Made available in DSpace on 2018-08-24T22:53:03Z (GMT). No. of bitstreams: 1 tese_3679_dissert_vesaofinal.pdf: 2234737 bytes, checksum: 7b99be2ff949fbc75aaf953220288ab6 (MD5) Previous issue date: 2009-08-19 / Os manguezais são constantemente atingidos por derramamentos de hidrocarbonetos de petróleo, provenientes das crescentes indústrias petroquímicas ou embarcações que transportam estes minerais brutos, ou como produtos derivados, dentre eles os diversos tipos de combustíveis. Por isso, estes ambientes são considerados ecossistemas vulneráveis e os mais sensíveis a derramamento de hidrocarbonetos de petróleo, devido à facilidade de o óleo aderir ao sedimento causando efeitos agudos e crônicos a toda biodiversidade. Sendo assim, uma das formas mais precisas de monitoramento biológico aos impactos antrópicos nos ecossistemas pode ser feitos através dos ensaios ecotoxicológicos agudos e ou crônicos, com organismos que sejam representativos ao ambiente. Com base nisso, para uma avaliação detalhada de um impacto por derrame de petróleo e derivados no manguezal, o presente trabalho buscou desenvolver uma metodologia de monitoramento do sedimento com estes contaminantes em etapa preliminar ocorrida em um mês e definitiva ocorrida em três meses, ambas com diferentes tipos de unidades experimentais (em recipientes Vaso e Lata/PET, respectivamente) fora do seu ambiente natural, por meio de ensaios ecotoxicológicos agudos, com a bactéria Vibrio fischeri. Os contaminantes testados foram o petróleo (16,9 ºAPI, pesado) e três frações provenientes deste mesmo hidrocarboneto, com densidades diferentes comparadas a alguns tipos de combustíveis, uma similar ao querosene (fração 2, 40,6 ºAPI, leve), outra similar ao óleo diesel (fração 6, 28 ºAPI, média) e a última similar ao óleo lubrificante (fração 12, 19,4 ºAPI, pesada). A toxicidade aguda apresentou relação com a densidade do óleo, pois foi sempre mais acentuada ao contaminante de grau API mais alto, onde também foi observada uma atenuação maior nos períodos experimentais, principalmente nas primeiras análises. Nos sedimentos contendo a fração 2 como contaminante apresentaram maior toxicidade aguda inicial, tanto para os experimentos preliminares quanto para os experimentos definitivos, de 81,12% e 75,46%, respectivamente.

M.Tech. / With the recent cholera outbreak in Zimbabwe and the outbreak taking a sub-regional dimension with cholera cases being reported from neighbouring countries like Botswana and South Africa, there was a need to monitor drinking water from environmental water sources as well as household water-storage containers at the point-of-use in rural communities. Although conventional culture-based microbiological methods for the identification of Vibrio species from environmental water samples are reliable, they require several days to complete (Khan and Cerniglia, 1994). Culture dependent and culture independent methods for the detection of Vibrio cholerae and Vibrio parahaemolyticus from water samples were optimised during the current study. With these methods, the occurrence and distribution of V. cholerae and V. parahaemolyticus in source waters as well as in household container stored-waters at the point-of-use in the Nwanedi Catchment, was determined. The culture based approach analyses involved the enrichment of water samples in alkaline peptone water (APW) for 18 hours at 37°C followed by culture on selective thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Typical colonies on TCBS agar were confirmed using the API 20NE as well as the two multiplex polymerase chain reactions (m-PCR). The culture independent PCR approach was done by filtering 100 ml of the water sample onto polycarbonate membranes followed by DNA extraction from the bacteria captured on the membranes using an adaptation of the in-house DNA extraction method used in the laboratory. This DNA was used as template for the m-PCR’s. For the culture based PCR detection, 100 ml water was filtered onto nitrocellulose membranes followed by 18 hours enrichment in APW. DNA was then extracted from the enrichment broth and subsequently used as template for the m-PCR’s. All water samples were analysed with all three methods to compare the results and determine the most effective method for the detection of the two-selected Vibrio species present in water samples. PCR analyses were performed using two m-PCR assays targeting the SodB (V. cholerae species), FlaE (V. parahaemolyticus species) and 16S