The microflora inhabiting the human gastrointestinal tract can be considered an essential 'metabolic organ', in a symbiotic relationship with its host. Due to the low cultivability and inappropriate sampling methodology the microflora is poorly explored and ill-defined. Preliminary, molecular-based research at the University of Waikato revealed the presence of 16S rRNA gene sequences originating from novel Mollicute-like species inhabiting the human GI tract. A ~830bp 'consensus' sequence representing these novel Mollicute-like sequences was classified within the Mollicute Genus Anaeroplasma the type species of which is Anaeroplasma abactoclasticum. It also displayed near exact matches with 16S rRNA sequences obtained from the human GI tract and matches of high similarity to those from the mouse GI tract in the NCBI database. This thesis describes an attempt to design and create primers that would amplify and characterize full-length versions of these Mollicute-like sequences from samples obtained from the mucosal surface of the human gastrointestinal tract. Primers sets targeted extended 5' and 3' versions of these novel 'known' sequences and were designed from sequence matches found in the preliminary work and other related sequences from the NCBI database. The attempt to amplify a full-length version of these novel Mollicute-like sequences was proven to be unsuccessful. No sequences were classified within the Genus Anaeroplasma, although 81% of amplicons from the 5' extending primer sets were classified within the same division as the Mollicutes, the Firmicutes, only 6% of the sequenced amplicons from the 3' extending primer set belonged to this division. Phylograms containing these 'relevant' sequences and the 'consensus' sequence grouped the 'consensus' sequence separately, indicating a lower relatedness than would have been seen if any of the amplicons contained the 'consensus' sequence.

Les plasmides pSci de Spiroplasma citri GII3 possèdent une structure mosaïque avec de nombreuses régions conservées. Des dérivés du plasmide pSci2 ont été produits par délétions successives et leur capacité de réplication a été évaluée. Le plus petit réplicon obtenu ne contient plus qu’une CDS (pE) et ses régions flanquantes. L’inactivation dans un vecteur navette du gène pE est suffisante pour abolir la réplication de ce plasmide dans S. citri. Des dérivés des pSci ont été introduits efficacement dans S. kunkelii et S. phoeniceum, deux spiroplasmes phytopathogènes pour lesquels aucun outil génétique n’était disponible jusqu’à présent. La stabilité des dérivés des pSci a également été évaluée par leur capacité à persister en l’absence de pression de sélection. L’instabilité ségrégationnelle des plasmides dans lesquels soj a été délété ou inactivé indique que la protéine de partition Soj/ParA est essentielle au maintien des plasmides pSci. L’incompatibilité sélective entre un plasmide pSci et ses dérivés a été exploitée pour produire une collection de souches possédant des profils plasmidiques différents. L’analyse des phénotypes d’acquisition et de transmission de plusieurs de ces mutants suggère que la CDS traG portée par le pSci6 est essentielle à la transmission de S. citri GII3. En revanche, les plasmides pSci1-5, codant les protéines adhésine-like ScARPs, ne sont indispensables ni à l’acquisition ni à la transmission. Dans le cadre de ce travail, plusieurs outils génétiques ont été adaptés aux mollicutes. Le promoteur Pxyl/tetO2 a été utilisé pour contrôler l’expression du gène de la spiraline chez S. citri et M. agalactiae. Enfin, un système de linéarisation de plasmide in vivo basé sur l’expression de l’endonucléase I-SceI a été utilisé pour l’élimination de plasmides pSci chez S. citri. / Plasmids pSci from Spiroplasma citri GII3 display a mosaic gene organization with highly conserved regions. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate. The smallest functional replicon consists of one single CDS (pE) and its flanking, intergenic regions. Furthermore, shuttle (S. citri/E. coli) plasmids, in which the pE gene was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein. S. citri plasmids were efficiently introduced into S. kunkelii and S. phoeniceum, two plant pathogenic spiroplasmas, the transformation of which had never been described before. Studying stability of various pSci-derived plasmids in the absence of selection pressure strongly suggests the occurrence of an active partition system involving the soj-like gene. Selective incompatibility between a given pSci plasmid and its derivatives was used to remove plasmids from the wild-type strain. As a result, a collection of S. citri GII3 mutants differing in their plasmid contents was produced. Experimental transmission of these mutants through injection to or ingestion by the leafhopper vector will provide new insights into the role of plasmid encoded determinants in the biology of S. citri. First data indicated that pSci6 traG is required for insect transmission of S. citri GII3. In contrast, pSci1-5, encoding adhesin-like proteins, are not essential for both transmission and acquisition. In the frame of this work, new genetic tools have been adapted for use in mollicutes. The tetracycline inducible promoter, Pxyl/tetO2, was used to control spiraline gene in S. citri and M. agalactiae. Also, an in vivo linearization system based on the expression of the I-SceI-endonuclease was used to remove pSci plasmids from S. citri.

