<p dir="ltr">Metabolic pathways are shaped by dynamic biotic interactions. My research delves into coevolution exemplified through two distinct projects that investigate the specialized metabolism of organisms as a consequence of biotic interactions. The first project focused on the remarkable metabolic adaptations of <i>Elysia crispata</i> morphotype clarki. This sea slug possesses the extraordinary ability to sequester and maintain functional chloroplasts (kleptoplasts) from the algae it consumes, allowing it to sustain photosynthetically active kleptoplasts for several months without feeding. To better understand the underlying molecular mechanism of this phenomenon, I generated a comprehensive 786 Mbp draft genome of <i>E. crispata</i> using a combination of ONT long reads and Illumina short reads. The resulting assembly provided a foundational resource for phylogenetic, gene family and gene expression analyses. This work advanced our understanding of the genetic underpinnings of kleptoplasty, shedding light on the evolution and maintenance of this unique metabolic strategy in sacoglossan sea slugs. I next investigated the transcriptional impacts of herbivory on maize (<i>Zea mays</i>) and green foxtail (<i>Setaria viridis</i>), induced by fall armyworm (<i>Spodoptera frugiperda</i>) and beet armyworm (<i>Spodoptera exigua</i>) feeding. This study aimed to contrast the defensive mechanisms of these grasses in response to each herbivore, and determined that green foxtail transcriptionally differentiates its responses to fall armyworm and beet armyworm herbivory. The fall armyworm has evolved a counter adaptation to lessen plant secondary metabolite production by producing a salivary protein (SFRP1) that suppresses jasmonate signaling. Investigation of the combinatorial effects of SFRP1 and beet armyworm herbivory determined the addition of endogenous SFRP1 during beet armyworm feeding is sufficient to reduce green foxtail defense responses. Results of this research shed light on host-pest reciprocal adaptations and the role of SFRP1 as an oral secretory protein. Coexpression analysis of maize and green foxtail transcriptomic responses to herbivory also identified putative genes involved in specialized metabolic pathways in green foxtail, providing insights into plant-insect interactions and potential solutions to herbivory in wild plant species. These findings highlight how gene diversification can contribute to pest resistance in grasses. Together, these seemingly unconnected projects underscore how biotic interactions influence metabolic processes across diverse organisms and reveal the fascinating intricacies of their adaptations to environmental challenges.</p>

Pseudomonas brassicacearum a la particularité de générer une diversité intraclonale aussi bien in vitro qu'en conditions naturelles dans la rhizosphère de plantes. Ce phénomène de variation phénotypique commun chez les bactéries est un processus d'adaptation aux environnements changeants. Des données de transcriptomique issues de puces à ADN, contenant aussi bien des séquences codantes que non codantes, nous ont permis d'identifier les gènes dont l'expression est altérée et surtout de relier ce phénomène à l'expression d'ARN non codants régulateurs (ARNnc) de type Rsm qui sont sous le contrôle du système à deux composants GacS/GacA. Nous avons montré que des mutations ponctuelles dans les gènes gacS ou gacA sont à l'origine de cette variation phénotypique et que l'expression de l'un des trois gènes rsmX, rsmY ou rsmZ permet de restaurer le phénotype de la souche sauvage. L'importance de ces ARNnc dans la survie de la bactérie aux fluctuations de son environnement est dénotée par la duplication de rsmX en un gène que nous avons nommé rsmX-2, dont la fonction a été validée. Nos données suggèrent une activation exclusive des gènes rsmX-1 et rsmX-2 par GacA et l'intervention de régulateurs additionnels dans le cas de rsmY et rsmZ. Au vu de la redondance fonctionnelle de ces quatre ARNnc, nous avons investigué leur niveau d'expression et leur stabilité dans différentes conditions de culture et montré des différences pour les quatre ARNnc. En réponse à une carence en nutriments, l'expression des ARNnc Rsm est fortement activée et atteint son maximum quand le ppGpp est détecté dans le milieu, suggérant un lien entre le système Gac/Rsm et la réponse « stringente ». / The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonisation under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as non-coding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small non-coding RNA (ncRNA) genes rsmX, rsmY and rsmZ, which was characterized by down-regulation. Naturally occurring mutations in the GacS/GacA two-component system accounted for phenotypic switching. The importance of these ncRNAs in the survival of the bacteria to changing environments is denoted by the duplication of rsmX gene, which we called rsmX-2 and whose function has been validated. Our data suggest an exclusive activation of rsmX-1 and rsmX-2 genes by GacA and the involvement of additional regulators in the case of rsmY and rsmZ. Given the functional redundancy of these ncRNAs, we investigated their expression level and stability in different culture conditions and showed differences for the four ncRNAs. In response to nutrient depletion, the four ncRNAs expression is strongly activated and reaches its maximum when the ppGpp is detected in bacterial cells, suggesting a link between the Gac/Rsm system and the "stringent" response. Determining the level of each Rsm ncRNA, which is defined by a balance between synthesis and degradation of each transcript, shows the maintenance of a very important pool of RsmZ compared to other ncRNAs.

No description available.