rRNA (Vibrio and Enterobacteriacea species) genes (Multiplex 1) and the V. cholerae O1 and V. cholerae O139 rfb genes, ctxA (cholera toxin) gene and 16S rRNA gene (Multiplex 2). The 16S rRNA primers were included in the Multiplex PCR’s as an internal control. The m-PCR assays were 100% specific for total and toxigenic V. cholerae and total V. parahaemolyticus when using target bacteria and various other non-target bacteria. The m-PCR assays when coupled with an 18 hours enrichment step could detect as few as 4-10 V. cholerae and V. parahaemolyticus cells in pure cultures as well as in spiked environmental water samples. Fifty water-storage containers and 56 environmental water samples (river, spring and borehole) from rural households in the Vhembe district of the Limpopo Province of South Africa were tested for the presence of selected Vibrio’s, using (1) the standard culture based approach, (2) PCR detection without enrichment and (3) PCR with a brief pre-enrichment. Container water samples were collected before [referred to as free volume (FV) of water] and after dislodging of the biofilm [referred to as dislodged biofilm (BD)] from the inner sidewalls of containers. Of the samples analysed with the standard cultured based technique combined with colony confirmation using m-PCR 1, 34 (12.8%) tested positive for the presence of V. cholerae (SodB gene), 2 (1.3%) for the presence of V. parahaemolyticus (FlaE gene) and all the samples tested positive for the 16S rRNA gene. In contrast, only 1 (0.6%) tested positive for the presence of V. cholerae and 0 (0%) for the presence of V. parahaemolyticus when the isolates were confirmed with API 20NE. With the culture dependant PCR method, 65 (41.7%) of the samples tested positive for the presence of V. cholerae, 3 (1.9%) for the presence of V. parahaemolyticus and all the samples tested positive for the 16S rRNA gene. Seventeen (10.9%) of the samples tested positive for the presence of V. cholerae (SodB) and 16S rRNA genes, 0 (0%) for the presence of V. parahaemolyticus (FlaE gene) with the culture independent direct PCR detection protocol. All the samples that tested positive for V. cholerae with any of the three methods were tested for the presence of toxigenic V. cholerae species with the second multiplex PCR. Six of the source water samples tested positive for V. cholerae O1 as well as the cholera toxin genes. Of the 56 source water samples, 14 (25%) were positive for V. cholerae and 0 (0%) were positive for V. parahaemolyticus with one or all of the methods. Six (10.7%) of the V. cholerae positive samples tested positive for V. cholerae O1 rfb gene, and ctxA gene (cholera toxin). Thirty (60%) of the 50 FV and 28 (56%) of the DB water samples tested positive for V. cholerae, and 3 (6%) of the FV and 0 (0%) of the DB samples tested positive for V. parahaemolyticus with one or all of the methods. None of the positive V. cholerae samples tested positive for the presence of toxigenic V. cholerae. The results presented suggest that the use of culture-based techniques alone is inadequate for detection of selected Vibrio’s in the environmental water samples and that such techniques are not enough to guarantee satisfactory protection of human health. The combination of filtration, enrichment, DNA extraction and m-PCR method provide a sensitive and specific method for the detection of V. cholerae and V. parahaemolyticus in environmental water samples. This method proved to be the most effective for detection and identification of selected Vibrio’s when compared to the culture based method and PCR without enrichment method. The inclusion of an enrichment period allows for the detection of culturable bacteria which is crucial as PCR detection does not give indications on the viability of the detected material. The enrichment period will also dilute any inhibitors for the m-PCR’s that may be present. Detection of V. cholerae and V. parahaemolyticus in the source water used by the population and in the water-storage containers indicates possible seeding of containers with Vibrio species from the source water. Furthermore, the detection of these organisms in DB samples indicates that these organisms attach to containers’ inner sidewalls, forming biofilms, further sustaining their occurrence and proliferation. The detection of V. cholerae and V. parahaemolyticus in household water-storage containers certainly places the consumers at risk of infection of diseases caused by these organisms.

