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Doctoral Thesis
DOI
10.11606/T.64.2012.tde-18012013-142454
Document
Author
Full name
Caroline Hoff Risseti
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2012
Supervisor
Committee
Fiore, Marli de Fatima (President)
Moraes, Luiz Alberto Beraldo de
Oliveira, Mariana Cabral de
Pinto Junior, Ernani
Sette, Lara Durães
Title in Portuguese
Moléculas bioativas e filogenia de isolados brasileiros de cianobactérias dos gêneros Dolichospermum, Sphaerospermopsis, Cuspidothrix, Cylindrospermopsis e Microcystis
Keywords in Portuguese
ELISA
Filogenia
LC-MS
NGS
PCR
Substâncias Bioativas
Abstract in Portuguese
O número crescente de descobertas de substâncias bioativas produzidas pelo metabolismo secundário de cianobactérias tem despertado o interesse de grupos de pesquisa no mundo todo com o objetivo comum de descrever e explorar estas moléculas e entender a sua biossíntese. No Brasil, as pesquisas sobre moléculas bioativas produzidas por linhagens de cianobactérias nativas são escassas. Neste trabalho, utilizando iniciadores específicos da PCR e sequenciamento, a presença de genes envolvidos na biossíntese da neurotoxina saxitoxina (STX) foi confirmada em representantes dos gêneros Dolichospermum, Sphaerospermopsis, Cuspidothrix e Cylindrospermopsis, enquanto que genes da citotoxina cilindrospermopsina (CYN) foram detectados somente em representantes de Cylindrospermopsis. Genes envolvidos com a produção dos inibidores enzimáticos, microviridina (MDN) e a cianobactina microciclamida (MCA) foram sequenciados em isolados do gênero Microcystis. Os genomas das linhagens de Cylindrospermopsis raciborskii CENA302 e CENA303 foram sequenciados usando a plataforma HiScan SQ (Ilumina) com biblioteca pareada 2 x 100 pb. O genoma da Sphaerospermopsis torques-reginae ITEP-024 foi sequenciado utilizando a plataforma Ion Torrent (Life Technologies) com tamanhos de fragmentos de até 200 pb. As tentativas de montagem ab initio dos genomas foram realizadas e o agrupamento gênico da saxitoxina (28 kb) da linhagem C. raciborskii CENA302 foi identificado e caracterizado. As análises filogenéticas das sequências de aminoácidos envolvidos com a biossíntese das moléculas bioativas avaliadas demonstraram que os isolados brasileiros de cianobactérias formam clados com elevado valor de reamostragem com sequências homólogas de cianobactérias conhecidas como produtoras dessas moléculas. Neste estudo é relatada pela primeira vez a presença de genes cyr em linhagens da América do Sul de C. raciborskii e a presença simultânea de genes cyr e sxt em uma única linhagem de C. raciborskii. Além disso, este é o primeiro estudo que relata a presença de genes envolvidos na biossíntese de MDN e MCA nas espécies de cianobactérias M. protocystis, M. panniformis e M. wesenbergii. Análises por espectrometria de massas acoplada a cromatografia líquida (LC-MS) e imunoensaio enzimático (ELISA) foram utilizadas a fim de detectar e identificar variantes estruturais das moléculas bioativas das cianobactérias que tiveram os genes biossintéticos sequenciados. A análise de LC-MS mostrou a produção das variantes GTX2, GTX3, STX e dc-STX pela linhagem C. raciborskii CENA302, enquanto que a linhagem C. raciborskii CENA305 apresentou as variantes NEO, C1 e dcGTX3. As quatro novas variantes de MCY, [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR e [D-Phe1]MC-LR, foram encontradas nas espécies M. panniformis SPC702 e M. protocystis SPC697. Este é o primeiro relato da produção de MCY por essas duas espécies de Microcystis. Dezesseis linhagens que ainda não possuíam as sequências do gene de RNAr 16S foram sequenciadas. O resultado da análise filogenética das sequências do gene de RNAr 16S foi coerente com as descrições morfológicas, sendo que todas as linhagens foram caracterizadas em nível de espécie. As informações geradas neste estudo contribuem para o aumento do conhecimento da diversidade metabólica dos isolados brasileiros de cianobactérias e trazem nova visão sobre a evolução dessas moléculas produzidas pelo metabolismo secundário
Title in English
Bioactive molecules and phylogeny of Brazilian cyanobacterial isolates from genera Dolichospermum, Sphaerospermopsis, Cuspidothrix, Cylindrospermopsis and Microcystis
Keywords in English
Bioactive compounds
ELISA
LC-MS
NGS
PCR
Phylogeny
Abstract in English
The growing numbers of discoveries of bioactive substances produced by cyanobacterial secondary metabolism has attracted the interest of research groups around the world with the common goal of describing and exploring these molecules and understanding their biosynthesis. In Brazil, researches on bioactive molecules produced by native cyanobacterial strains are scarce. In this work, using specific PCR primers and sequencing, the presence of genes involved in the biosynthesis of the neurotoxin saxitoxin (STX) was confirmed in representatives of the genera Dolichospermum, Sphaerospermopsis, Cuspidothrix and Cylindrospermopsis, while genes of the cytotoxin cylindrospermopsin (CYN) were detected only in representatives of Cylindrospermopsis. Genes involved in the production of protease inhibitors, microviridin (MDN) and the cianobactin microciclamide (MCA), were sequenced in isolates of the genus Microcystis. The genomes of Cylindrospermopsis raciborskii strains CENA302 and CENA303 were sequenced using the high-throughput platform HiScan SQ (Illumina) as paired-ends 2 x 100 bp. The Sphaerospermopsis torques-reginae ITEP-024 genome was sequenced using the high-throughput platform Ion Torrent (Life Technologies) with fragment sizes up to 200 bp. Attempts of ab initio genomes assembly were performed and the 28 kb saxitoxin gene cluster of C. raciborskii strains CENA302 was identified and characterized. Phylogenetic analyses of amino acid sequences involved in the biosynthesis of the bioactive molecules evaluated showed that the Brazilian cyanobacterial isolates formed clades with high bootstrap values with homologous sequences of known cyanobacterial producers of these molecules. In this study is reported for the first time the presence of cyr genes in South America strains of C. raciborskii and the simultaneous presence of cyr and sxt genes in a single C. raciborskii strain. Furthermore, this is the first study reporting the presence of genes involved in the biosynthesis of MDN and MCA in the cyanobacterial species M. protocystis, M. panniformis e M. wesenbergii. Analyses by mass spectrometry coupled to liquid chromatography (LC-MS) and enzyme immunoassay (ELISA) were used to detect and identify structural variants of bioactive molecules of the cyanobacteria that had the biosynthetic genes sequenced. Analysis of LC-MS showed the production of the variants GTX2, GTX3, STX and dc-STX by the C. raciborskii strain CENA302, whereas the strain C. raciborskii CENA305 presented the variants NEO, C1 and dcGTX3. The new four MCY variants [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR and [D-Phe1]MC-LR were found in the species M. panniformis SPC702 and M. protocystis SPC697. This is the first report of the MCY production by these two species of Microcystis. Sixteen strains that still lacked the 16S rRNA gene sequences were sequenced. The result of the phylogenetic analysis of 16S rRNA gene sequences was consistent with the morphological descriptions, and all strains were characterized to species level. The informations generated in this study contribute to the increase of knowledge on metabolic diversity of Brazilian cyanobacterial strains and bring new insight into the evolution of these molecules produced by secondary metabolism
 
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Publishing Date
2013-02-26
 
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