Notre connaissance de la diversité virale associée aux microorganismes procaryotiques issus des sources hydrothermales océaniques profondes demeure encore limitée. Peu d’études se sont intéressées à l’abondance virale ou à l’impact des virus sur la mortalité microbienne au sein de ces écosystèmes. Le nombre de virus caractérisés, issus de ces environnements, reste faible. Deux virus, PAV1 et TPV1, associés à des archées hyperthermophiles anaérobies appartenant à l’ordre des Thermococcales ont été décrits dans notre laboratoire. Afin de poursuivre nos recherches sur la diversité virale infectant les microorganismes hydrothermaux marins, l’ordre bactérien des Thermotogales a été ciblé. Cet ordre est composé de bactéries chimioorganotrophes anaérobies pour la plupart hyper/thermophiles. Elles partagent la même niche écologique que les Thermococcales et sont métaboliquement proches. De nombreux transferts latéraux de gènes ont, par ailleurs, contribué à l’histoire évolutive des Thermotogales, subodorant l’implication potentielle de virus. La présence de séquences CRISPR a également été rapportée au sein de plusieurs génomes de Thermotogales, suggérant que les Thermotogales sont ou ont certainement déjà été exposées à des infections virales. Pour autant, à ce jour, les seuls éléments génétiques mobiles à avoir réellement été décrits chez les Thermotogales sont 3 miniplasmides et aucun virus. Une cinquantaine de souches de Thermotogales provenant majoritairement de la collection de notre laboratoire (Souchothèque de Bretagne et Collection Ifremer) a été passée au crible quant à la présence d’éventuels bactériovirus associés. A l’issue de ce criblage, des éléments à ADN extra-chromosomiques, incluant 2 plasmides et 7 bactériovirus (du type Siphoviridae) ont été découverts au sein de souches appartenant aux genres Thermosipho et Marnitoga. Des analyses préliminaires ont été réalisées sur ces différents éléments et l’un des nouveaux systèmes hôte/virus a été caractérisé en détail. MPV1 (Marinitoga piezophila virus 1) est un siphovirus-like tempéré isolé d’une bactérie piezophile, il constitue le premier bactériovirus associé à l’ordre des Thermotogales. La souche hôte est piezophile mais aisément cultivable à pression atmosphérique au terme de plusieurs repiquages. Si l’essentiel des analyses a été mené à pression atmosphérique, la production virale s’est avérée tout à fait effective à pression hydrostatique. Nous avons réalisé les analyses de la séquence complète du génome MPV1 (43,7 kb, extrait des capsides virales purifiées) et sa comparaison avec le provirus présent au sein du génome séquencé de Marinitoga piezophila KA3. Les analyses de ce génome viral ont suggéré une proximité évolutive de MPV1 avec les bactériovirus de Firmicutes. Nous avons également mis en évidence que le bactériovirus partage son hôte avec un élément génétique extra chromosomique circulaire de 13,3 kb (pMP1). Ce « ménage à 3 » est surprenant dans le sens où l’élément de 13,3 kb, contenant 13 ORF de fonctions majoritairement inconnues, utilise les capsides virales afin de se propager. Ceci pourrait, ainsi, illustrer un nouvel exemple de piratage moléculaire. / Our knowledge of the viral diversity associated to procaryotic microorganisms inhabiting the deep sea hydrothermal vents is still limited. Only few studies have focused on viral abundance and impact on microbial mortality within these ecosystems. A limited number of viruses from these environments were isolated and characterized. Two viruses, PAV1 and TPV1, associated to hyperthermophilic anaerobic Archaea, Thermococcales order, have ever been described in our laboratory. The topic of this phD thesis was to extend our investigation to other deep sea vent microorganisms in order to deepen our knowledge on the marine hydrothermal virosphere. We decided to focus more precisely on the bacterial order of Thermotogales. This order is composed of anaerobic chemoorganotrophic bacteria that are, for almost, hyper/thermophilic. They share the same ecological niche as the Thermococcales and are metabolically close. Numerous lateral gene transfers have contributed to the evolutionary history of the Thermotogales, implying the potential involvement of viruses. The presence of CRISPRs has also been reported in many genomes, suggesting that Thermotogales certainly are or have been exposed to viral infections. However, up till now, only 3 miniplasmids have been described within Thermotogales and no viruses. Fifty strains of Thermotogales, mostly from the LM2E culture collection (Ifremer and “UBOCC”), were screened for the presence of potential bacteriovirus. Extrachromosomal DNA elements, including 2 plasmids and 7 bacterioviruses (siphovirus-like), were discovered amongst strains belonging to both Thermosipho and Marinitoga genera. Preliminary studies were performed on these elements and one of the new virus-host systems was characterized in details. MPV1 (Marinitoga piezophila virus 1) is a temperate siphovirus-like isolated from a piezophilic bacterium, it is the first bacteriovirus associated to the Thermotogales order. This host strain is piezophilic but easily cultivable at atmospheric pressure after several subcultures. Whether most experiments were performed at atmospheric pressure, the viral production appeared to be effective at hydrostatic pressure. We reported the analyses of the complete sequence of the MPV1 genome (43.7 kb, extracted from purified virions) and its comparison to the provirus present in the sequenced genome of Marinitoga piezophila KA3. Analyses of the viral genome suggested a close evolutionary relationship of MPV1 to Firmicutes bacterioviruses .We also reported that this bacteriovirus shares its host with a circular extrachromosomal genetic element of 13.3 kb (pMP1). This ‘ménage à trois’ is surprising in the sense where the 13.3kb element, that contains 13 ORFs of mostly unknown function, uses the viral capsid to propagate. Therefore, it would likely correspond to a new example of molecular piracy.

Os tratamentos convencionais para carcinoma medular de tireóide (CMT) metastático são insatisfatórios. Tanto a quimioterapia quanto a radioterapia são pouco eficazes para a doença avançada. Portanto, a terapia gênica é uma promissora opção. Trabalhos de construção de vetores plasmidiais ou adenovirais específicos para cultura de células de carcinoma medular de tireóide e/ou animais têm demonstrado resultados encorajadores, conseguindo significativa redução do tumor. O objetivo deste trabalho foi construir e avaliar a eficácia do plasmídio pTCPtkevIL-12 contendo o gene da timidina quinase (HSV-tk) e da interleucina 12 otimizada/evolved (evIL-12), ambos sob controle do promotor da calcitonina modificado (TCP), visando terapia gênica do CMT. A associação entre um gene ?suicida? (TK) e um gene imunomodulador (IL12) é sabidamente sinérgica, o que motivou o emprego destes dois genes no vetor terapêutico. Por melhoramento genético, obteve-se recentemente a IL-12 otimizada/evolved, com elevada capacidade em induzir resposta imune. O promotor TCP é mais forte e mais específico que o promotor de calcitonina natural , e já foi usado em diversos trabalhos em CMT. Para determinar a atividade biológica das interleucinas 12 (evIL-12 e mIL-12), os sobrenadantes das culturas de células TT transfectadas foram utilizados para avaliar proliferação linfocitária e estimulação de células dendríticas (DCs). As células TT (carcinoma medular humano de tireóide) foram transfectadas com o plasmídio pTCPtkevIL-12 ou pTCPmIL-12 (plasmídio com o gene da IL-12 murina, sob controle do promotor TCP). A proliferação linfocitária foi quantificada por citometria de fluxo e a diferenciação de linfócitos T para um padrão Th1 foi verificada através da dosagem de IFN-? e IL-4 por ELISA. A avaliação do perfil fenotípico das DCs, cultivadas com sobrenadante contendo mIL-12 ou evIL-12 durante a diferenciação, foi feita através de marcação com anticorpos das moléculas de membrana marcadoras de maturação CD80, CD83, CD86 e CD40, com leitura também por citometria de fluxo. Também foi avaliada e comprovada a capacidade do plasmídio pTCPevIL-12 de promover apoptose induzida pelo sistema suicida timidina quinase/ganciclovir, nas células TT transfectadas. Os experimentos de proliferação linfocitária verificaram que ambas IL-12 promoveram intensa proliferação linfocitária, em similar magnitude. A principal função da IL12, todavia, não é estimular proliferação linfocitária, mas sim, induzir fortemente diferenciação para Th1, fundamental para uma eficiente resposta anti-tumoral. Foi observado que ambas IL-12 proporcionaram forte resposta Th1. Porém, a evIL-12 mostrou-se superior à mIL-12 na diferenciação dos linfócitos T para o padrão Th1. Os experimentos que avaliaram a capacidade da IL-12 maturar DCs diferenciadas, mostraram um aumento na expressão de CD40 nas DCs sob estímulo de ambas IL-12, porém a expressão foi discretamente maior com evIL-12 que com mIL-12 . Não foi observada alteração na expressão das outras proteínas marcadoras de maturação (CD80, CD83, CD86). Comparando-se o