In recent years, mortalities of unknown aetiology have occurred in Pacific oyster aquaculture in Baynes Sound, BC during the summer. Field studies were conducted to examine environmental, reproductive and microbial factors that could be contributing to these mortalities. In 2017, oysters were observed at three sites from July 5 to September 15. Each intertidal site had three modules containing seven stacked trays with 80 oysters per tray. Final mortalities ranged from 9.3 ± 1.9 to 38.8 ± 4.9% per module. The mortality per module correlated significantly with gonad length and the proportion of oysters that were female in a multiple linear regression model (R2=0.824, p=0.002). Vibrio aestuarianus, a well-documented pathogen of farmed Pacific oysters in France, was well represented in bacterial cultures from intertidal oysters in 2017 based on recA gene sequencing of 158 bacterial isolates. In 2018, juvenile Pacific oysters were monitored to characterize the onset of a summer mortality event in suspended culture. From May 11 to September 17, data on shell size, reproductive development, environmental conditions, and the microbial community of gill tissue was tracked at culture densities of 150, 300, 450, and 600 oysters tray-1. The onset of mortality was associated with a period of rapid growth, reproductive development, and elevated temperatures. Cumulative mortality per tray ranged from 34 to 75%, with the highest density trays having significantly lower mortality (p=0.023), smaller shell width (p=0.001), smaller shell length (p=0.002) and smaller gonad length (p=0.049) than the lowest density trays in a linear mixed-effects regression. Histology of oysters from August 12, during the mortality event, showed a mixed microbial infection in peripheral gill tissue. High-throughput sequencing of the 16S rRNA gene and qPCR of V. aestuarianus using species-specific recA primers suggest V. aestuarianus is temporally associated with summer mortality. Mortalities observed in 2017 and 2018 occurred in different age classes and with different oyster culture techniques, but all were associated with elevated water temperature, increased reproductive effort, and the presence of V. aestuarianus. / Graduate / 2021-08-06

A rapid assay kit for Vibrio cholerae (Vc) was developed to detect and quantify Vc cells in oyster samples within 24 h. The kit, formulated within a two -phase (liquid and solid) 96-well plate, can detect biomarker expression of Vc when the enrichment broth and incubation temperature are optimized. The kit showed 91 % selectivity and 92 % specificity when tested with 23 inclusive Vc and 106 exclusive non-Vc strains. The kit was further optimized using 47 samples of oysters, clams, and soil. There was no significant difference in most probable number between the kit, conventional PCR and BAX PCR regardless of agar heating method (autoclaved vs. boiled). The kit’s limit of detection was below 5 cfu/g. The kit is a reliable method for the detection of V. cholerae in bivalve samples.

Vibrio vulnificus, an emergent human pathogen, causes septicemia with a mortality rate over 50%. Additionally, symptom onset occurs rapidly, with the incubation time for ingestion cases being around 26 hours. This, combined with the severity of symptoms has led to V. vulnificus being considered the deadliest seafood-associated pathogen, claiming responsibility for 95% of seafood-related deaths. Currently, the molecular mechanisms through which some strains of this bacteria emerge to become pathogens are unknown. The main focus of this study is to expand upon the base of knowledge surrounding this question through comparing virulence phenotypes in environmentally collected strains of V. vulnificus. Specifically, this study will evaluate the pathogenic potential of environmental isolates collected from water and oyster sources in Lee County, Florida, in lieu of the outbreak that occurred in October 2022. To test this, a variety of assays were performed. First, a phylogenetic tree was built to establish the relationships between strains. Next, to study in vitro responses, serum resistance assays and sialic acid growth curves were performed. Then, to further classify the pathogenic potential of these environmental strains, they were tested against THP-1 monocytes differentiated into macrophages for their ability to resist phagocytosis and induce apoptosis. This study found differential responses amongst the environmental isolates, with some exhibiting significant pathogenic potential and others being sensitive to all tested assays. Understanding which strains emerge as pathogens will help determine the prevalence of key virulence factors within natural populations of bacteria and provide critical data on the phenotypic outcomes of differing genotypes.