Nostoc é um gênero cianobacteriano com distribuição ubíqua que tem importância em diversos ecossistemas. Contudo, poucos genomas estão atualmente disponíveis para esse gênero. Enquanto Nostoc spp. são as cianobactérias mais comumente relatadas em relações simbióticas com fungos, animais, plantas e outros organismos, associações com outros micro organismos não receberam atenção similar. Como consequência das fortes interações entre cianobactérias e heterótrofos, culturas não axênicas são geralmente obtidas no isolamento dessas bactérias, o que proporciona uma oportunidade interessante para o desenvolvimento tanto de estudos genômicos quanto metagenômicos. Este trabalho teve como objetivo investigar as características genômicas e funcionais da linhagem Nostoc sp. CENA67, isolada de terra preta antropogênica, bem como estudar sua comunidade associada. Para esse fim, células de uma cultura não axênica de Nostoc sp. CENA67 foram sequenciadas com as plataformas MiSeq e Ion PGM e analisados com ferramentas genômicas e metagenômicas. A linhagem CENA67 de fato pertence à família Nostocaceae e possui algumas características em comum com cianobactérias do gênero Nostoc, porém diverge em certos aspectos morfológicos e filogenéticos do grupo típico de Nostoc, sugerindo que seja representante de um novo táxon. Além disso, seu genoma apresenta diferenças em relação aos genomas atualmente disponíveis para cianobactérias relacionadas ao gênero. A mineração desse genoma revelou 31 agrupamentos gênicos hipoteticamente relacionados à síntese de metabólitos secundários, a maioria dos quais não mostrou similaridade significativa com agrupamentos conhecidos. A análise de um agrupamento gênico de microviridina desvendou uma maior diversidade de genes para precursores dessa molécula do que se acreditava anteriormente, sugerindo que um número considerável de variantes ainda está a ser descoberta. A análise taxonômica da comunidade associada confirmou a dominância de cianobactérias na cultura, mas também revelou a presença de grande número de gêneros microbianos que normalmente são capazes de fixar nitrogênio atmosférico e estabelecer simbiose com plantas, incluindo Mesorhizobium, Sinorhizobium e Starkeya, entre outros. Rascunhos genômicos foram obtidos para Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia lata e Hyphomicrobium nitrativorans. Todavia, genes para fixação de nitrogênio não foram detectados nesses genomas, apesar de serem encontrados no genoma da cianobactéria e no metagenoma da comunidade, o que sugere que algumas populações podem estar sob pressão de seleção para a perda da capacidade de fixação de nitrogênio, provavelmente devido a este nutriente estar sendo fornecido pelo organismo mais abundante nesta comunidade, a cianobactéria. A análise funcional indicou vias exclusivas tanto à cianobactéria quanto à comunidade associada, e sugeriu a complementariedade de certos metabolismos. Os resultados possibilitam o aumento do conhecimento sobre a diversidade molecular e química do filo Cyanobacteria e levantam possíveis interações com micro organismos simbiontes / Nostoc is a cyanobacterial genus with ubiquitous distribution that is important in several ecosystems. However, few genomes are currently available for this genus. While Nostoc spp. are the most commonly reported cyanobacteria in symbiotic relationship with fungi, animals, plants, and other organisms, associations with other microorganisms have not received similar attention. As a consequence of tight interactions between cyanobacteria and heterotrophs, non-axenic cultures are usually achieved in the isolation of these bacteria, which provides an interesting opportunity for carrying out both genomic as metagenomic studies. This work aimed to investigate the genomic and functional characteristics of the strain Nostoc sp. CENA67, isolated from anthropogenic dark earth, and to study its associated community. For this purpose, cells from a non-axenic culture of Nostoc sp. CENA67 were sequenced with the platforms MiSeq and Ion PGM and analyzed with genomic and metagenomic tools. The strain CENA67 indeed belongs to the family Nostocaceae and presents some characteristics in common with cyanobacteria of the genus Nostoc, but diverges in certain morphological and phylogenetic aspects of the typical Nostoc group, suggesting that it is a representative of a new taxon. In addition, its genome presents differences in relation to the genomes currently available for cyanobacteria related to this genus. Genome mining revealed 31 gene clusters hypothetically related to the synthesis of secondary metabolites, most of which did not show significant similarity to known clusters. The analysis of a microviridin gene cluster unveiled a larger diversity of precursor genes for this molecule than was previously believed, suggesting that a considerable number of variants is still to be found. The taxonomic analysis of the associated community confirmed the dominance of cyanobacteria in the culture, but also revealed the presence of a great number of microbial genera that are usually capable of fixing atmospheric nitrogen and establishing symbiosis with plants, including Mesorhizobium, Sinorhizobium, and Starkeya, among others. Genomic drafts were obtained for Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia lata, and Hyphomicrobium nitrativorans. Nevertheless, genes for nitrogen fixation were not detected in these genomes, despite being found in the cyanobacterial genome and the community metagenome, suggesting that some populations might be under selection pressure for the loss of the ability to fix nitrogen, probably due to this nutrient being provided for the most abundant organism in this culture, the cyanobacterium. Functional analysis indicated pathways exclusive both to the cyanobacterium as to the associated community, and suggested the complementarity of certain metabolisms. The results allow the increase of the knowledge about the molecular and chemical diversity of the phylum Cyanobacteria and raise possible interactions with symbiotic microorganisms