sobrenadante das células TT transfectadas com o plasmídio pTCPtkevIL-12 antes e após adição de GCV, verificou-se que ele se tornou mais eficiente para induzir expressão de CD40 nas células dendríticas após a adição do GCV. O incremento de expressão de CD40 nas DCs após tratamento com GCV poderia explicar, ao menos em parte, o efeito anti-tumoral sinérgico observado com a expressão simultânea dos genes timidina quinase e IL-12, já descritos em estudos in vivo, na literatura. Considerando-se que a evIL-12 mostrou-se mais eficiente em promover diferenciação dos linfócitos T para padrão Th1 e em promover uma maturação/ativação de melhor qualidade das células dendríticas diferenciadas (maior expressão de CD40), poderia-se afirmar que esta IL- 12 é extremamente imunogênica, superior inclusive à IL-12 murina , a única utilizada até o momento em terapia gênica de CMT. Os resultados satisfatórios alcançados neste trabalho oferecem perspectivas de aplicação futura ao plasmídio terapêutico pTCPtkevIL-12 para uso em terapia gênica em CMT. O plasmídio poderia ser utilizado em aplicação intra-tumoral e em estimulação de células dendríticas. A vacinoterapia com células dendríticas estimuladas com sobrenadante de células TT transfectadas com pTCPtkevIL-12 e tratadas com ganciclovir, devido a elevada expressão de CD40, pode ser uma esperança de um tratamento mais eficaz das metástases do CMT. Diversos estudos afirmam haver uma correlação direta entre maior expressão de CD40 e maior regressão do tumor primário e das metástases. / Present treatments of advanced and metastatic medullary thyroid carcinoma (MTC) are unsatisfactory. Tissue-specific cancer gene therapy is a promising alternative approach. IL-12 gene is a good citokyne to be used in gene therapy because it appears to be the most effective immunomodulatory gene. Literature data shows synergism in the association of two antitumor methods: suicide gene thymidine kinase (HSV-tk) and interleukin genes; therefore, they should ideally be together in the vector. The evolved interleukin12, obtained by DNA shuffling, is believed to elicit more antitumoral immune response than the human IL-12. None of them has been tested in MTC, only the murine IL-12 has been employed in MTC gene therapy. To explore a more efficient multi-gene antitumor treatment, development and evaluation of a plasmid expressing both herpes simplex virus thymidine kinase type 1 (HSVtk) and evolved interleukin-12 (evIL-12) under the control of modified calcitonin promoter (TCP) were done in this study. TCP promoter is more specific and efficient than the natural calcitonin promoter CT/CGRP, and has already been used in several studies. To verify IL-12 biological activity, lymphocyte proliferation and dendritic cell stimulation after IL-12 were studied. TT cells (human MTC) were transfected by pTCPtkevIL-12 plasmid or by pTCPmIL-12 (plasmid containing murine IL-12 gene, under control of TCP promoter). Lymphocyte proliferation was analysed by flow cytometry, and Th1 response was assessed by IFN-? and IL-4 ELISA measurement. The phenothypic analysis of dendritic cells (DCs) by flow cytometry was based on the expression of the maturative surface markers CD80, CD83, CD86 and CD40. Also, plasmid pTCPtkevIL-12 ability to promote TT transfected cells apoptosis, through the suicide system HSV-tk/ganciclovir, was evaluated and confirmed. Both IL-12 elicited similar intense lymphocyte proliferation. Nevertheless, the main IL-12 function is not to stimulate lymphocyte proliferation but induce Th1 immune response, which is essential for efficient anti-tumour response. Both IL-12, showed great Th1 response; however fortunately, ev-IL12 was superior to mIL-12 in promoting T cell Th1 response. Flow cytometry analysis of DCs revealed significant higher expression of CD40 surface molecule after differentiated DCs were exposed to TT transfected cells, either with pTCPtkevIL-12 or pTCPmIL-12, supernatants. DCs stimulated with supernatants containing evIL-12 were 36.91% CD40+, whereas when stimulated by supernatants containing mIL-12 were 14.74% CD40+. The other maturative markers (CD80, CD83, CD86) remained with the same expression level. Moreover, TT pTCPtkevIL-12- transfected cells supernatants, showed a even higher ability of increasing CD40 expression in DCs after treatment with GCV. At least partially, this increase in dendritic cells CD40 expression could explain the synergism observed with the simultaneous expression of thymidine kinase and interleukin genes. Outstanding lymphocyte proliferation and dendritic cell stimulation were achieved by the evIL-12 secreted in the supernatants, confirming that this interleukin 12 is really very potent. The good results achieved by the present study justify further experiments with this therapeutic plasmid. It could be used intra-tumorally and to stimulate/mature dendritic cells. Vaccine with DCs stimulated by TT pTCPtkevIL-12-transfected cells after GCV treatment supernatants, due to higher CD40 expression, could be very suitable for the treatment of MTC metastasis. Several studies show better primary tumor and metastasis regression in the presence of high levels of CD40 expression.

Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese. / Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.

As micobactérias não tuberculosas (NTM) podem ser encontradas em diversos ambientes, como solo, sistemas aquáticos naturais, sistemas de abastecimento de água potável e também em eucariotos. As micobactérias de crescimento rápido são um subgrupo de NTM que têm prevalência significativa em infecções relacionadas a traumas cutâneos, procedimentos cirúrgicos vídeo-assistidos e em infecções pulmonares, sendo Mycobacterium abscessus a espécie mais relevante desse grupo. Durante um pseudo-surto de infecções pulmonares pós-broncoscopia, ocorrido na cidade de São Paulo em 2007, cepas de M. abscessus subsp. bolletii (\"M. massiliense\") pertencentes ao clone epidêmico BRA100 foram detectadas em amostras coletadas do único broncoscópio usado e em amostras de lavado broncoalveolar. Todas as cepas, com exceção da F1660, apresentaram tolerância ao glutaraldeído, uma característica marcante do clone BRA100. Foi realizado o sequenciamento completo do genoma utilizando-se o sistema Illumina com metodologia de pares casados e as sequências obtidas foram comparadas, com enfoque na análise de genes potencialmente envolvidos na tolerância ao glutaraldeído. Encontramos em ambas as cepas cromossomos com 4,6 Mpb, com 64% de conteúdo GC. As sequências de nucleotídeos possuem 99% de identidade entre si. Ambos os cromossomos possuem 4.616 sequências codificantes de proteínas. Elementos de profago, como proteínas de bacteriófagos, estão presentes nos cromossomos. Bem como diversos genes associados à resistência a antibióticos e biocidas. Foram localizados operons mce, associados com a capacidade de sobrevivência em amebas e invasão de macrófagos e componentes da família T7SS, relacionadas à virulência em diversas espécies de micobactérias, como M. tuberculosis e também à transferência genética horizontal em M. smegmatis. Adicionalmente, foi detectada em ambas as cepas uma estrutura extracromossômica circular, de 97 Kbp, com 62,7% de conteúdo GC e 121 sequências codificantes. Embora a maioria das proteínas preditas tenha função desconhecida, foi possível encontrar um bloco de genes que pertencem ao sistema de secreção do tipo sete (T7SS), similar ao encontrado em plasmídeos de micobactérias de crescimento lento, sugerindo a troca de material genético entre essas espécies. Apenas na cepa tolerante ao glutaraldeído - F1725 - foi detectado um plasmídeo do grupo IncP, designado pBRA100, que contém genes que codificam resistência à kanamicina, amônio quaternário, sulfonamidas e estreptomicina, e um novo gene fosI, descrito neste estudo, que confere resistência à fosfomicina. Não foram encontradas nos cromossomos diferenças que possam justificar a tolerância ao glutaraldeído. A diferença marcante entre os dois genomas é a presença do plasmídeo pBRA100 na cepa tolerante ao glutaraldeído / Non-tuberculous mycobacteria (NTM) can be found in diverse environments as soil, water plant, natural water systems as well as in Eukaryotes. Among the NTM group, rapid growth mycobacteria (RGM) have a substantial prevalence in infections following cutaneous trauma, lung infection and after video assisted surgeries. Mycobacterium abscessus is considered to be the most relevant pathogen pertaining to the RGM group. During a pseudooutbreak of lung infections occurred in the city of São Paulo in 2007, strains from M. abscessus subsp. bolletii (\"M. massiliense\") pertaining to the BRA100 clone were detected in samples from the biopsy channel of the only bronchoscope in use as well as from bronchoalveolar lavage. All the strains but F1660 exhibited tolerance to glutaraldehyde, which is a remarking characteristic of the BRA100 clone. We report here the complete genome sequences for the strains F1725 and F1660, respectively tolerant and susceptible to glutaraldehyde. The genomes of both strains consist of