Vibrio parahaemolyticus is considered to be an emerging, yet understudied, human pathogen. The V. parahaemolyticus BB22OP genome was sequenced to allow for a comparative analysis between the genome of BB22OP and another previously sequenced, pathogenic strain of V. parahaemolyticus, RIMD2210633. V. parahaemolyticus BB22OP is interesting because it exhibits a spontaneous phenotypic switch in colony morphology due to the loss of a functional OpaR; this also influences virulence. OpaR is the major quorum-sensing regulator in V. parahaemolyticus homologous to LuxR from V. harveyi. When opaR is removed from the RIMD2210633 genome, the same phenotypic switch is not seen indicating a difference between the quorum-sensing systems in these two strains. Understanding the regulatory variation in these two strains has the potential to provide key insights into the control of pathogenesis in this organism. Initially, the BB22OP genome sequencing results aligned into 125 contigs. The genome has now been assembled into two distinct chromosomes with only two gaps remaining to be filled. These gaps are located in the integron region, which is difficult to assemble due to its structure. The integron is a series of gene cassettes separated by inverted repeats that facilitate recombination events that build the integron. The integron region is further evidence of genetic differences between the two strains. The integron in the RIMD2210633 strain is comprised of 69 gene cassettes, while the BB22OP integron contains at least 86 gene cassettes. There are 313 genes novel to the BB22OP genome, which could result in the phenotypic differences seen in these two strains. Additionally five of the 313 genes are predicted to be transcriptional regulators indicating the potential for differential gene regulation. Further comparative analysis will likely reveal more phenotypic divergence between the physiology of RIMD2210633 and BB22OP. Additionally, the CsrA regulatory network was explored in RIMD2210633. CsrA was first characterized in E. coli as a global regulator of carbon storage and metabolism. RIMD2210633 contains a CsrA homolog and was predicted to contain four CsrA-regulating sRNAs (CsrB1-3 and CsrC), and this work confirmed that these sRNAs regulate CsrA in the same manner as in E. coli. CsrA and the same CsrA-regulating sRNAs were found in the BB22OP genome as well. Since CsrA is known to regulate glycogen production, a qualitative iodine-staining plate assay and a quantitative glycogen assay were used to indirectly measure CsrA activity in the presence and absence of individual regulatory sRNAs. The RIMD2210633 CsrA, CsrB1, CsrB2, CsrB3 and CsrC were shown to have the predicted physiological role in recombinant E. coli, with higher glycogen levels observed when CsrA was active and lower levels when each of the sRNAs was overexpressed. CsrA is also known to regulate biofilm production and virulence factors. In an attempt to develop a screening method for potential CsrA targets, a transcriptional/translational fusion system was developed. Transcriptional and translational fusions to β-galactosidase were created to PdksA, PglgC1 and PtoxR from RIMD2210633. CsrA or CsrB2 was overexpressed in recombinant E. coli containing each of the fusion constructs in order to see what happens to the gene expression from these promoters at low and high CsrA activity levels. Surprisingly, changing the activity levels of CsrA impacted both transcriptional and translational levels making the results of the assay difficult to interpret. Collectively these efforts have enhanced our understanding of V. parahaemolyticus. In particular, the sequencing of BB22OP has allowed for a comparative analysis between the BB22OP and RIMD2210633 strains. These strains have remarkably conserved genomes despite the phenotypic differences they exhibit. It appears there is variation in the quorum-sensing systems of these two strains. Further analysis will reveal how the quorum-sensing regulons differ and how this impacts the virulence of these two pathogenic V. parahaemolyticus strains. / Master of Science