A esterigmatocistina (ST) é uma micotoxina policetônica produzida por diferentes espécies de Aspergillus e outros gêneros fúngicos como Bipolaris e Chaetomium. Esta toxina é caracterizada como uma bifuranoxantona com fórmula molecular C18H12O6, freqüentemente encontrada como contaminante de diversos produtos alimentícios como sementes oleaginosas e cereais. Possui propriedades carcinogênicas, toxigênicas, mutagênicas e teratogênicas, entretanto, ela é menos tóxica do que a aflatoxina. O fungo Emericella nidulans (Eidam) Vuillemin, cujo anamorfo é o Aspergillus nidulans (Eidam) Winter, foi usado como modelo genético para a investigação de genes envolvidos na via biossintética da esterigmatocistina. Linhagens estoque de Utrecht (originárias de Glasgow) foram analisadas quanto à produção de ST e, entre elas, somente a linhagem UT196 [yA2 (I); metA17 (II); piroA4 (IV)] apresentou produção de 4 ppm de ST (stc+), enquanto que as linhagens UT448 e UT184 mostraram-se não produtoras dessa micotoxina (stc). Embora a linhagem UT196 seja muito bem caracterizada geneticamente, este foi o primeiro relato da produção de ST nessa linhagem. Os índices de segregação alélica e todas as freqüências de recombinação entre os marcadores genéticos ligados e não ligados foram determinados tanto pelo cruzamento meiótico UT448 (stc) x UT196 (stc+) como pelo cruzamento mitótico UT448//UT184. 175 segregantes meióticos e 140 segregantes mitóticos foram analisados quanto à produção de ST, aos marcadores de auxotrofia e de resistência a acriflavina. Essas linhagens cruzadas apresentavam vários marcadores em heterozigose e isso permitiu o mapeamento de um gene estrutural da ST (stcZ+), localizado no braço esquerdo do cromossomo I, 4% distante do gene da riboflavina (riboA1). Como um subproduto deste trabalho, foi detectado um pigmento vermelho, de Rf = 0,90, em todos os segregantes meióticos e mitóticos, produtores ou não de ST, indicando tratar-se, provavelmente, de um pigmento policetônico produzido pelos ascosporos do fungo. A baixa expressão da produção de ST em 13 segregantes meióticos e a alta expressão dessa toxina nos diplóides UT448//UT196 e UT448//UT184 (40 ppm) permitiram concluir a existência de um fator regulador, compreendido na região w-met do cromossomo II, responsável pela expressão do gene estrutural stcZ+. A análise desses genes através do ciclo parassexual sugeriu um comportamento epigenético típico envolvendo esses genes. Com base nos dados obtidos, hipóteses para explicar o controle da expressão desses genes e suas inter-relações foram aqui apresentadas. / Sterigmatocystin (ST) is a polyketide produced by different species of Aspergillus as well as by other fungus genera such as Bipolaris and Chaetomium. This toxin is characterized as a bifuranoxanthone, whose molecular formula is C18H12O6 and which is frequently found as a contaminant in several food products such as oil-seed grains and cereals. It has carcinogenic, toxigenic, mutagenic and teratogenic properties; however, it is less toxic than aflatoxin. The fungus Emericella nidulans (Eidam) Vuillemin, whose anamorph is Aspergillus nidulans (Eidam) Winter, was used as a genetic model to investigate the genes involved in the sterigmatocystin biosynthetic pathway. Strains from Utrecht stocks (originally from Glasgow) were analyzed in order to detect ST production and, among them, only the UT196 strain [yA2 (I); metA17 (II); piroA4 (IV)] showed the production of 4 ppm of ST (stc+), whereas the UT448 and UT184 strains showed to be nonproducers of such toxin (stc). Although the UT196 strain is very well characterized genetically, this has been the first report on its production of ST. The allelic segregation rates and all the recombination frequencies between linked and non-linked genetic markers were determined by both the meiotic crossing UT448 (stc) x UT196 (stc+) and mitotic crossing UT448//UT184. 175 meiotic segregants and 140 mitotic segregants were analyzed as to ST production, auxotrophy markers and resistance to acriflavine. These crossed strains presented various markers in heterozygous configuration, which allowed to map a structural gene of ST (stcZ+) located on the left arm of chromosome I, 4% distant from the riboflavin gene (riboA1). As a byproduct of this work, a red pigment of 0.90 Rf was detected in all meiotic and mitotic segregants, whether they were ST producers or not, which indicated that was probably a polyketide produced by the fungus ascopores. The low expression of ST production in 13 meiotic segregants and the high expression of such toxin in the UT448//UT196 and UT448//UT184 diploids (40 ppm) allowed to conclude that a regulating factor existed in the w-met region of chromosome II, which is responsible for the expression of the structural gene stcZ+. The analyses of those genes through the parasexual cycle suggested a typical epigenetic behavior which involved them. Based on the data obtained, hypotheses to explain the expression control of these genes as well as their inter-relationships were here presented.

La carotte présente un grand intérêt nutritionnel comme source alimentaire en caroténoïdes. Pourtant, la connaissance des mécanismes d’accumulation de ces composés est un enjeu majeur. Si le déterminisme génétique a été relativement bien étudié, l’impact de stress sur l’accumulation des caroténoïdes chez la carotte reste méconnu. Ce travail de thèse vise donc à déterminer (i) l’impact de stress biotique et abiotique appliqués individuellement ou en combinaison sur les teneurs en caroténoïdes dans les feuilles et racines de carotte,(ii) les mécanismes de régulation pouvant expliquer ces variations (iii) si le métabolisme secondaire est affecté spécifiquement, indépendamment du métabolisme primaire.Les résultats mettent en évidence un effet négatif des différentes conditions de stress, en particulier appliquées en combinaison, sur les teneurs en caroténoïdes dans les feuilles et les racines de carotte, mais dépendent du génotype. La régulation transcriptionnelle au niveau de la voie de biosynthèse des caroténoïdes ne peut expliquer qu’en partie les variations de teneurs. Les variations de teneurs en chlorophylles des feuilles et sucres des racines sont corrélées à celles des teneurs en caroténoïdes, suggérant des mécanismes communs de régulation.Ce travail montre que l’impact de stress en culture, et en particulier leur combinaison, est une composante importante de l’élaboration de la qualité nutritionnelle. Les travaux doivent être poursuivis afin d’établir un schéma plus précis de la régulation de l’accumulation des caroténoïdes chez la carotte. / Carrot presents a high nutritional interest as a carotenoid intake source. However, knowledge about accumulation mechanisms of these compounds is a major issue. While genetic determinism was relatively well studied, the impact of stresses on carotenoid accumulation in carrot remains unknown. This thesis work aims to determine (i) the impact of biotic and abiotic stresses applied individually or in combination on carotenoid contents in carrot leaves and roots, (ii) the regulation mechanisms which could explainthese variations and (iii) if secondary metabolism is specifically affected independently from primarymetabolism. Results bring to light a negative effect of the different stress conditions, particularly applied in combination, on carotenoid contents in carrot leaves and roots but depend on genotypes. Transcriptional regulation based on carotenoid biosynthetic genes can only partially explain contentvariations. Chlorophyll content variations in leaves and sugar content variations in roots are correlated to those of carotenoids suggesting common regulation mechanisms. This work shows that the impact of stress on culture, and particularly in combination, is an important determinism of nutritional quality. Further works need to be performed to establish a more precise regulation network pattern of carotenoid accumulation in carrot.