a 4.6 Mpb chromosome with 4,616 coding sequences and high GC content of 64%. The chromosomes encode diverse proteins related to antibiotic and biocide resistance. Operons involved in virulence, as mce, are present as well as components from T7SS family, related to virulence in Mycobacterium tuberculosis and in horizontal gene transfer in M. smegmatis. Both strains harbored a circular extra-chromosomal structure, with T7SS system elements related to those found in plasmids from slow growing mycobacteria. It is circular, has 97 kbp and 121 open reading frames. Additionally, only F1725 strain has an IncP multidrug resistant plasmid, named pBRA100. It confers resistance to kanamycin, quaternary ammonium, sulfamethoxazole and streptomycin and also has new fosfomycin resistance gene fos I. No differences were found in the chromosomes that could justify tolerance to glutaraldehyde. The remarkable difference between the two genomes is the presence of plasmid pBRA100 in the glutaraldehyde tolerant strain

Methods are presented to acquire data from analytical ultracentrifugation experiments by computer using the absorption optical scanning system of the Beckman Model-E ultracentrifuge. A computer program was written which analyzes sedimentation velocity experiments by the van Holde - Weischet method and by the second moment method. The van Holde - Weischet method allows a high resolution analysis of sedimentation velocity data by eliminating the effects of diffusion on the shape of the moving boundary to provide sedimentation coefficients for a heterogeneous composition of a sample. The second moment method obtains the sedimentation coefficient by calculating the second moment point, by which the sedimentation coefficient is defined. Since it is impractical to manually analyze sedimentation velocity data by this method, these computer programs make an important analysis method available to the researcher. Using this computer program, it is now possible to analyze data to a higher resolution and accuracy than manual analysis of stripchart recordings would permit. Moreover, the time required for the analysis is greatly reduced. Data from sedimentation equilibrium experiments are analyzed by x² minimization. Further, a program was written for the acquisition of data to measure diffusion coefficients from quasi elastic light scattering experiments with a Langley Ford correlator. The analysis of autocorrelation spectra from light scattering experiments is performed by the Levenberg - Marquardt method, which allows fitting of data to nonlinear models. The model used allows the analysis of multicomponent systems by fitting to a sum of exponentials and a baseline. Traditional analysis of autocorrelation data by hand was limited to least squares fitting of the data to a linear model of one component without an optimized baseline, often an unrealistic approximation of the system. Analysis of autocorrelation data by nonlinear curve fitting increases both the accuracy and amount of data that can be analyzed. The development of the PPOL-1 208-n series of plasmids and of the miniplasmid pMX is described. These plasmids were designed to allow studies of in vitro transcription and chromatin structure after reconstitution with histones. The plasmids themselves were analyzed by sedimentation and diffusion studies using the computer programs. Sedimentation data is presented which suggests a new method for rapid estimation of S₀ (the sedimentation coefficient at zero concentration) for molecules which show a concentration dependency of the sedimentation coefficient. This is accomplished by linearly extrapolating van Holde Weischet distributions to zero concentration. Manual analysis of sedimentation velocity experiments to determine nonideality contributions required several experiments, computer analysis can provide this information in a single experiment due to the increased resolution of the method. Diffusion data for this plasmid DNA is used to demonstrate the feasibility of the multicomponent analysis presented here. Also, sedimentation measurements were carried out on reconstituted chromatin and on the effects of ethidium bromide on reconstituted chromatin. The programs were used to demonstrate significant changes in chromatin structure upon ethidium bromide binding. These changes involved the reduction of S of reconstituted plasmids upon addition of ethidium bromide as well as a reduction of heterogeneity of the sample. The data indicates the possibility of a forced exchange of nucleosomes between plasmids, as well as conformational changes in the chromatin structure. / Graduation date: 1993