The World Health Organization (WHO) states that 30,000 children under the age of five die each day worldwide. Around a quarter of these die from diarrheal disease caused by microbial infection. In addition to this high mortality rate, there are data emerging on the morbidity effects of diarrheal disease, for example a few episodes of diarrhea in the first two years of life can remove 10 IQ points and lead to growth deficiency. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is a serious problem in third world countries, where sanitary and hygiene infrastructure is very poor, and claims several thousand lives every year. In order to better understand the pathogenicity regulation in V. cholerae, structural and functional investigations of a hypothetical protein family present in pathogenicity islands and a transcriptional regulator protein for DNA-binding were investigated. Two adjacent genes, vc1804 and vc1805, encode hypothetical proteins within the Vibrio pathogenicity island-2 (VPI-2) of Vibrio cholerae, and are part of a cluster of genes only present in pathogenic strains of the bacterium. Paralogous adjacent genes, vc0508 and vc0509, are also present within a second pathogenicity island, the Vibrio seventh pandemic island-2 (VSP-2), of V. cholerae O1 El Tor and O139 serogroup isolates. Sequence similarity suggests that the VC0508, VC0509, VC1804 and VC1805 proteins will share a similar fold. The crystal structures of VC0508, VC0509 and VC1805 have been determined to a resolution of 1.9, 2.4 and 2.1 Å, respectively. Several recombinant constructs of vc1804 were made, but no soluble proteins were expressed. This hypothetical protein family reveals structural homology to human mitochondrial protein p32. Human p32 is a promiscuous protein known to bind to a variety of partners including the globular head component of C1q. We have shown that VC1805 binds to C1q. One possibility is that VC1805 is involved in adherence of the bacterium to membrane-bound C1q in the gut. To explore the roles of VC0508, VC0509, VC1804 and VC1805 in vivo, gene knockout and animal model studies of those proteins are underway. The ferric uptake regulator (Fur), a metal-dependent DNA-binding protein, acts as both a repressor and activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, and this opens up the potential of Fur as a drug target for cholera. The first crystal structure of a Fur protein, from Pseudomonas aeruginosa, revealed a dimeric molecule with each monomer containing a dimerization domain, a helical DNA-binding domain and two metal binding sites: Zn1 is proposed to be a regulatory Fe-binding site, and Zn2 is proposed to be a structural Zn-binding site. Here we present the crystal structure of V. cholerae Fur (VcFur) that reveals a very different orientation of the DNA-binding domains. Accompanying these structural changes are alterations in the amino acids coordinating the zinc at the Zn2 site, and this lends support to this being the site regulated by iron. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including the much-studied E. coli Fur. An analysis of the metal binding properties shows that like other Fur proteins, VcFur can be activated by a range of divalent metals. EPR spectroscopy measurements of the movements of the DNA-binding domain, in the presence of DNA and different metals, are underway.

