Lactococcus lactis subsp. cremoris Ropy 352 and L. lactis subsp. cremoris Hollandicus produce an exopolysaccharide (EPS) that imparts commercially desirable textural and rheological properties to fermented milk products. This ropy phenotype is expressed under specific environmental conditions. A mucoid EPS phenotype, also expressed under specific environmental conditions, but not involved in the fermentation of ropy milk was identified. The two EPS phenotypes can be expressed individually or concurrently. Genetic regulators involved in expression of the EPS phenotypes were sought. DNA probes and polyclonal antiserum specific to two regulators of EPS in Escherichia coli, Lon protease and RcsA protein, were used to probe ropy and non-ropy strains of L. lactis. The two ropy strains of L. lactis subsp. cremoris, Ropy 352 and Hollandicus, expressed significantly less of the Lon protein than non-ropy strains. Southern and Western blot analysis was extended to a number of Gram negative and Gram positive bacteria. All of the Gram negative bacteria probed contained DNA sequences that hybridized to the Ion and rcsA gene probes, and all of these bacteria has at least one protein that reacted with antiserum to E. coli Lon and RcsA proteins. Two of the Gram positive bacteria contained DNA sequences that hybridized to the E. coli rcsA probe. None of the other Gram positive organisms contained DNA sequences that hybridized to the rcsA or the Ion probes. However, all the Gram positive bacteria contained one high molecular weight protein that reacted with Lon antiserum. In addition, Streptococcus salivarius expressed a protein that reacted with RcsA antiserum. In the course of this study, a second RcsA protein was identified in E. coll. The two RcsA proteins are expressed from one rcsA gene. One RcsA protein is not the proteolytic product of the other RcsA protein. Limited peptide digest profiles of each RcsA protein reveals almost identical peptides indicating the two proteins share a high degree of homology but are not identical. Ferguson plot analysis strongly suggests that the two RcsA proteins differ by size not by charge. Neither RcsA protein can be detected in cells mutant for Ion and rcsB. / Graduation date: 1997

Thesis (Ph. D.)--Victoria University (Melbourne, Vic.), 2009. / Includes bibliographical references.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-05T12:48:56Z No. of bitstreams: 1 DISSERTAÇÃO DIANA ALVES MATOS.pdf: 2032501 bytes, checksum: ff9cc795950425acd0e99b3bfde3d670 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-03T15:26:37Z (GMT) No. of bitstreams: 1 DISSERTAÇÃO DIANA ALVES MATOS.pdf: 2032501 bytes, checksum: ff9cc795950425acd0e99b3bfde3d670 (MD5) / Made available in DSpace on 2017-07-03T15:26:37Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO DIANA ALVES MATOS.pdf: 2032501 bytes, checksum: ff9cc795950425acd0e99b3bfde3d670 (MD5) / CAPES / Os exopolissacarídeos se tornaram objeto de estudo em diversas áreas da biotecnologia, uma vez que estes apresentam propriedades químicas e físicas que permitem uma vasta aplicação industrial, em áreas como aplicações em várias indústrias, tais como, a farmacêutica, petrolífera, de cosméticos e de alimentos. O aumento da demanda por polímeros naturais para várias aplicações industriais nos últimos anos levou a um aumento de interesse na produção de exopolissacarídeo (EPS) por micro-organismos. Alguns exopolissacarídeos microbianos já estão disponíveis comercialmente como xantana, dextrana, gelana, alginato, levana, entre outros, mas ainda são poucos em relação à quantidade de polímeros que vem sendo descoberta. A busca por produtos biodegradáveis configura uma prioridade nos estudos com polímeros de aplicação industrial e as bactérias se destacam pela rapidez e facilidade de manipulação. As bactérias do gênero Vibrio apresentam-se numa vasta distribuição em ambiente marinho e são poucos os estudos relacionados à produção de exopolissacarídeos e suas aplicações. Dessa forma o objetivo deste estudo foi buscar exopolissacarídeos produzidos pelos isolados CCMB 01, CCMB 17, CCMB 18, CCMB 22 e CCMB 65 pertencentes ao gênero Vibrio sp. a fim de analisar possível aplicação industrial. Os isolados foram triados em meio TCBS Agar, quatro dos isolados cresceram amarelados no meio indicando fermentação de sacarose e apenas um não fermentou sacarose (colônias verdes). Os isolados foram testados em três meios de produção diferentes, Zobell, SWN e SWN