The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, serves as a model organism to study the circadian clock in plants and animals. Rhodopsins are blue/green-light photoreceptors also found in C. reinhardtii. Chlamyrhodopsin (COP), the most abundant eyespot protein, was reported to have no role in the phototactic and photophobic responses in C. reinhardtii. Its function is yet unknown. In the present study, we hypothesized that the function of COP is to mediate entrainment of the circadian clock by light. In order to test this hypothesis, a C. reinhardtii selection marker conferring resistance to the antibiotic paromomycin was cloned into a COP RNAi construct obtained from another lab. Firstly, the COP RNAi construct was restriction digested to linearize it. The linearized plasmid was then blunt ended with T4 DNA polymerase and dephosphorylated with phosphatase. The linearized fragment was ligated with the paromomycin resistance marker obtained by restriction digestion of the plasmid containing it and transformed into E.coli. The recombinant clones obtained were confirmed by restriction digests. Fusion regions and the orientation of the insert in the recombinant plasmid were confirmed by sequencing. An attempt was made to transform C. reinhardtii with the construct, but this was not successful. Future studies will be required to optimize the C. reinhardtii transformation method. Transformants with reduced COP amounts can then be tested for defects in resetting the clock after light pulses. This will determine whether chlamyrhodopsin is involved in the circadian input pathway or not. The results of the complete project are expected to contribute to our understanding of the circadian clock of many other organisms including humans.

No description available.

Bacteria are social organisms which participate in multiple cooperative and group behaviours. They moreover have peculiar genetic systems, as they often bear mobile genetic elements like plasmids, molecular symbionts that are the cause of widespread horizontal gene transfer and play a large role in bacterial evolution. Both cooperation and horizontal transfer have consequences for human health: cooperative behaviours are very often involved in the virulence of pathogens, and horizontal gene transfer leads to the spread of antibiotic resistance. The evolution of plasmid transfer has mainly been analyzed in terms of infectious benefits for selfish mobile elements. However, chromosomal genes can also modulate horizontal transfer. A huge diversity in transfer rates is observed among bacterial isolates, suggesting a complex co-evolution between plasmids and hosts. Moreover, plasmids are enriched in genes involved in social behaviours, and so could play a key role in bacterial cooperative behaviours. We study here the coevolution of gene mobility and sociality in bacteria. To investigate the selective pressures acting on plasmid transfer and public good production, we use both mathematical modelling and a synthetic system that we constructed where we can independently control public good cooperation and plasmid conjugation in Escherichia coli. We first show experimentally that horizontal transfer allows the specific maintenance of public good alleles in a structured population by increasing relatedness at the gene-level. We further demonstrate experimentally and theoretically that this in turn allows for second-order selection of transfer ability: when cooperation is needed, alleles promoting donor and recipient abilities for public good traits can be selected both on the plasmid and on the chromosome in structured populations. Moreover, donor ability for private good traits can also be selected on the chromosome, provided that transfer happens towards kin. The interactions between transfer and cooperation can finally lead to an association between transfer and public good production alleles, explaining the high frequency of genes related to cooperation that are located on plasmids. Globally, these results provide insight into the mechanisms maintaining cooperation in bacteria, and may suggest ways to target cooperative virulence.

This master thesis is focused on the role of horizontal gene transfer in disseminating tetracycline resistance among bacteria in farm soils. In the experimental part, plasmids carrying antibiotic resistance, were exogenously isolated in biparental matings with cattle manure and Escherichia coli K-12 CV601 gfp recipients.