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria.

Conclusions: Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.
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http://dx.doi.org/10.1186/s12864-021-07939-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406957PMC
August 2021

Biosynthesis of Chlorinated Lactylates in sp. LEGE 00249.

J Nat Prod 2021 02 14;84(2):278-286. Epub 2021 Jan 14.

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Avenida General Norton de Matos, s/n, 4450-208 Matosinhos, Portugal.

Lactylates are an important group of molecules in the food and cosmetic industries. A series of natural halogenated 1-lactylates, chlorosphaerolactylates (-), were recently reported from sp. LEGE 00249. Here, we identify the biosynthetic gene cluster, containing all the necessary functionalities for the biosynthesis of the natural lactylates, based on in silico analyses. Using a combination of stable isotope incorporation experiments and bioinformatic analysis, we propose that dodecanoic acid and pyruvate are the key building blocks in the biosynthesis of -. We additionally report minor analogues of these molecules with varying alkyl chains. This work paves the way to accessing industrially relevant lactylates through pathway engineering.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923214PMC
February 2021

Absence of Cyanotoxins in Llayta, Edible Nostocaceae Colonies from the Andes Highlands.

Toxins (Basel) 2020 06 9;12(6). Epub 2020 Jun 9.

Centro Interdisciplinar de Investigação Marinha e Ambiental- CIIMAR, 4450-208 Matosinhos, Portugal.

Edible Llayta are cyanobacterial colonies consumed in the Andes highlands. Llayta and four isolated cyanobacteria strains were tested for cyanotoxins (microcystin, nodularin, cylindrospermopsin, saxitoxin and β-N-methylamino-L-alanine-BMAA) using molecular and chemical methods. All isolates were free of target genes involved in toxin biosynthesis. Only DNA from Llayta amplified the E gene. Presence of microcystin-LR and BMAA in Llayta extracts was discarded by LC/MS analyses. The analysed Llayta colonies have an incomplete microcystin biosynthetic pathway and are a safe food ingredient.
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http://dx.doi.org/10.3390/toxins12060382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354591PMC
June 2020

Chlorosphaerolactylates A-D: Natural Lactylates of Chlorinated Fatty Acids Isolated from the Cyanobacterium sp. LEGE 00249.

J Nat Prod 2020 06 1;83(6):1885-1890. Epub 2020 Jun 1.

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal.

Four natural lactylates of chlorinated fatty acids, chlorosphaerolactylates A-D (-), were isolated from the methanolic extract of the cyanobacterium sp. LEGE 00249 through a combination of bioassay-guided and MS-guided approaches. Compounds - are esters of (mono-, di-, or tri)chlorinated lauric acid and lactic acid, whose structures were assigned on the basis of spectrometric and spectroscopic methods inclusive of 1D and 2D NMR experiments. High-resolution mass-spectrometry data sets also demonstrated the existence of other minor components that were identified as chlorosphaero(bis)lactylate analogues. The chlorosphaerolactylates were tested for potential antibacterial, antifungal, and antibiofilm properties using bacterial and fungal clinical isolates. Compounds - showed a weak inhibitory effect on the growth of S54F9 and SMI416, as well as on the biofilm formation of coagulase-negative FI31.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00072DOI Listing
June 2020

First Detection of Microcystin-LR in the Amazon River at the Drinking Water Treatment Plant of the Municipality of Macapá, Brazil.

Toxins (Basel) 2019 11 15;11(11). Epub 2019 Nov 15.

Bionorte Post-Graduate Program, UNIFAP, Federal University of Amapá, Macapá, 68903-419 Amapá, Brazil.

Human poisoning by microcystin has been recorded in many countries, including Brazil, where fatal cases have already occurred. The Amazon River is the main source of drinking water in municipalities such as Macapá, where there is no monitoring of cyanobacteria and cyanotoxins. This study investigated the presence of cyanobacteria and cyanotoxins in samples from a drinking water treatment plant (DWTP) that catches water from the Amazon River. The toxin analyses employed ELISA, LC/MS, and molecular screening for genes involved in the production of cyanotoxins. The sampling was carried out monthly from April 2015 to April 2016 at the intake (raw water) and exit (treated water) of the DWTP. This study reports the first detection of microcystin-LR (MC-LR) in the Amazon River, the world's largest river, and in its treated water destined for drinking water purposes in Macapá, Brazil. The cyanobacterial density and MC-LR concentration were both low during the year. However, showed a density peak (± 900 cells mL) in the quarter of June-August 2015, when MC-LR was registered (2.1 µg L). Statistical analyses indicate that may produce the microcystin.
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http://dx.doi.org/10.3390/toxins11110669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891726PMC
November 2019

The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae, Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica.

J Phycol 2020 02 17;56(1):208-216. Epub 2019 Nov 17.

Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, 4450-208, Matosinhos, Portugal.

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.
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http://dx.doi.org/10.1111/jpy.12940DOI Listing
February 2020

Inhibition of Bacterial and Fungal Biofilm Formation by 675 Extracts from Microalgae and Cyanobacteria.

Antibiotics (Basel) 2019 Jun 12;8(2). Epub 2019 Jun 12.