Η ρύπανση από τα χημικά λιπάσματα είναι έντονη στα ελληνικά εδάφη (ιδιαίτερα από τα νιτρικά άλατα) και επομένως είναι κρίσιμη η μείωση των εισροών αγροχημικών στο περιβάλλον. Η μείωση της λίπανσης αυξάνει την συγκέντρωση αντιοξειδωτικών ουσιών στους φυτικούς ιστούς. Πολλές επιστημονικές μελέτες μέχρι σήμερα συσχετίζουν την διατροφή με φυτικά προϊόντα υψηλής διατροφικής αξίας που περιέχουν φαινολικές-αντιοξειδωτικές ουσίες με την πρόληψη καρδιαγγειακών παθήσεων, πολλών μορφών καρκίνου αλλά και την γήρανση. Έτσι η έρευνα αυτή είχε διπλό στόχο: τη μείωση της ρύπανσης του περιβάλλοντος μέσω της μείωσης των λιπασμάτων που χορηγούνται στις καλλιέργειες, και την παραγωγή φυτικών προϊόντων υψηλής βιολογικής και διατροφικής αξίας, αφού θα παρέχουν μεγαλύτερες ποσότητες αντιοξειδωτικών ουσιών στον ανθρώπινο οργανισμό. Επίσης θα πρέπει να αναφερθεί ότι η μείωση των ποσοτήτων των λιπασμάτων που θα απαιτούνται για την καλλιέργεια των φυτών θα επιφέρει και οικονομικό όφελος προς τους καλλιεργητές και τους καταναλωτές, αφού θα μειωθεί το κόστος παραγωγής των φυτικών προϊόντων. Για το σκοπό αυτό μελετήθηκε για πρώτη φορά η επίδραση της μεταβολής της συγκέντρωσης των χορηγούμενων νιτρικών ιόντων μέσω του θρεπτικού διαλύματος υδροπονικής καλλιέργειας στο ρυθμό παραγωγής πολυφαινολικών ενώσεων σε λαχανικά ευρείας κατανάλωσης (παντζάρι και μαρούλι) και διαπιστώθηκε ότι η μεγιστοποίηση της παραγωγής των φυσικών αντιοξειδωτικών παραγόντων είναι δυνατή μέσω του ελέγχου της αζωτούχου θρέψης. Παράλληλα, διαπιστώθηκε και ποσοτικοποιήθηκε η ύπαρξη ενός κρίσιμου σημείου στη συγκέντρωση του χορηγούμενου αζώτου που ενεργοποιεί τον δευτερογενή μεταβολισμό των καλλιεργούμενων φυτών παντζαριού και μαρουλιού, αυξάνοντας σημαντικά το ρυθμό παραγωγής των ιδιαίτερα ευεργετικών για την υγεία φυτοχημικών ενώσεων όπως φαινολικών και μπετακυανινών. Η αύξηση όχι μόνο της περιεκτικότητας αλλά και της ενεργότητας των αντιοξειδωτικών παραγόντων επιβεβαιώθηκε με τη χρήση προηγμένων μεθόδων προσδιορισμού όπως το EPR. Τέλος προσδιορίστηκαν και ποσοτικοποιήθηκαν με σύγχρονη μέθοδο φασματομετρίας μαζών (LC-MS/MS) στα διαφορετικά μέρη των φυτών παντζαριού και στα φύλλα του μαρουλιού συνολικά επτά διαφορετικές πολυφαινολικές ενώσεις. / The pollution from chemical fertilizers is pronounced in Greek soils (particularly nitrates) and is therefore critical to reduce inputs of agrochemicals in the environment. The decrease of fertilization increases the concentration of antioxidants in plant tissues. Many scientific studies to date relate the diet with plant products of high nutritional value containing phenolic-antioxidants with the prevention of cardiovascular disease, number of cancers and aging. So the aims of the present research are: to reduce environmental pollution by reducing fertilizer applied to crops, and the production of crops with high biological and nutritional value, providing greater amounts of antioxidants in the human body. It should also be noted that the decrease in the quantities of fertilizer that will be required for the cultivation of plants will also lead to economic benefits to farmers and consumers, as it will reduce the cost of production of plant products. For this purpose the effect of varying concentration of nitrate granted via hydroponic nutrient solution, in the production rate of polyphenolic compounds in vegetables (lettuce and beetroot) was studied. It was found that maximizing the production of natural antioxidants is possible through the control of nitrogen nutrition. Also, it was ascertained that a critical point in the concentration of the administered nitrogen exists below which the secondary metabolism of the studied crops i.e. beetroot and lettuce is activated, significantly increasing the rate of production of the highly beneficial for the health compounds such as phenolic phytochemicals and betacyanins. The augmentation of the activity of antioxidants was confirmed by using advanced methods such as EPR. Finally seven different polyphenolic compounds were identified and quantified by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), in different plant parts of beetroot and lettuce leaves.

El principal objectiu d'aquest treball ha estat estudiar la producció de metabòlits amb activitat antibiòtica per soques de l'espècie Pseudomonas fluorescens de la col·lecció EPS, i també avaluar la seva potencialitat com a agents de biocontrol. Es va disposar també de diverses soques de P. fluorescens, cedides per altres investigadors, que van utilitzar-se com a referència perquè algunes són actives en control biològic i produeixen metabòlits secundaris d'interès en el biocontrol de malalties de plantes.La present memòria s'estructura en cinc capítols, que són, introducció al control biològic, descripció de l'etapa de selecció de soques i cerca dels metabòlits produïts, estudi de la producció d'HCN per la soca EPS288, estudi de la producció de l'antibiòtic 2,4-diacetilfloroglucinol (DAPG), i finalment, el darrer capítol, on s'ha estudiat la producció de DAPG sobre material vegetal i la capacitat de colonitzar arrels per diverses soques d'interès.En l'etapa de prospecció, va demostrar-se que un 37% del total de les soques de la col·lecció EPS produïen HCN, totes de l'espècie P. fluorescens, i un 90% d'aquestes provenien de les arrels de plantes. Es va confirmar la producció dels metabòlits secundaris 2,4-diacetilfloroglucinol, àcid fenazina-1-carboxílic, i pirrolnitrina, per diverses soques de la col·lecció EPS seleccionades mitjançant tècniques moleculars. Així, de la col·lecció EPS, les soques EPS317 i EPS808 produeixen DAPG, la EPS263 àcid fenazina-1-carboxílic i pirrolnitrina i, EPS894, EPS895, EPS945 produeixen àcid fenazina-1-carboxílic. La producció d'HCN es va estudiar més exhaustivament en la soca EPS288, seleccionada per la seva activitat antifúngica i candidata a agent de biocontrol contra Stemphylium vesicarium, causant de la estemfiliosi de la perera, i contra Penicillium expansum, causant de la podridura blava en conservació de fruita a postcollita. Per aquest estudi, es va dissenyar i validar un sistema per recollir l'HCN a partir de cultius en medi líquid. S'ha demostrat que la temperatura d'incubació, la concentració cel·lular de sembra i la composició del medi de cultiu afectaven a la producció d'HCN. Els medis complexos i la glicina n'afavorien la síntesi i la font de carboni no afectava. La soca EPS288 va produir més HCN que P. fluorescens CHA0, soca de referència productora d'HCN i descrita com a activa en processos de biocontrol de fongs fitopatògens. En l'estudi de la producció de DAPG, les soques de la col·lecció EPS i de referència, es van comparar en diversos medis de cultiu estudiant l'efecte de la complexitat i consistència del medi, i l'addició de ferro o de glucosa. Va demostrar-se que la producció de DAPG depèn principalment de la soca i de les característiques del medi de cultiu. La glucosa estimula la producció, mentre que el ferro pràcticament no