Spiroplasma citri est un mollicute phytopathogène transmis de plante à plante par des cicadelles du genre Circulifer selon un mode persistant circulant-multipliant. Les franchissements de l’épithélium intestinal et des glandes salivaires sont basés sur un mécanisme d’endocytose/exocytose. Ce processus d’invasion met en jeu, au sein de complexes protéiques, des protéines bactériennes spécifiques qui reconnaissent des motifs déterminés présents à la surface des cellules eucaryotes. La recherche de protéines de l’insecte vecteur interagissant avec S. citri a notamment conduit à l’identification de l’actine. Cette interaction a pu être confirmée à la fois in vitro et in vivo. L’interaction de l’actine avec son partenaire chez S. citri, qui s’est avéré être la phosphoglycérate kinase (PGK), est impliquée dans l’internalisation du spiroplasme dans les cellules de l’insecte. La région minimale de liaison à l’actine de la PGK a également été déterminée.La réalisation d’un mutant de S. citri dépourvu de PGK a été entreprise avec le plasmide navette pGOT mais n’a pas permis la sélection de spiroplasmes mutés dans ce gène essentiel. Néanmoins, la recherche de l’évènement de recombinaison chez les clones obtenus a permis de mettre en évidence la mutation de deux gènes chez ces derniers.L'ensemble des protéines impliquées dans la transmission du spiroplasme, identifiées au préalable chez ce dernier, n’étant pas essentielle au cours de ce processus, une étude préliminaire des complexes multi-protéiques contenant plusieurs de ces protéines a été réalisée. L’identification de complexes impliquant à la fois la PGK, la P32 et les ScARPs pourrait permettre de mieux comprendre les mécanismes régissant la vection de S. citri par son insecte. / Spiroplasma citri is a phytopathogenic mollicute transmitted from plant to plant by leafhoppers of the genus Circulifer in a persistent and propagative manner on condition to cross the intestinal and salivary glands barriers of the insect. These crossings are based on a endocytosis/exocytosis mechanism which involves bacterial protein complexes in order to recognize specific patterns on the surface of eukaryotic cells.Specific molecular interactions between S. citri proteins and those of C. haematoceps were investigated using far Western technology and had notably led to the identification of actin. This interaction has been confirmed both in vitro and in vivo. The interaction of actin with his partner in S. citri, which has been identified as the phosphoglycerate kinase (PGK), is involved in the internalization of spiroplasma into the insect cells. The minimal actin-binding region of PGK was also determined.The realization of a S. citri PGK mutant was carried out using the shuttle plasmid pGOT but the selection of spiroplasmas mutated in this essential gene failed. Nevertheless, findings in the localization of recombination events in S. citri chromosome, allowed us to identify mutations in two genes that could be tested in our experimental transmission system.The set of proteins involved in the spiroplasmal transmission, previously identified as non essential proteins in the invasion process, prompted us to study the role of multi-protein complexes containing several of these proteins. Identification of complexes involving both the PGK, the P32 and ScARPs should enable us to better understand mechanisms governing the transmission of S. citri by its insect.