ISGlobal, Hospital Clínic-Universitat de Barcelona, 08036 Barcelona, Spain.

Bacterial biofilms are complex biological systems that are difficult to eradicate at a medical, industrial, or environmental level. Biofilms confer bacteria protection against external factors and antimicrobial treatments. Taking into account that about 80% of human infections are caused by bacterial biofilms, the eradication of these structures is a great priority. Biofilms are resistant to old-generation antibiotics, which has led to the search for new antimicrobials from different sources, including deep oceans/seas. In this study, 675 extracts obtained from 225 cyanobacteria and microalgae species (11 phyla and 6 samples belonging to unknown group) were obtained from different culture collections: The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC), the Coimbra Collection of Algae (ACOI) from Portugal, and the Roscoff Culture Collection (RCC) from France. The largest number of samples was made up of the microalgae phylum (270) followed by Cyanobacteria (261). To obtain a large range of new bioactive compounds, a method involving three consecutive extractions (hexane, ethyl acetate, and methanol) was used. The antibiofilm activity of extracts was determined against seven different bacterial species and two strains in terms of minimal biofilm inhibitory concentration (MBIC). The highest biofilm inhibition rates (%) were achieved against and . , , and Cyanobacteria were the most effective against all microorganisms. In particular, extracts of phylum presented the lowest MBIC and MBIC values for all the strains except .
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http://dx.doi.org/10.3390/antibiotics8020077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628188PMC
June 2019

Structure of Hierridin C, Synthesis of Hierridins B and C, and Evidence for Prevalent Alkylresorcinol Biosynthesis in Picocyanobacteria.

J Nat Prod 2019 02 4;82(2):393-402. Epub 2019 Feb 4.

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR) , University of Porto , Avenida General Norton de Matos, s/n , 4450-208 Matosinhos , Portugal.

Small, single-celled planktonic cyanobacteria are ubiquitous in the world's oceans yet tend not to be perceived as secondary metabolite-rich organisms. Here we report the isolation and structure elucidation of hierridin C, a minor metabolite obtained from the cultured picocyanobacterium Cyanobium sp. LEGE 06113. We describe a simple, straightforward synthetic route to the scarcely produced hierridins that relies on a key regioselective halogenation step. In addition, we show that these compounds originate from a type III PKS pathway and that similar biosynthetic gene clusters are found in a variety of bacterial genomes, most notably those of the globally distributed picocyanobacteria genera Prochlorococcus, Cyanobium and Synechococcus.
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http://dx.doi.org/10.1021/acs.jnatprod.8b01038DOI Listing
February 2019

GST transcriptional changes induced by a toxic Microcystis aeruginosa strain in two bivalve species during exposure and recovery phases.

Ecotoxicology 2018 Nov 21;27(9):1272-1280. Epub 2018 Sep 21.

CIIMAR/CIMAR - Interdisciplinary Centre of Marine and Environmental Research, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450-208, Matosinhos, Portugal.

Previous studies have demonstrated the modulation of glutathione transferases (GSTs) induced by microcystin (MC) alone or in combination with other cyanobacterial secondary metabolites in bivalves. However, interspecies information about which and how GST isoforms are affected by these secondary metabolites is still scarce, especially considering the dynamic process involving their uptake and elimination routes. In this context, the role of GSTs gene expression changes in response to a toxic Microcystis aeruginosa extract were examined for Mytilus galloprovincialis and Ruditapes philippinarum during exposure and recovery phases. The expression levels of sigma 1, sigma 2, pi and mu-class GST genes were analyzed in the hepatopancreas of both bivalve species during cyanobacteria extract exposure (24 h) and post-exposure (24 and 72 h). Only a significant induction of sigma 1-class GST expression was observed for R. philippinarum upon 24-hour exposure of both bivalve species to Microcystis extract. During the recovery phase, GST transcriptional changes for M. galloprovincialis were characterized by an early induction (24 h) of sigma 1 and sigma 2 transcripts. On the other hand, GST transcriptional changes for R. philippinarum during post-exposure phase were characterized by an early induction (24 h) of sigma 1 and mu transcripts and a later induction (72 h) of the four analyzed GST transcripts. Such differences reflect variable GST response mechanisms to cope with MC-producing cyanobacterial blooms exposure between these two bivalve species, revealing a higher sensitivity of R. philippinarum to Microcystis-induced stress than M. galloprovincialis. The results also suggest a much higher level of activity of the GST detoxification system during the recovery phase compared to the period of the stress exposure for both bivalve species.
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http://dx.doi.org/10.1007/s10646-018-1980-yDOI Listing
November 2018

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

J Appl Phycol 2018 6;30(3):1437-1451. Epub 2018 Jan 6.

1Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), Terminal de Cruzeiros do Porto de Leixões, University of Porto, 4450-208 Matosinhos, Portugal.

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.
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http://dx.doi.org/10.1007/s10811-017-1369-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5982461PMC
January 2018

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

Front Microbiol 2017 30;8:1233. Epub 2017 Jun 30.

Faculty of Sciences, University of PortoPorto, Portugal.

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 Araruama's 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.
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http://dx.doi.org/10.3389/fmicb.2017.01233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5492833PMC
June 2017
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