afecta, i en general, el medi sòlid i complex estimula la producció de DAPG. Tanmateix, aquests efectes varien en alguna de les soques assajades donant lloc a comportaments singulars. En el seguiment del creixement amb un sistema automàtic es va comprovar que la velocitat específica de creixement i la concentració cel·lular assolida al final del cultiu, estaven condicionades per la composició del medi de cultiu. En les proves d'antagonisme vers fitopatògens que foren seleccionats com a indicadors, va observar-se que tant l'antagonisme in vitro com la inhibició d'infeccions sobre material vegetal estaven parcialment relacionades amb la producció dels metabòlits secundaris estudiats. La promoció del creixement de portaempelts per aquestes soques depenia de la soca i de l'hoste, però no es pogué establir una relació causa-efecte amb el metabòlits produïts. També es va comprovar que algunes de les soques podien sobreviure en ferides de pomes i de peres, on produïren DAPG.Mutants resistents a rifampicina de diverses soques de la col·lecció EPS i de referència es van inocular en llavors de pomera i de tomatera que es van sembrar i incubar en condicions controlades. Es va fer el seguiment de la població bacteriana total i resistent a rifampicina present a les arrels durant 72 dies. Totes les soques van colonitzar les arrels de les plantes, mantenint una elevada població durant 37 dies, cap d'elles va estimular el creixement ni mostrar efectes fitotòxics, no afectant tampoc signicativament a la població bacteriana espontània de les arrels. La soca EPS808, una de les seleccionades pel treball, va aconseguir uns nivells de producció de DAPG, una velocitat de creixement i una supervivència relativa a les arrels similar a altres soques de referència descrites com a bons agents de biocontrol. En conseqüència, se la considera una candidata a agent de biocontrol que hauria de ser objecte de futurs estudis d'eficàcia. / El principal objetivo de este trabajo ha sido estudiar la producción de metabolitos con actividad antibiótica por cepas de la especie Pseudomonas fluorescens de la colección EPS, y también evaluar su potencialidad como agentes de biocontrol. Se dispuso de varias cepas de P. fluorescens, cedidas por otros investigadores, que se utilizaron como referencia ya que algunas de estas son activas en el control biológico, y producen metabolitos secundarios de interés para el biocontrol de enfermedades de plantas.La presente memoria se estructura en cinco capítulos, revisión bibliográfica del control biológico, descripción de la etapa de selección de cepas y de los metabolitos producidos, estudio de la producción de HCN por la cepa EPS288, estudio de la producción de 2,4-diacetilfloroglucinol(DAPG), y finalmente, el último capítulo, donde se ha estudiado la producción de DAPG sobre material vegetal y la capacidad de colonizar las raíces de diversas plantas. En la etapa de prospección se demostró que el 37% del total de las cepas de la colección EPS producían HCN, todas ellas de la especie P. fluorescens, de las cuales el 90% había sido aislada de las raíces de plantas. Se confirmó la producción de los metabolitos secundarios 2,4-diacetilfloroglucinol, ácido fenazina-1-carboxílico y pirrolnitrina, por diversas cepas de la colección EPS seleccionadas mediante técnicas moleculares. De la colección EPS las cepas EPS317 y EPS808 producen DAPG, la EPS263 ácido fenazina-1-carboxílico y pirrolnitrina y las cepas EPS894, EPS895 y EPS945 producen ácido fenazina-1-carboxílico. La producción de HCN se estudió en detalle por la cepa EPS288, esta había sido seleccionada por su actividad antifúngica y es candidata a agente de biocontrol contra Stemphylium vesicarium, causante de la estemfiliosis del peral, y contra Penicillium expansum, causante de la enfermedad del moho azul en la conservación de la fruta en poscosecha. Para este estudio se diseñó y se validó un sistema para la recogida del HCN que se desprende de cultivos en medio líquido. La temperatura de incubación, la concentración en la siembra y la composición del medio de cultivo afectaban a la producción de HCN. Los medios de cultivo complejos y la glicina estimulaban su síntesis, mientras que la fuente de carbono no afectaba. La cepa EPS288 produjo más HCN que la P. fluorescens CHA0, cepa de referencia productora de HCN y descrita como activa en procesos de control biológico de hongos fitopatógenos.En el estudio de la producción de DAPG, las cepas de la colección EPS y de referencia, se compararon en diferentes medios de cultivo estudiando el efecto de la complejidad y la consistencia del medio de cultivo, y de la adición de hierro o de glucosa. Se demostró que la producción de DAPG depende principalmente de la cepa y de la composición del medio de cultivo. La glucosa estimula la producción, mientras que el hierro casi no tiene efecto. Sin embargo, estos efectos varían en alguna de las cepas ensayadas dando lugar a comportamientos singulares.El seguimiento del crecimiento mediante un sistema automático permitió verificar que la velocidad específica de crecimiento y la concentración celular en la fase estacionaria estaban condicionadas por la composición del medio de cultivo. En las pruebas de antagonismo frente a hongos fitopatógenos seleccionados como indicadores, se observó que el antagonismo in vitro y la inhibición de infecciones sobre material vegetal correlacionaban parcialmente con la producción de los metabolitos secundarios estudiados. La promoción del crecimiento de portainjertos con esas cepas era función del huésped y de la cepa. Se comprobó que algunas de las cepas sobrevivían en heridas de manzanas y de peras dónde sintetizaban DAPG. Mutantes resistentes a rifampicina de diversas cepas de la colección EPS y de referencia se inocularon en semillas de manzano y de tomate las cuales se sembraron e incubaron en ambiente controlado. La población bacteriana en las raíces de las plantas se monitorizó durante un periodo de 72 días. Todas las cepas colonizaron las raíces de las plantas manteniendo una elevada población durante, al menos, 37 días. Ninguna de la cepas estimuló el crecimiento ni mostró fitotoxicidad, y no afectaron significativamente a la población bacteriana espontánea de las raíces.La cepa EPS808, una de las seleccionadas para el presente trabajo, demostró una producción de DAPG, velocidad específica de crecimiento y una supervivencia relativa en las raíces, similares a otras cepas de referencia descritas como buenos agentes de biocontrol. Por lo tanto, se considera a esta cepa como una candidata a agente de biocontrol, que tendría que ser objeto de futuros estudios de eficacia. / The main objective of this work is to study the production of biologically active secondary metabolites produced by Pseudomonas fluorescens strains from EPS collection. Moreover, their potential as biocontrol agents was also evaluated. We used P. fluorescens strains, provided by foreign scientists, that were utilised as a reference