Frequentemente, fitoplasmas têm sido associados com doenças em cucurbitáceas, tanto cultivadas, como silvestres e daninhas. Em abobrinha-de-moita cultivada no Vale do Ribeira, Estado de São Paulo, foi observado superbrotamento de hastes, malformação da parte aérea e folhas deformadas e enrugadas, sugerindo uma possível infecção por fitoplasmas. A associação de fitoplasmas com morangueiro parece ser rara no Brasil. No entanto, nos municípios de Domingos Martins e Santa Maria de Jetibá, Estado do Espírito Santo, foram observadas plantas de morango apresentando expressiva filodia de frutos, sintoma suspeito de infecção por fitoplasmas. O presente trabalho teve por objetivos detectar e identificar molecularmente os possíveis fitoplasmas associados às plantas doentes. O DNA total das amostras, extraído com um kit comercial ou pelo protocolo 2X CTAB, foi amplificado por dupla PCR com os primers universais P1/Tint e R16F2n/R16R2. Os produtos amplificados com o primeiro par de primers foram reamplificados usando os pares R16(I)F1/R16(I)R1 e R16(III)F2/R16(III)R1, visando a identificação e os produtos de dupla PCR, amplificados com o segundo par de primers, utilizados na análise por RFLP e no sequenciamento. Para as análises filogenéticas adotaram-se seqüências nucleotídicas de fitoplasmas pertencentes a diversos grupos. Observações feitas ao microscópio eletrônico de transmissão revelaram a presença de corpúsculos pleomórficos típicos de fitoplasmas no floema de morangueiro. Por dupla PCR com os primers universais, foi consistentemente detectada a presença de fitoplasmas nos tecidos das plantas doentes. Com o uso dos primers específicos foi possível a identificação de um fitoplasma afiliado ao grupo 16SrIII em abobrinha-de-moita e de fitoplasmas afiliados aos grupos 16SrI e 16SrIII em morangueiro. A análise por RFLP confirmou os resultados obtidos com os primers específicos e demonstrou a presença de um fitoplasma afiliado ao grupo 16SrXIII nas amostras de morangueiro. O sequenciamento permitiu a obtenção de duas seqüências a partir das amostras de morangueiro. A primeira seqüência nucleotídica, SFP-Br1 (EU719107), apresentou 99% de similaridade com as seqüências dos fitoplasmas afiliados ao grupo 16SrIII e a análise filogenética mostrou um maior relacionamento com o subgrupo 16SrIII-B. A segunda seqüência nucleotídica, SFP-Br-3 (EU719109), apresentou 98% de similaridade com as seqüências dos fitoplasmas afiliados ao grupo 16SrXIII. A análise filogenética mostrou este fitoplasma como um representante do grupo 16SrXIII, porém localizado em um novo ramo, diverso daqueles conhecidos para representantes deste grupo. Os coeficientes de similaridade calculados para SFP-Br3 e para os subgrupos 16SrXIII-A, 16SrXIII-B e 16SrXIII-C variaram de 0,91 a 0,96, o que distinguiu o fitoplasma presente no morangueiro dos outros subgrupos de fitoplasmas afiliados ao grupo 16SrXIII já existentes. Assim, através de análise molecular, foi possível relatar a abobrinha-de-moita como um novo hospedeiro de fitoplasmas do grupo 16SrIII, bem como detectar e identificar distintos fitoplasmas nas plantas de morango, inclusive um novo representante, aqui denominado de 16SrXIII-D. / Phytoplasmas have been associated with some diseases of cucurbit as cultivated as sylvester and weed species. Symptoms of witches broom and leaf malformation were observed in summer squash cultivated in the Vale do Ribeira, São Paulo State, suggesting infection by phytoplasma. The association of phytoplasmas with strawberry diseases seems to be rare in Brazil. Nevertheless, in Domingos Martins and Santa Maria de Jetibá cities, located in Espirito Santo State, strawberries plants with expressive fruit phyllody were observed. This kind of symptom also suggested infection associated to phytoplasma. So, the present work was conducted in order to detect and identify possible phytoplasmas associated to simptomatic plants through molecular analysis. The DNA, extracted with a commercial kit or through 2X CTAB protocol, was amplified by nested PCR with universal primers P1/Tint and R16F2n/R16R2. The amplified products with the first primers pairs were reamplified using the primers R16(I)F1/R16(I)R1 and R16(III)F2/R16(III)R1 for specific