suplementado com 1% de glicose. Os maiores valores de produção foram no meio SWN, de baixo custo, alcançando 7,9 g/L. Os EPSs extraídos apresentaram baixa massa molecular e consequentemente baixa viscosidade. Os espectros de infravermelho apresentaram bandas representando a presença de anéis de galactose e manose, grupo hidroxila, presença de amina e grupo haleto de alquila que são comuns a outros EPSs descritos pela literatura. / The exopolysaccharides have become the object of study in different fields of biotechnology, since they exhibit chemical and physical properties that allow industrial applications such as the pharmaceutical, petroleum, cosmetics and foods. The increase in demand for natural polymers for various industrial applications in recent years has led to an increased interest in the production of exopolysaccharides (EPS) by microorganisms. Some microbial exopolysaccharides are already commercially available as xanthan gum, dextran, gellan, alginate, levan, etc., but there are few compared to the amount of polymers has been discovered. The search for biodegradable products sets a priority in studies with industrial application of polymers and bacteria are characterized by speed and ease of handling. The bacteria Vibrio species have shown up in a wide distribution in the marine environment and there are few studies related to the production of exopolysaccharides and their applications. Thus, the aim of this study is to look for exopolysaccharides produced by isolated CCMB 01, CCMB 17, CCMB 18, CCMB 22 and CCMB 65 belonging to the genus Vibrio sp. to analyze possible industrial application. The isolates were screened on TCBS Agar medium, four of the isolates grew yellow in the middle indicating fermentation of sucrose and not just a fermented sucrose (green colonies). The strains were tested at three different production facilities, Zobell, SWN SWN and supplemented with 1% glucose. The greatest production amounts were the SWN medium (up to 7.9 g / L of production) that is a low-cost way. The extracted EPSs showed low molecular mass and consequently low viscosity. Infrared spectra showed bands representing the presence of galactose and mannose rings, hydroxyl group, the presence of amine and alkyl halide groups that are common to other EPSs described in the literature

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-06T13:04:01Z No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-29T15:06:56Z (GMT) No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / Made available in DSpace on 2017-06-29T15:06:56Z (GMT). No. of bitstreams: 1 Dissertação Final - Jamille Almeida.pdf: 1302682 bytes, checksum: 996b2da859b03a0f196518660811cde6 (MD5) / CAPES / Os polissacarídeos microbianos estão sendo muito utilizados atualmente por causa das vantagens em relação aos provenientes de outras fontes. Muitos são sintetizados por bactérias pertencentes à família Sphingomonadaceae como gelana, ramsana, welana, diutana, entre outras. Apesar da quantidade de polissacarídeos existentes, a descoberta de novos polissacarídeos microbianos é importante, tendo em vista a sua vasta aplicabilidade industrial, como espessantes, emulsificantes, estabilizantes e quelantes. Além disso, há a possibilidade de propriedades mais vantajosas e maior produção bacteriana. Este trabalho teve como objetivo selecionar linhagens bacterianas nativas de ambiente marinho produtoras de exopolissacarídeos e caracterizá-los. Neste contexto, a otimização da composição dos meios de cultivo e condições de processo podem modificar a produção, com possibilidade de aplicação industrial. Quatro bactérias foram selecionadas a partir da Coleção de Cultura Microbiana do Instituto de Ciências da Saúde pela resistência ao meio ágar nutriente contendo o antibiótico estreptomicina nas concentrações 100 e 200 μg.mL-1, sendo posteriormente identificadas por análise molecular como pertencentes aos gêneros Sphingomonas sp., Sphingobium sp. e Bacillus sp. A produção dos polímeros sintetizados por essas bactérias foi realizada em meio de cultivo, com alteração da fonte de carbono (sacarose ou glicerina bruta). A quantidade dos exopolissacarídeos sintetizados pelas bactérias pertencentes aos gêneros Sphingomonas sp. e Bacillus sp foi de 0,2 g.L-1 independente da fonte de carbono utilizada. O polímero produzido por Sphingobium sp. foi de 0,1 g.L-1 no meio contendo sacarose e 0,2 g.L-1 no meio com glicerina bruta. A CCMICS SB 22 não produziu exopolissacarídeo