because these strains are active as biocontrol agents and produce secondary metabolites involved in biological control. The present work is divided into five chapters which deal with an introduction to biological control, the selection of strains, the detection and identification of metabolites produced by these bacteria, the study of HCN production by EPS288 strain, the study of 2,4-diacetylphloroglucinol (DAPG) production, and finally, the DAPG production based on fruits and plants, and root colonisation by selected strains.It was shown that 37% of isolates in EPS collection produced cyanide, all were P. fluorescens, and 90% of them were isolated at roots of different plants. The secondary metabolites 2,4-diacetilphloroglucinol (DAPG), phenazine-1-carboxilic acid (PCA) and pyrrolnitrin were identified and its production by some EPS collection strains, selected with molecular techniques, was demonstrated. The strains EPS317 and EPS808 produce DAPG, EPS263 produces phenazine-1-carboxilic acid and pyrrolnitrin, and the strains EPS894, EPS895, EPS945 produce phenazine-1-carboxilic acid.The production of cyanide acid by P. fluorescens EPS288 was studied more exhaustively. This strain was selected as a biocontrol agent due to its antifungal activity against Stemphylium vesicarium, that causes brown spot on pear, and Penicillium expansum, that causes the blue mould of fruits during postharvest. A device to collect cyanide produced by bacteria from broth cultures was set up and validated. The incubation temperature, the initial cell density and the composition of growth media clearly affected HCN production by EPS288 strain. Complex media and glycine stimulated HCN production, however, it was not affected by carbon source in defined growth media. The EPS288 strain produced more cyanide in Castric growth media than P. fluorescens CHA0, a reference strain employed in biological control of fungal pathogens. The reference strains and those from EPS collection which produced DAPG were compared considering the factors complexity and consistence of growth media. The effect of iron and glucose addition were also evaluated. The strain and growth media characteristics were the predominant factors in the DAPG production. Glucose increased DAPG production, while iron addition had no considerable effect, and as general rule, solid and complex media stimulated DAPG production. However these effects are variable depending on the strain. The monitoring of bacterial growth and DAPG production with an automated absorbance reader showed that the growth rate and cellular yield were affected by growth media.In antagonism tests against selected fungal pathogens, the in vitro inhibition and inhibition of infections over organic material were, in part, correlated with secondary metabolite production. The rootstock growth promotion caused by some of these strains was dependent on the strain and the host, but a cause-effect relationship with antibiotics produced could not be clearly established. Some strains were able to colonise and produce DAPG in wounds on apples and pears. Rifampicine resistant spontaneous mutants of the reference and of EPS collection strains were isolated and inoculated on apple and tomato seeds, which were sown and grown under a controlled conditions. The evolution of inoculated strains and the whole bacterial population at the roots were monitored during 72 days. All selected strains efficiently colonised plant roots at high levels for at least 37 days, and nor phytotoxic either plant growth promotion was observed by any strain. Moreover, they did not significantly affected the spontaneous bacterial population at roots.The EPS808 strain exhibited a 2,4-diacetilphloroglucinol production level, a specific growth rate and a relative root population as high as the reference strains that are referenced to be good biocontrol agents. Thus we conclude that this strain can be considered as a candidate for biological control agent, but further efficacy studies are required.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Biotechnology is an important tool that is used to isolate and characterise genes. It is also used to produce clones that are genetically and phenotypically similar. Many Arabidopsis thaliana transcription factors have been isolated and characterised, but many have yet to be fully described. MYB proteins are members of a super-family of multifunctional transcription factors that can also interact with other transcription factors in the control of pathways. To date, more than 126 AtMYBs have been identified, but most have not been fully characterised, particularly in terms of the molecular role(s) they play in plants. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 and MYB32 have been reported to be negative regulators of general phenylpropanoid metabolism. It has been reported that the five transcription factors mentioned above are likely to negatively regulate flavonoid biosynthesis, even though they may have different target genes. Studies on AtMYB13, AtMYB14 and AtMYB15 reported that they are likely regulators of general phenylpropanoid metabolism. The mentioned roles of the eight AtMYB transcription factors means that they can be manipulated in order to see what effect they have on primary and secondary metabolites in plants. The transcription factors ligated into the pUBI510-GRFCA vector were then used to transform sugarcane callus (Chapter 3). Sugarcane produces sucrose which makes up 70% of the sugar produced in the world, making sugarcane a commercially important and profitable plant. The sugarcane callus was transformed via particle bombardment. The transcription factors AtMYB3, AtMYB6, AtMYB7, AtMYB13 and AtMYB32 were successfully incorporated into the genomic DNA of the sugarcane callus. The data obtained for callus over-expressing AtMYB3, AtMYB13 and AtMYB32 on solid media and the callus in liquid media were contradictory (i.e callus on solid media producing more sucrose than the wildtype whereas the same transgenic line will poduce less sucrose that the wildtype in liquid media or vice versa). However, AtMYB13 transgenic lines produced more sucrose than the wildtype. Transgenic lines of AtMYB7 all produced less sucrose as compared to the wildtype both on solid and in liquid media. The transcription factors which resulted in increased production of starch when over-expressed were AtMYB7 and AtMYB13. The data obtained for AtMYB6 transgenic lines was highly inconsistent in lines grown on same media and across the two media. The effects of these transcription factors in the overall metabolism of the sugarcane callus, either on MSC3 solid or liquid media, could not be fully determined from the GC-MS analysis as there was no consistent phenotypic effect between different transgenic lines for any of the MYB transcription factors used. In Chapter 4, a micropropagation strategy was developed and phytochemicals and their biological activities were determined