identification; and the products of nested PCR, amplified from second primers pair, were submitted to RFLP analysis and to sequencing. For phylogenetic analysis were included sequences of phytoplasmas belonging to many groups. Results showed that pleomorphic bodies were present in the phloem of diseased strawberries plants, by using electronic microscopy. The amplifications by nested PCR with universal primers allowed the detection of phytoplasmas in the tissues of symptomatic strawberry and squash plants. PCR assays with specific primers revealed a phytoplasma of 16SrIII group in summer squash and phytoplasmas of 16SrI and 16SrIII groups in strawberry plants. RFLP analysis confirmed the occurrence of a member of 16SrIII group in summer squash and demonstrated the presence of phytoplasmas affiliated to 16SrI, 16SrIII and 16SrXIII groups in strawberry samples. The sequencing allowed the attainment of two sequences from strawberries samples. The first sequence, designated SFP-Br1 (EU719107) representing a phytoplasma affiliated to 16SrIII group, showed 99% of similarity with other sequences of phytoplasmas affiliated with this group. The phylogenetic analysis showed that this phytoplasma was more related with the subgroup 16SrIII-B. The second sequence, designated SFP-Br3 (EU719109) representing a phytoplasma affiliated to 16SrXIII group, showed 98% of similarity with members of this group. The phylogenetic analysis revealed this phytoplasma belonging to 16SrXIII group, but located on a new different branch from those previously known. The similarity coefficient indicated variations between SFP-Br3 and the three phytoplasmas affiliated to 16SrXIII-A, 16SrXIII-B and 16SrXIII-C sub-groups, varying on 0.91 to 0.96, distinguishing the strawberry phytoplasma from the other sub-groups of phytoplasma affiliated with the 16SrXIII group already existing. This way, through molecular analysis, it was possible to relate summer squash as a new host of 16SrIII group phytoplasma, as well as detecting and identifying distinct phytoplasmas on strawberry plants and characterize a new 16SrXIII-D subgroup.

Frequentemente, fitoplasmas têm sido associados com doenças em cucurbitáceas, tanto cultivadas, como silvestres e daninhas. Em abobrinha-de-moita cultivada no Vale do Ribeira, Estado de São Paulo, foi observado superbrotamento de hastes, malformação da parte aérea e folhas deformadas e enrugadas, sugerindo uma possível infecção por fitoplasmas. A associação de fitoplasmas com morangueiro parece ser rara no Brasil. No entanto, nos municípios de Domingos Martins e Santa Maria de Jetibá, Estado do Espírito Santo, foram observadas plantas de morango apresentando expressiva filodia de frutos, sintoma suspeito de infecção por fitoplasmas. O presente trabalho teve por objetivos detectar e identificar molecularmente os possíveis fitoplasmas associados às plantas doentes. O DNA total das amostras, extraído com um kit comercial ou pelo protocolo 2X CTAB, foi amplificado por dupla PCR com os primers universais P1/Tint e R16F2n/R16R2. Os produtos amplificados com o primeiro par de primers foram reamplificados usando os pares R16(I)F1/R16(I)R1 e R16(III)F2/R16(III)R1, visando a identificação e os produtos de dupla PCR, amplificados com o segundo par de primers, utilizados na análise por RFLP e no sequenciamento. Para as análises filogenéticas adotaram-se seqüências nucleotídicas de fitoplasmas pertencentes a diversos grupos. Observações feitas ao microscópio eletrônico de transmissão revelaram a presença de corpúsculos pleomórficos típicos de fitoplasmas no floema de morangueiro. Por dupla PCR com os primers universais, foi consistentemente detectada a presença de fitoplasmas nos tecidos das plantas doentes. Com o uso dos primers específicos foi possível a identificação de um fitoplasma afiliado ao grupo 16SrIII em abobrinha-de-moita e de fitoplasmas afiliados aos grupos 16SrI e 16SrIII em morangueiro. A análise por RFLP confirmou os resultados obtidos com os primers específicos e demonstrou a presença de um fitoplasma afiliado ao grupo 16SrXIII nas amostras de morangueiro. O sequenciamento permitiu a obtenção de duas seqüências a partir das amostras de morangueiro. A primeira seqüência