no meio contendo sacarose, enquanto que com a glicerina bruta foi de 0,2 g.L-1. As viscosidades dos exopolissacarídeos produzidos pelas quatro linhagens estudadas não apresentaram diferença entre si. A massa molecular do exopolissacarídeo produzido por Sphingobium sp. foi de 1,13 x 103 Daltons. Os outros polímeros não tiveram a massa molecular determinada por não apresentarem solubilidade em água. / The microbial polysaccharides are currently used being much because of advantages over from other sources. Most of those which are being studied are synthesized by bacteria of Sphingomonadaceae family, like gelan, rhamsan, welan, diutan, among others. Despite the amount of existing polysaccharides, the discovery of new polysaccharides is important, in view of its wide industrial applicability, such as thickeners, emulsifiers, stabilizers, and binders. Furthermore, there is the possibility of further advantageous properties and increased bacterial production. This work aimed to select native bacterial strains of exopolysaccharides-producing marine environment and characterize them. In this context, optimization of the composition of culture media and process conditions may change the production, with the possibility of industrial application. Four bacteria were selected from the Microbial Culture Collection of Sciences Institute of Health the resistance to the nutrient agar containing the antibiotic streptomycin in concentrations 100 and 200 μg.mL-1, subsequently identified by molecular analysis as belonging to the Sphingomonas sp., Sphingobium sp. and Bacillus sp. genres. The production of polymers synthesized by those bacteria was held in the culture medium, by changing the carbon source (sucrose or crude glycerin). The quantity of synthesized exopolysaccharides by the bacteria belonging to the Sphingomonas sp. and Bacillus sp genres was 0,2 g.L-1 regardless of the carbon source used. The polymer produced by Sphingobium sp. was 0,1 g.L-1 in the medium containing sucrose and 0,2 g.L-1 in the medium with crude glycerin. The CCMICS SB 22 produced no exopolysaccharide in the medium containing sucrose, while with crude glycerin was 0,2 g.L-1. The viscosities of exopolysaccharides produced by the four strains studied did not differ among themselves. The molecular mass of the exopolysaccharide produced by Sphingobium sp. was 1,13 x 10³ Daltons. The others polymers did not have the molecular mass determined for not showing solubility in water.

Orientador: André Henrique Rosa / Resumo: When introduced in environment, manufactured nanoparticles (NPs) can interact with biotic and abiotic molecules yielding a transformed NP usually coated with these molecules (natural coating). These different forms of the NPs should be considered as new materials because them distinct properties from the released NPs forms. In fact, these in situ transformations of the NPs have relevant impacts on their toxicological effects, having new or additional risks still not studied. This project aims to determine, understand and predict the impact of natural molecules in the transformations and bio-effects of Ag NPs, widely used as antibacterial agents. Despite the growing interest and use of these NPs, the gathered knowledge on their environmental consequences is still scarce, since the large majority of the studies do not consider the effect of the presence of natural coatings around the particles. In fact, most of the studies do not even determine the effects of the presence of manufactured coatings. Generally, NPs manufacturers add ionic or polymeric coatings to improve their mobility and stabilization in terms of size. Although the possible effects of these manufactured coatings in the NPs behavior there is not systematically studied about their impact. This project aims to overcome the current uncertainty about the environmental safety of manufactured coated NPs, and explore the impact of natural molecules on their environmental risk. The project objectives are: i) characterize... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Au cours de la dernière décennie, la tendance « clean label » ou naturalité a été considérée comme tendance prédominante parmi les consommateurs. Les additifs alimentaires dans les yogourts sont utilisés pour conserver et améliorer les propriétés souhaitables comme la texture, la viscosité, la rétention d'eau et la sensation en bouche. L'utilisation des bactéries lactiques productrices d'exopolysaccharides pendant la fermentation suscite un intérêt technologique, car elles pourraient remplacer l'utilisation des additifs alimentaires dans les yogourts. Les différentes caractéristiques des exopolysaccharides déterminent leur capacité d'interaction avec les protéines dans le yogourt, et donc, leur capacité d'agir comme agents viscosifiants et stabilisants. Jusqu'à présent, aucune étude n'a été réalisée sur l'utilisation de cultures d'appoint productrices d'exopolysaccharides, de l'espèce Weissella cibaria, ajoutés en combinaison avec des ferments dans la production des yogourts. Ce travail avait pour but d'évaluer les propriétés rhéologiques des laits fermentés en utilisant des cultures d'appoint productrices d'exopolysaccharides en coopération avec des ferments commerciaux utilisées dans le yogourt. Du lait écrémé reconstitué avec deux concentrations de sucrose (0% et 2%) a été utilisé pour la fermentation. Deux ferments commerciaux (Yo-Mix 433 et YoFlex Premium 3.0) en combinaison avec deux souches de Weissella cibaria (Hani-535 et Hani-536) productrices d'exopolysaccharides ont été utilisés. Les souches de W. cibaria ont été aussi évaluées dans un système laitier acidifié chimiquement avec du glucono-delta-lactone. La cinétique de la diminution du pH, la mesure de caractère filant, le dénombrement bactérien, la consistance, la thixotropie et la synérèse du produit ont été évalués. Les résultats ont démontré que l'utilisation de W. cibaria comme cultures d'appoint productrices d'exopolysaccharides, dans les conditions choisies, n'ont pas permis d'améliorer la consistance ni le caractère filant du yogourt. Néanmoins, la culture d'appoint et les cultures commerciales n'ont pas eu d'effet inhibiteur, car la croissance des souches et le taux d'acidification n'ont pas été affectés. En plus, l'évaluation de la thixotropie du produit a démontré une différence significative dans les yogourts avec la culture d'appoint. Ce comportement a expliqué une meilleure récupération de la structure gélifiée après le cisaillement et une meilleure stabilité au cisaillement en comparaison avec les yogourts contrôles (sans la culture d'appoint) grâce une possible production d'exopolysaccharides. / Over the past decade, the "clean label" trend has raised popularity among consumers. The food additives in yogurts are used to maintain and improve the desirable properties such as texture, viscosity, water retention and mouthfeel. There is a technological interest in the use of exopolysaccharide-producing lactic acid bacteria during fermentation that could replace the use of food additives in yogurt. The different characteristics of exopolysaccharide determine their ability to interact with proteins in yogurt, and therefore, their ability to act as viscosifying and stabilizing. To our knowledge, no studies have been carried out on the use of adjunct cultures exopolysaccharide-producers, from the species Weissella cibaria, added in combination with commercial cultures in the production of yogurt. The aim of this work was to evaluate the rheological properties of fermented milks using exopolysaccharide-producing adjunct cultures in cooperation with commercial cultures used in yogurt. Reconstituted skim milks with two concentrations of sucrose (0% and 2%) were used for fermentation. Two commercial cultures (Yo-Mix 433 and YoFlex Premium 3.0) in combination with two exopolysaccharide-producing strains of Weissella cibaria (Hani-535 and Hani-536) were used. The W. cibaria strains were also evaluated in a dairy system chemically acidified with glucono-delta-lactone (GDL). The kinetics of decreasing pH, the ropiness measurement, the bacterial count, the consistency, the thixotropy, and the measurement of syneresis of the product were evaluated. The results showed that the use of exopolysaccharide-producing adjuncts of W. cibaria, under the selected conditions, were not able to improve the consistency or the ropiness of the yogurt. Nevertheless, the adjuncts and the commercial cultures did not have an inhibitory effect because the growth of the strains and the acidification rate were not affected. In addition, the evaluation of the thixotropy of the product showed a significant difference in yogurts with the adjunct culture. This behavior explained a better recovery of the gel structure after shearing and a better shear stability compared to control yogurts (without the adjunct culture) due to a possible production of exopolysaccharides.