Neste estudo, 26 amostras de ostras (Crassostrea gigas) comercializadas na cidade de São Paulo e em alguns pontos do litoral de São Paulo, e 36 amostras de mexilhões (Perna perna) colhidas mensalmente em 3 pontos do litoral de Ubatuba - SP, foram submetidas à pesquisa de vibrios potencialmente patogênicos. As amostras desses moluscos eram submetidas a enriquecimento em água peptonada alcalina sem cloreto de sódio e com 1 por cento de cloreto de sódio, e GSTB. O isolamento foi realizado em ágar TCBS. Colônias sacarose positivas e negativas, sugestivas de espécies de Vibrio foram identificadas presuntivamente em meio de ágar ferro de Kligler, sendo confirmadas através de provas bioqufmicas complementares. Uma parte das amostras de vibrios potencialmente patogênicos isoladas foi submetida ao teste de Dean e teste de alça ligada em íleo de coelhos. Os vibrios potencialmente patogênicos encontrados em amostras de ostras foram V. alginolyticus (81 por cento ), V.parahaemolyticus (77 por cento ), V. cholerae não 0:1 (31 por cento ), V. fluvialis (27 por cento ), V. furnissii (19 por cento ), V. mimicus (12 por cento ) e V. vulnificus (12 por cento ) e em amostras de mexilhões foram V. alginolyticus(97 por cento ), V. parahaemolyticus(75 por cento ), V. fluvialis (47 por cento ), V. vulnificus (11 por cento ), V. cholerae não 0:1 (6 por cento ), V. furnissii (6 por cento ) e V. mimicus (6 por cento ). Observou-se acúmulo de fluido em alça ligada de íleo de coelho entre 0,25 e 0,49 ml/cm em 6,9 por cento das amostras, entre 0,5 e 0,99 ml/cm em 15,6 por cento e maior ou igual a 1 ml/cm em 15,1 por cento , e/ou intestino de camundongos lactentes (Teste de Dean) em 26,6 por cento das amostras testadas, confirmando o elevado potencial desses microrganismos em causar gastrenterite. Verificou-se ausência de variação sazonal e também, de correlação entre os vibrios potencialmente patogênicos isolados e os indicadores de contaminação fecal, confirmando que a presença desses microrganismos ocorre de forma autóctone e que, as condições climáticas foram favoráveis à sobrevivência dessas espécies em todas as épocas do ano. Considerando-se os resultados obtidos no presente estudo e o fato de que ostras e mexilhões são habitualmente ingeridos crus ou insuficientemente cozidos, pode-se concluir que sua ingestão constitui-se em um determinado grau de risco para a saúde do consumidor. / In this work, 26 oysters samples (Crassostrea gigas), found in the market of São Paulo city and some coastal areas of São Paulo State, and 36 mussels samples (Perna perna), that were collected monthly in 3 coastal areas of Ubatuba city - SP., were analyzed for the potential patogenic vibrios occurrence. Samples were enriched in alcalin peptone water with (1 per cent ) and without sodium cloride and GSTB. Isolation was performed on TCBS agar. suspect sacharosis positive and negative colonies, resembling vibrio species, were presumptively identified on Kligler iron agar, and confirmed by complementary biochemical tests. Some of this potential patogenic vibrios were submitted to suckling mouse assay and rabbit ileal loop assay. Potential patogenic vibrios isolated from oyster samples were: V. alginolyticus (81 per cent ), V. parahaemolyticus (77 per cent ), V. cholerae non 0:1 (31 per cent ), V. fluvialis (27 per cent ) I V. furnissii (19 per cent ), V. mimicus (12 per cent ) and V. vulnificus (12 per cent ) and from mussels samples were: V. a.1ginolyticus (97 per cent ), V. parabaemolyticus (75 per cent ), V. fluvialis (47 per cent ), V. vulnificus (11 per cent ), V. cholerae non 0:1 (6 per cent ), V. furnissii (6 per cent ) and V. mimicus (6 per cent ). It was found 6,9 per cent of samples between 0,25 and 0,49 ml/cm of fluid accumulation in ileal loop assay, 15,6 per cent between 0,5 and 0,99 ml/cm and 15,1 per cent was equal or higher than 1 ml/cm. Among the samples assayed for suckling mouse 26,6 per cent were positive. These results confirm the high potential of these microrganisms to induce gastroenteritis. Seasonal variation as well as correlation between the potential patogenic vibrios isolated and the fecal contamination indicators were not found, confirming that the presence of such microrganisms occurs autochthonously and that the climate conditions were favourable to these species survival during the whole year. with the results of this work and considering that oyster and mussels are usually ingested raw or insufficiently cooked, the conclusion is that the ingestion of such mollusks presents a certain degree of risk for the consumer\'s health.

