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How to Cite Complete Issue More Information About This Article Revista de Biología Tropical ISSN: 0034-7744 ISSN: 0034-7744 Universidad de Costa Rica Barboza, Giuseppe F. O.; Gorlach-Lira, Krystyna; Sassi, Cristiane F. C.; Sassi, Roberto Microcystins production and antibacterial activity of cyanobacterial strains of Synechocystis, Synechococcus and Romeria from water and coral reef organisms (Brazil) Revista de Biología Tropical, vol. 65, no. 3, 2017, pp. 890-899 Universidad de Costa Rica DOI: 10.15517/rbt.v65i3.29437 Available in: http://www.redalyc.org/articulo.oa?id=44954192005 How to cite Complete issue Scientific Information System Redalyc More information about this article Network of Scientific Journals from Latin America and the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Project academic non-profit, developed under the open access initiative Microcystins production and antibacterial activity of cyanobacterial strains of Synechocystis, Synechococcus and Romeria from water and coral reef organisms (Brazil) Giuseppe F. O. Barboza1, Krystyna Gorlach-Lira1*, Cristiane F. C. Sassi2 & Roberto Sassi2 1. Federal University of Paraiba, Department of Molecular Biology, Laboratory of Biology of Microorganisms, Cidade Universitária, João Pessoa, 58059-900, Paraíba, Brazil; [email protected], [email protected] 2. Federal University of Paraiba, Department of Systematics and Ecology, Laboratory of Reef Environments and Biotechnology of Microalgae, Cidade Universitária, João Pessoa, 58059-900, Paraíba, Brazil; [email protected], [email protected] * Correspondence Received 04-II-2017. Corrected 27-III-2017. Accepted 28-IV-2017. Abstract: Cyanobacteria are widely distributed in terrestrial, freshwater and marine environments, and over the past decades have been recognized as a powerful source of bioactive compounds. In this study, some cyano- bacterial strains were isolated from samples of seawater, brackish water and tissue of reef benthic invertebrates (zoanthid Protopalythoa variabilis, the sponges Cynachrella sp. and Haliclona sp., the coral Siderastrea stel- lata, and ascidians), collected at the states of Paraíba and Rio Grande do Norte (Northeast of Brazil), during the period between July 2010 and February 2014. After standard isolation methods, the cultivation of the strains was carried out in acclimatized culture chamber (25 °C) under constant aeration, for 15 days at 12-hour pho- toperiod, using Conway and BG11 media made with filtered seawater. The cyanobacterial cells were analysed for the microcystin production by the ELISA technique and their ethanolic and methanolic extracts for the antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa by the agar well diffusion method. The detection of the mcyB gene, one of the genes related to the microcystin synthesis, was done by the Polymerase Chain Reaction (PCR) technique. The majority of the eighteen cyanobacterial strains belonged to Synechococcaceae Family. The genera of Synechocystis, Synechococcus and Romeria were represented by ten, six and two strains, respectively. The production of microcystins was observed in five strains belonging to the genus Synechocystis. The presence of mcyB gene was detected in 12 strains of cyanobacteria: Synechocystis (three strains), Synechococcus (six strains) and Romeria (two strains). Only one strain (Synechocystis aquatilis) showed both the microcystin production and the mcyB gene presence. The antibacterial activity was observed for one strain of Romeria gracilis, one strain of Synechocystis aquatilis and two strains of Synechococcus sp. The ethanolic extracts of R. gracilis strain and two Synechococcus spp. strains inhibited the growth of P. aeru- ginosa. Among methanolic extracts of cyanobacteria, only one strain of S. aquatilis showed activity against S. aureus, and one R. gracilis strain against P. aeruginosa. Some cyanobacterial strains studied were positive for the microcystin production and antibacterial activity against pathogenic bacteria S. aureus and P. aeuruginosa, and may be further explored for additional biotechnological applications. Rev. Biol. Trop. 65 (3): 890-899. Epub 2017 September 01. Key words: Synechococcales, extracts, mcyB, cyanobacteria. Cyanobacteria or blue-green algae are In the last decades, cyanobacteria have photosynthetic prokaryotes widely distributed been gaining attention in ecology, biochemistry, in almost all habitats, from aquatic marine and physiology and molecular biology, because of freshwater ones to terrestrial environments, their high potential for antibiotics and pharma- being also associated with various marine cologically active compounds production (Car- organisms such as corals and sponges (Glas et dozo et al., 2007; Al-Wathnani, Ara, Thamaz, al., 2010; Paerl & Paul, 2011). Al-Dayel, & Bakir, 2012). The exploitation 890 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (3): 890-899, September 2017 of natural cyanobacterial products can result in MATERIALS AND METHODS the discovery of new compounds (lipopeptides, amino acids, fatty acids, macrolides) with anti- Isolation and identification of marine protozoal, antiviral, antibacterial, antifungal, cyanobacteria: Cyanobacteria were isolated antitumoral, cytotoxic, and other biological from samples of seawater, brackish water of the activities (Ehrenreich, Waterbury, & Webb, river mouth, and tissue of reef benthic inverte- 2005; Singh, Tiwari, Rai, & Mohapatra, 2011; brates: the zoanthid Protopalythoa variabilis, Costa et al., 2012). Among the toxins produced the sponges Cinachyrella sp. and Haliclona by cyanobacteria, the microcystins have been sp., the coral Siderastrea stellata, and ascidian (Ascidiacea) collected at the states of Paraíba the most commonly found in blooms around and Rio Grande do Norte, Northeastern Brazil. the world, and they are produced by sev- The water and reef organism samples eral cyanobacterial genera such as Microcystis, were collected between July 2010 and Febru- Anabaena, Nodularia, Oscillatoria, Nostoc, ary 2014 at the Northeast coast of Brazil. The Cylindrospermopsis, Aphanizomenon, Plankto- samples of coral Siderastrea stellata, ascidian thrix, Anabaenopsis, Synechocystis, Lyngbya, and sponge Cinachyrella sp. were collected in and others (Siqueira & Oliveira-Filho, 2005; a coral reef of Cabo Branco Beach (7°08’50” Bortoli & Pinto, 2015). The toxic strains can S - 34°47’51” W), João Pessoa, Paraíba State, be identified by the presence of mcy gene while the samples of sponge Haliclona sp. were encoding the polyketide synthases and peptide collected in the coral reefs of Carapibus (7°17’ synthetases involved in the biosynthesis of 59.14” S - 34°47’45” W), Conde, Paraiba State. microcystins (Ross, Santiago-Vázquez, & Paul, The seawater were obtained from a coral reef 2006; Dyble, Fahnenstiel, Litaker, Millie, & of Cabo Branco Beach (7°08’50” S - 34°47’51” Tester, 2008). Among the mcy genes, a region W) (João Pessoa), Cabedelo Beach (Cabedelo) of the mcyB, has been often used as a molecu- and Acau Beach (Acau), Paraiba State. The lar marker for the detection of microcystin brackish water samples were collected at Inter- producers (Bittencourt-Oliveira, 2003; Dyble mares Lagoon (07º02’52” S - 34º51’34” W), et al., 2008; Bittencourt-Oliveira, Oliveira, João Pessoa, Paraiba State, Mamanguape River & Pinto, 2011). estuary (6°47’19” S - 34°59’22” W), Maman- The importance of secondary metabolites guape, Paraiba State, Bucatú River estuary of cyanobacteria and unicellular algae with (7º18’19.85”S - 34º47’47.14” W), Conde, antimicrobial properties has been extensively Paraiba State, and Pirangi River estuary, Rio revised by Senhorinho, Ross and Scott (2015). Grande do Norte State. Additionally, the antibacterial and antifungal The 500 mL water samples were collected activity of cyanobacteria exometabolites has using sterilized bottles, and the samples of reef been previouly reported by Volk and Furkert organisms were placed in plastic bags contain- (2006) and Ramos et al. (2015). In this work, ing seawater, and were sealed hermetically. The we isolated and identified the cyanobacteria samples were transported on ice to the Labora- belonging to the genera Synechocystis, Syn- tory of Reef Environments and Biotechnology echococcus and Romeria from seawater and of Microalgae of Federal University of Paraíba, reef benthic invertebrates (sponges, corals and João Pessoa, Paraíba State. ascidians) from the Brazilian Northeastern coast The Siderastrea spp. tissue was extracted and we evaluated their capacity for microcys- using a high-pressure jet of sterile seawater tin production and antibacterial activity. (Waterpik®) according to the protocol of Costa, Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (3): 890-899, September 2017 891 Sassi, and Gorlach-Lira (2008). The zoanthid, the Federal University of Paraiba. The culture sponges and ascidian samples (5.0 g) were of the strains was carried out in bottom flat fragmented and macerated with the steril por- flasks containing 5 L of filtered seawater with celain pestle and mortar with the addition of 2 Conway and BG11 medium. The cultures were mL of filtered seawater. incubated in acclimatized culture chamber (25 The aliquots of seawater (2 mL) or tis- °C) under constant aeration provided by Resun sue samples of each reef invertebrate (2 mL), AOC2 minicompressor for 15 days with light- were transferred to 250 mL autoclaved flasks ing system of 12-hours photoperiod. Then, containing Conway medium (Walne, 1970) or these were centrifuged at 3 500 g, 25
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