nucleotídica, SFP-Br1 (EU719107), apresentou 99% de similaridade com as seqüências dos fitoplasmas afiliados ao grupo 16SrIII e a análise filogenética mostrou um maior relacionamento com o subgrupo 16SrIII-B. A segunda seqüência nucleotídica, SFP-Br-3 (EU719109), apresentou 98% de similaridade com as seqüências dos fitoplasmas afiliados ao grupo 16SrXIII. A análise filogenética mostrou este fitoplasma como um representante do grupo 16SrXIII, porém localizado em um novo ramo, diverso daqueles conhecidos para representantes deste grupo. Os coeficientes de similaridade calculados para SFP-Br3 e para os subgrupos 16SrXIII-A, 16SrXIII-B e 16SrXIII-C variaram de 0,91 a 0,96, o que distinguiu o fitoplasma presente no morangueiro dos outros subgrupos de fitoplasmas afiliados ao grupo 16SrXIII já existentes. Assim, através de análise molecular, foi possível relatar a abobrinha-de-moita como um novo hospedeiro de fitoplasmas do grupo 16SrIII, bem como detectar e identificar distintos fitoplasmas nas plantas de morango, inclusive um novo representante, aqui denominado de 16SrXIII-D. / Phytoplasmas have been associated with some diseases of cucurbit as cultivated as sylvester and weed species. Symptoms of witches broom and leaf malformation were observed in summer squash cultivated in the Vale do Ribeira, São Paulo State, suggesting infection by phytoplasma. The association of phytoplasmas with strawberry diseases seems to be rare in Brazil. Nevertheless, in Domingos Martins and Santa Maria de Jetibá cities, located in Espirito Santo State, strawberries plants with expressive fruit phyllody were observed. This kind of symptom also suggested infection associated to phytoplasma. So, the present work was conducted in order to detect and identify possible phytoplasmas associated to simptomatic plants through molecular analysis. The DNA, extracted with a commercial kit or through 2X CTAB protocol, was amplified by nested PCR with universal primers P1/Tint and R16F2n/R16R2. The amplified products with the first primers pairs were reamplified using the primers R16(I)F1/R16(I)R1 and R16(III)F2/R16(III)R1 for specific identification; and the products of nested PCR, amplified from second primers pair, were submitted to RFLP analysis and to sequencing. For phylogenetic analysis were included sequences of phytoplasmas belonging to many groups. Results showed that pleomorphic bodies were present in the phloem of diseased strawberries plants, by using electronic microscopy. The amplifications by nested PCR with universal primers allowed the detection of phytoplasmas in the tissues of symptomatic strawberry and squash plants. PCR assays with specific primers revealed a phytoplasma of 16SrIII group in summer squash and phytoplasmas of 16SrI and 16SrIII groups in strawberry plants. RFLP analysis confirmed the occurrence of a member of 16SrIII group in summer squash and demonstrated the presence of phytoplasmas affiliated to 16SrI, 16SrIII and 16SrXIII groups in strawberry samples. The sequencing allowed the attainment of two sequences from strawberries samples. The first sequence, designated SFP-Br1 (EU719107) representing a phytoplasma affiliated to 16SrIII group, showed 99% of similarity with other sequences of phytoplasmas affiliated with this group. The phylogenetic analysis showed that this phytoplasma was more related with the subgroup 16SrIII-B. The second sequence, designated SFP-Br3 (EU719109) representing a phytoplasma affiliated to 16SrXIII group, showed 98% of similarity with members of this group. The phylogenetic analysis revealed this phytoplasma belonging to 16SrXIII group, but located on a new different branch from those previously known. The similarity coefficient indicated variations between SFP-Br3 and the three phytoplasmas affiliated to 16SrXIII-A, 16SrXIII-B and 16SrXIII-C sub-groups, varying on 0.91 to 0.96, distinguishing the strawberry phytoplasma from the other sub-groups of phytoplasma affiliated with the 16SrXIII group already existing. This way, through molecular analysis, it was possible to relate summer squash as a new host of 16SrIII group phytoplasma, as well as detecting and identifying distinct phytoplasmas on strawberry plants and characterize a new 16SrXIII-D subgroup.