for the medicinal plant Salvia repens. Salvia plants have been found to be medicinally important due to the secondary metabolites, particularly the essential oils that they produce. The plant extracts have been found to have many biological activities such as antibacterial, anti-inflammatory, antioxidant and anticancer activities. Salvia repens was successfully germinated in vitro,with 60% germination being achieved in MS media containing 1x10-5 times diluted smoke water following scarification for 12 min in 75% (v/v) H2SO4. Success rates of 100% were achieved in the hardening off process when the seedlings were moved into the greenhouse. Germination of S. repens ex vitro was 100% in an autoclaved soil mixture of 1:1 (v/v) sand and vermiculite. Importantly the medicinal value of S. repens produced in vitro or ex vitro was not lost as the GC-MS metabolite analysis showed that the plants produced the chemicals that are medicinally important. Metabolite extracts of S. repens were for the first time reported to be active against fungi with MIC values lower than 1 mg/ml over 4-5 d period against four Fusarium spp. tested. Lastly (Chapter 5), transcription factors AtMYB6 and AtMYB13 were used to trasnform Salvia stenophylla via Agrobacterium-mediated transformation, in order to determine whether the over-expression of these transcription factors could up-regulate the production of medicinally and commercially important secondary metabolites in S. stenophylla. Whilst both A. tumefaciens and A. rhizogenes strains were utilised for the transformation procedure, transformation was only achieved using A. rhizogenes and no transformants could be generated from the A. tumefaciens-treated material. Transgenic hairy roots did not produce any of the medicinally important metabolites. The GC-MS analysis of the transgenic root material identified mainly sugars and other primary metabolites. / AFRIKAANSE OPSOMMING: Biotegnologie is 'n belangrike instrument wat gebruik kan word om gene te isoleer en te karakteriseer. Dit word ook gebruik om klone wat geneties en fenotipies identies is te produseer. Baie Arabidopsis thaliana transkripsiefaktore is al geïsoleer en gekarakteriseer, maar baie moet nog volledig beskryf word. MYB proteïene is lede van 'n super-familie van multifunksionele transkripsiefaktore wat ook interaksie het met ander transkripsiefaktore tydens die beheer van metaboliese weë. Tot op hede is meer as 126 AtMYBs geïdentifiseer, maar die meeste is nie volledig gekarakteriseer nie, veral nie ten opsigtigte van die molekulêre rol(le) wat hulle in plante speel nie. Arabidopsis thaliana MYB3, MYB6, MYB7, MYB8 en MYB32 is gevind om negatiewe reguleerders van algemene fenielpropanoied-metabolisme te wees. Daar is ook berig dat dié vyf transkripsiefaktore moontlik flavenoied-biosintese negatief kan reguleer, selfs al kan hulle verskillende teikengene hê. Studies op AtMYB13, AtMYB14 en AtMYB15 het berig dat hulle waarskynlik reguleerders van algemene fenielpropanoied-metabolisme is. Die genoemde rolle van die agt AtMYB transkripsiefaktore beteken dat hulle gemanipuleer kan word om te bepaal watter effek hulle op primêre en sekondêre metaboliete in plante het. Die transkripsiefaktore, wat in die pUBI510-GRFCA vektor geligeer was, is toe gebruik om suikerriet-kallus te transformeer (Hoofstuk 3). Suikerriet vervaardig sukrose wat tot 70% van die suiker wat in die wêreld geproduseer word opmaak. Dít maak suikerriet 'n kommersieel belangrike en winsgewende plant. Die suikerriet-kallus is getransformeer deur middel van partikel-bombardering. Die transkripsiefaktore AtMYB3, AtMYB6, AtMYB7, AtMYB13 en AtMYB32 was suksesvol in die DNA van die suikerriet-kallus opgeneem. Data wat verkry was vir kallus wat AtMYB3, AtMYB13 en AtMYB32 ooruitgedruk het op soliede media en kallus in vloeibare medium was teenstrydig (m.a.w. kallus op soilede media wat meer sukrose as die wildetipe op soliede media geproduseer het, terwyl dieselfde transgeniese lyn minder sukrose as die wildetipe geproduseer het in vloeibare medium, en anders om). Nietemin, het AtMYB13 transgeniese lyne meer sukrose geproduseer as die wildetipe. Transgeniese lyne van AtMYB7 het almal minder sukrose geproduseer as die wildetipem op beide soliede en vloiebare media. Die transkriopsiefaktore wat gelei het tot 'n styging in stysel produksie wanneer hulle ooruitgedruk was was AtMYB7 en AtMYB13. Data wat verkry is van die AtMYB6 transgeniese lyne was hoogs veranderlik in lyne wat op dieselfde medium gegroie was en oor die twee media. Die effek van hierdie transkripsiefaktore op die algehele metabolisme van die suikerriet-kallus, hetsy op MSC3 soliede of vloeibaremedia, kon egter nie van die GC-MS analise ten volle bepaal word aangesien daar geen konsekwente fenotipiese effek tussen die verskillende transgeniese lyne vir enige van die gebruikte MYB transkripsiefaktore was nie. In Hoofstuk 4 was ‘n mikropropagerings strategie ontwikkel. Fitochemikalieë en hul biologiese aktiwiteite was ook bepaal vir die medisinale plant Salvia repens. Salvia plante is gevind om medisinaal belangrik te wees as gevolg van die sekondêre metaboliete, veral die essensiële olies, wat hulle produseer. Dit is ook bevind dat die plant-ekstrakte baie biologiese aktiwiteite soos anti-bakteriese, anti-inflammatoriese, anti-oksidant en anti-kanker aktiwiteite het. Salvia repens is suksesvol ontkiem in vitro, met 60% ontkieming wat bereik is in MS media met 1x10-5 maal verdunde rook-water na insnyding vir 12 min in 75% (v/v) H2SO4. Suksessyfers van 100% was behaal in die afhardingsproses wanneer die saailinge na die glashuis verskuif was. Ontkieming van S. repens ex vitro was 100% in 'n geoutoklaveerde grondmengsel van 1:1 (v/v) sand en vermikuliet. Gewigtig het die medisinale waarde van S. repens wat in vitro of ex vitro geproduseer was nie verlore gegaan nie. Die GC-MS data metaboliete analise het aangetoon dat die plante die medisinaal belangrike chemikalieë geproduseer het. Metaboliet-ekstrakte van S. repens was vir die eerste keer na berig aktief teen swamme, met MIK waardes laer as 1mg/ml oor ‘n tydperk van 4-5 d, teen vier Fusarium spp wat getoets was. Laastens (Hoofstuk 5), transkripsiefaktore AtMYB6 en AtMYB13 was gebruik om Salvia stenophylla te transformeer deur Agrobacterium-bemiddelde transformasie, om sodoende te bepaal of die ooruitdrukking van hierdie transkripsiefaktore die produksie van medisinale en kommersieël-belangrike sekondêre metaboliete in S. stenophylla kan verhoog. Alhoewel beide A. tumefaciens en A. rhizogenes stamme gebruik was vir die transformasie proses, kon transformasie slegs deur die gebruik van A. rhizogenes bereik word. Geen transformante kon gegenereer word vanuit die A. tumefaciens behandelde materiaal nie. Transgeniese harigewortels het geen van die medisinaal belangrike metaboliete vervaardig nie. Die GC-MS analise van die transgeniese wortel materiaal het hoofsaaklik suikers en ander primêre metaboliete geïdentifiseer.