L’objectif de cette thèse est de définir la chorégraphie de ségrégation des deux chromosomes circulaires de Vibrio cholerae, c’est à dire le positionnement de l’information génétique au cours de la croissance de la cellule, ainsi que les mécanismes dirigeant ces ségrégations. Il a longtemps été supposé que les bactéries étaient trop petites pour avoir une organisation intra-cellulaire, et le manque de techniques appropriées ne permettait pas d’infirmer cette hypothèse. Or la taille des chromosomes comparée à celle de la bactérie impose une compaction et aujourd’hui, de nouvelles techniques de microscopie et d’analyse génétique permettent d’affirmer que les chromosomes bactériens étudiés jusqu’à maintenant ont tous une organisation et une chorégraphie de ségrégation précises et différentes selon les espèces. Toutes les espèces étudiées à ce jour ont un chromosome circulaire unique : la réplication du chromosome commence à une origine unique bidirectionnelle, les deux fourches de réplication se déplacent le long des deux bras de réplication (ou réplichores) et finissent la réplication au terminus, diamétralement à l’opposée de l’origine de réplication sur la carte du chromosome. Peu d’espèces ont été étudiées, et Vibrio cholerae émerge progressivement comme un nouveau modèle : son génome est réparti sur deux chromosomes, et la chorégraphie de plusieurs chromosomes dans une cellule n’a jamais été décrite. De plus, cette espèce semble être au croisement évolutif entre Caulobacter crescentus et Escherichia coli : Vibrio cholerae a d’une part une morphologie en croissant, des systèmes de partition aux origines et un positionnement de l’origine du chromosome I, semblables à C. crescentus, et d’autre part un système de compaction du terminus et un set de gènes impliqués dans la maintenance du chromosome ayant co-évolué, qu’on ne retrouve que dans peu d’espèces proches d’E. coli. Une autre caractéristique intéressante de V. cholerae est que le chromosome II semble avoir été acquis récemment et n’est donc peut être pas gouverné par les mêmes mécanismes que le chromosome I, comme en témoignent le positionnement de son origine et son terminus, inédits pour des chromosomes bactériens. Parmi les Vibrios (environ 60 espèces principalement retrouvées dans les environnements aquatiques), certaines espèces sont des pathogènes dévastateurs pour les poissons, le corail, les crustacés ou les fruits de mer. Mais la plus documentée est Vibrio cholerae, car elle provoque chez l’Humain une maladie provoquée par l’ingestion d’eau contaminée qui peut être mortelle si le patient n’est pas réhydraté à temps. Bien que facilement traitable, le choléra fait encore de nombreuses victimes dans les pays en développement où les structures de santé et les règles d’hygiène font parfois défaut. Ainsi l’étude de Vibrio cholerae présente un intérêt médical, mais également par extension aux autres Vibrios, un intérêt environnemental non négligeable. / The aim of this thesis is to define the segregation choreography of the two circular chromosomes of Vibrio cholerae, which is the positionning of the genetic information during cell growth, as well as the mecanisms directing those segregations. It was supposed for a long time that bacteria were too small to have a intra-cellular organization and the lack of appropriate tools could not prove this hypothesis wrong. The size of the chromosomes compared to the size of the cell means there has to be a compaction and today, new tools for microscopy and genetic analysis allow us to affirm that all bacterial chromosomes studied so far have an organization and a segregation choreography which are precise and different between specie. Most bacterial specie studied to this day have a unique circular chromosome : the replication of the chromosome starts at a unique and bidirectionnal origin, both replication forks move along the two replication arms (or replichores) and end the replication at the terminus which is diametrically to the opposite of the origin on the chromosome map. A few specie have been studied, and Vibrio cholerae progressively emerges as a new model : its genome is divided between two chromosomes, and the choreography of several chromosomes in a cell has never been described. Moreover, this species seems to be at the crossover between Caulobacter crescentus and Escherichia coli : Vibrio cholerae as on one hand, a crescent shape, partition systems positionned at both origins and a positionning of the chromosome I origin similar to C. crescentus, and on the other hand a compaction system of the terminus and a set of genes involved on the maintenance of chromosomes that one only finds in very few specie closely related to E. coli. An other interesting characteristic of V. cholerae is that the chromosome II seems to have been acquired recently and thus might not be governed by the same mecanisms as the chromosome I, as shown by the positionning of its origin and terminus which are completely new to bacterial chromosomes. Among Vibrios (about 60 species mostly found in aquatic environments), some species are devastating pathogens for fish, coral, crustacean and shellfish. But the most documented one is Vibrio cholerae, because it induces a disease in humans caused by the ingestion of contaminated water, which can be deadly if the patient is not rehydrated on time. Although easily treatable, cholera still makes a lot of victims in developing countries where health structures and basic hygiene sometimes lack dramatically. The study of Vibrio cholerae has a medical interest, but also by extention to other Vibrios, a non-negligible environmental interest.