Nostoc é um gênero cianobacteriano com distribuição ubíqua que tem importância em diversos ecossistemas. Contudo, poucos genomas estão atualmente disponíveis para esse gênero. Enquanto Nostoc spp. são as cianobactérias mais comumente relatadas em relações simbióticas com fungos, animais, plantas e outros organismos, associações com outros micro organismos não receberam atenção similar. Como consequência das fortes interações entre cianobactérias e heterótrofos, culturas não axênicas são geralmente obtidas no isolamento dessas bactérias, o que proporciona uma oportunidade interessante para o desenvolvimento tanto de estudos genômicos quanto metagenômicos. Este trabalho teve como objetivo investigar as características genômicas e funcionais da linhagem Nostoc sp. CENA67, isolada de terra preta antropogênica, bem como estudar sua comunidade associada. Para esse fim, células de uma cultura não axênica de Nostoc sp. CENA67 foram sequenciadas com as plataformas MiSeq e Ion PGM e analisados com ferramentas genômicas e metagenômicas. A linhagem CENA67 de fato pertence à família Nostocaceae e possui algumas características em comum com cianobactérias do gênero Nostoc, porém diverge em certos aspectos morfológicos e filogenéticos do grupo típico de Nostoc, sugerindo que seja representante de um novo táxon. Além disso, seu genoma apresenta diferenças em relação aos genomas atualmente disponíveis para cianobactérias relacionadas ao gênero. A mineração desse genoma revelou 31 agrupamentos gênicos hipoteticamente relacionados à síntese de metabólitos secundários, a maioria dos quais não mostrou similaridade significativa com agrupamentos conhecidos. A análise de um agrupamento gênico de microviridina desvendou uma maior diversidade de genes para precursores dessa molécula do que se acreditava anteriormente, sugerindo que um número considerável de variantes ainda está a ser descoberta. A análise taxonômica da comunidade associada confirmou a dominância de cianobactérias na cultura, mas também revelou a presença de grande número de gêneros microbianos que normalmente são capazes de fixar nitrogênio atmosférico e estabelecer simbiose com plantas, incluindo Mesorhizobium, Sinorhizobium e Starkeya, entre outros. Rascunhos genômicos foram obtidos para Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia lata e Hyphomicrobium nitrativorans. Todavia, genes para fixação de nitrogênio não foram detectados nesses genomas, apesar de serem encontrados no genoma da cianobactéria e no metagenoma da comunidade, o que sugere que algumas populações podem estar sob pressão de seleção para a perda da capacidade de fixação de nitrogênio, provavelmente devido a este nutriente estar sendo fornecido pelo organismo mais abundante nesta comunidade, a cianobactéria. A análise funcional indicou vias exclusivas tanto à cianobactéria quanto à comunidade associada, e sugeriu a complementariedade de certos metabolismos. Os resultados possibilitam o aumento do conhecimento sobre a diversidade molecular e química do filo Cyanobacteria e levantam possíveis interações com micro organismos simbiontes / Nostoc is a cyanobacterial genus with ubiquitous distribution that is important in several ecosystems. However, few genomes are currently available for this genus. While Nostoc spp. are the most commonly reported cyanobacteria in symbiotic relationship with fungi, animals, plants, and other organisms, associations with other microorganisms have not received similar attention. As a consequence of tight interactions between cyanobacteria and heterotrophs, non-axenic cultures are usually achieved in the isolation of these bacteria, which provides an interesting opportunity for carrying out both genomic as metagenomic studies. This work aimed to investigate the genomic and functional characteristics of the strain Nostoc sp. CENA67, isolated from anthropogenic dark earth, and to study its associated community. For this purpose, cells from a non-axenic culture of Nostoc sp. CENA67 were sequenced with the platforms MiSeq and Ion PGM and analyzed with genomic and metagenomic tools. The strain CENA67 indeed belongs to the family Nostocaceae and presents some characteristics in common with cyanobacteria of the genus Nostoc, but diverges in certain morphological and phylogenetic aspects of the typical Nostoc group, suggesting that it is a representative of a new taxon. In addition, its genome presents differences in relation to the genomes currently available for cyanobacteria related to this genus. Genome mining revealed 31 gene clusters hypothetically related to the synthesis of secondary metabolites, most of which did not show significant similarity to known clusters. The analysis of a microviridin gene cluster unveiled a larger diversity of precursor genes for this molecule than was previously believed, suggesting that a considerable number of variants is still to be found. The taxonomic analysis of the associated community confirmed the dominance of cyanobacteria in the culture, but also revealed the presence of a great number of microbial genera that are usually capable of fixing atmospheric nitrogen and establishing symbiosis with plants, including Mesorhizobium, Sinorhizobium, and Starkeya, among others. Genomic drafts were obtained for Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia lata, and Hyphomicrobium nitrativorans. Nevertheless, genes for nitrogen fixation were not detected in these genomes, despite being found in the cyanobacterial genome and the community metagenome, suggesting that some populations might be under selection pressure for the loss of the ability to fix nitrogen, probably due to this nutrient being provided for the most abundant organism in this culture, the cyanobacterium. Functional analysis indicated pathways exclusive both to the cyanobacterium as to the associated community, and suggested the complementarity of certain metabolisms. The results allow the increase of the knowledge about the molecular and chemical diversity of the phylum Cyanobacteria and raise possible interactions with symbiotic microorganisms