Vibrio splendidus est une bacterie Gram négative du genre Vibrio. Les Vibrios sont des bactéries motiles, en forme de virgule, vivant essentiellement dans les ecosystèmes marins. Depuis une quinzaine d'années, Vibrio splendidus a été associé a des épisodes estivaux de mortalité de naissains d'huitres en France, jusqu'à compromettre l'économie ostréicole. Cependant on sait encore peu de choses sur l’évolution de cette espèce, sa capacité d'adaptation, et ses mécanismes de virulence. De nombreux travaux au cours de ces dernières années ont souligné l'importance des petits ARN non codants (sRNA) dans la régulation des gènes bactériens, en particulier en réponse à l'environnement ainsi qu'au cours de la pathogenèse. Nous avons réalisé une étude de transcriptomique par séquençage à haut débit afin d'établir un répertoire des sRNA chez V. splendidus. Cette analyse transcriptomique nous a permis d'identifier des centaines de sRNAs putatifs, dont la majorité sont spécifiques de cette espèce, une minorité résultant de transferts horizontaux avec d'autres bactéries marines, comme les Shewanellaceae. Les données transcriptomiques ont montré la présence chez V. splendidus de 4 copies du petit ARN CsrB, contrairement aux autres Vibrios qui en ont trois ou deux. Nous avons pu montrer que ces CsrBs jouent un role important dans la physiologie cellulaire en contrôlant la production et/ou la sécrétion de deux proteases jouant un role dans la virulence. De plus, nous avons montré que le réseau de régulation impliquant ces CsrBs est significativement différent des autres Vibrios, indiquant que la présence d'une copie supplémentaire a sans doute entraine un remodelage de ce réseau de régulation. / Vibrio splendidus is a Gram negative bacterium of the genus Vibrio. The Vibrios are motile, comma-shaped bacteria, living mainly in marine ecosystems. Over the past fifteenyears, Vibrio splendidus has been associated with oyster summer mortality episodes inFrance, generating heavy economic losses. However, still little is known about the evolutionof this species, its adaptive capacity, and the mechanisms of virulence. Many works in recentyears have stressed the importance of small noncoding RNAs (sRNAs) in bacterial generegulation, particularly in response to the environment as well as during pathogenesis. Wedid a transcriptomic study by high-throughput sequencing in order to explore the sRNArepertoire in V. splendidus. This transcriptomic analysis allowed us to identify hundreds ofputative sRNAs, the majority of which being specific of this species, a minority resulting fromhorizontal transfers with other marine bacteria, such as the Shewanellaceae. Transcriptomicdata showed the presence of 4 copies of the small RNA CsrB in V. splendidus, unlike otherVibrios which have two or three. We have shown that these CsrBs play an important role incell physiology by controlling the production and/or secretion of two proteases playing a rolein virulence. In addition, we have shown that the regulatory network involving these CsrBs issignificantly different from other Vibrios, indicating that the presence of an extra copy hasindeed resulted in a reshaping of the regulatory network.