Au cours de la dernière décennie, la tendance « clean label » ou naturalité a été considérée comme tendance prédominante parmi les consommateurs. Les additifs alimentaires dans les yogourts sont utilisés pour conserver et améliorer les propriétés souhaitables comme la texture, la viscosité, la rétention d'eau et la sensation en bouche. L'utilisation des bactéries lactiques productrices d'exopolysaccharides pendant la fermentation suscite un intérêt technologique, car elles pourraient remplacer l'utilisation des additifs alimentaires dans les yogourts. Les différentes caractéristiques des exopolysaccharides déterminent leur capacité d'interaction avec les protéines dans le yogourt, et donc, leur capacité d'agir comme agents viscosifiants et stabilisants. Jusqu'à présent, aucune étude n'a été réalisée sur l'utilisation de cultures d'appoint productrices d'exopolysaccharides, de l'espèce Weissella cibaria, ajoutés en combinaison avec des ferments dans la production des yogourts. Ce travail avait pour but d'évaluer les propriétés rhéologiques des laits fermentés en utilisant des cultures d'appoint productrices d'exopolysaccharides en coopération avec des ferments commerciaux utilisées dans le yogourt. Du lait écrémé reconstitué avec deux concentrations de sucrose (0% et 2%) a été utilisé pour la fermentation. Deux ferments commerciaux (Yo-Mix 433 et YoFlex Premium 3.0) en combinaison avec deux souches de Weissella cibaria (Hani-535 et Hani-536) productrices d'exopolysaccharides ont été utilisés. Les souches de W. cibaria ont été aussi évaluées dans un système laitier acidifié chimiquement avec du glucono-delta-lactone. La cinétique de la diminution du pH, la mesure de caractère filant, le dénombrement bactérien, la consistance, la thixotropie et la synérèse du produit ont été évalués. Les résultats ont démontré que l'utilisation de W. cibaria comme cultures d'appoint productrices d'exopolysaccharides, dans les conditions choisies, n'ont pas permis d'améliorer la consistance ni le caractère filant du yogourt. Néanmoins, la culture d'appoint et les cultures commerciales n'ont pas eu d'effet inhibiteur, car la croissance des souches et le taux d'acidification n'ont pas été affectés. En plus, l'évaluation de la thixotropie du produit a démontré une différence significative dans les yogourts avec la culture d'appoint. Ce comportement a expliqué une meilleure récupération de la structure gélifiée après le cisaillement et une meilleure stabilité au cisaillement en comparaison avec les yogourts contrôles (sans la culture d'appoint) grâce une possible production d'exopolysaccharides. / Over the past decade, the "clean label" trend has raised popularity among consumers. The food additives in yogurts are used to maintain and improve the desirable properties such as texture, viscosity, water retention and mouthfeel. There is a technological interest in the use of exopolysaccharide-producing lactic acid bacteria during fermentation that could replace the use of food additives in yogurt. The different characteristics of exopolysaccharide determine their ability to interact with proteins in yogurt, and therefore, their ability to act as viscosifying and stabilizing. To our knowledge, no studies have been carried out on the use of adjunct cultures exopolysaccharide-producers, from the species Weissella cibaria, added in combination with commercial cultures in the production of yogurt. The aim of this work was to evaluate the rheological properties of fermented milks using exopolysaccharide-producing adjunct cultures in cooperation with commercial cultures used in yogurt. Reconstituted skim milks with two concentrations of sucrose (0% and 2%) were used for fermentation. Two commercial cultures (Yo-Mix 433 and YoFlex Premium 3.0) in combination with two exopolysaccharide-producing strains of Weissella cibaria (Hani-535 and Hani-536) were used. The W. cibaria strains were also evaluated in a dairy system chemically acidified with glucono-delta-lactone (GDL). The kinetics of decreasing pH, the ropiness measurement, the bacterial count, the consistency, the thixotropy, and the measurement of syneresis of the product were evaluated. The results showed that the use of exopolysaccharide-producing adjuncts of W. cibaria, under the selected conditions, were not able to improve the consistency or the ropiness of the yogurt. Nevertheless, the adjuncts and the commercial cultures did not have an inhibitory effect because the growth of the strains and the acidification rate were not affected. In addition, the evaluation of the thixotropy of the product showed a significant difference in yogurts with the adjunct culture. This behavior explained a better recovery of the gel structure after shearing and a better shear stability compared to control yogurts (without the adjunct culture) due to a possible production of exopolysaccharides.

Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers. / Ph. D.

Lorsqu'un biofilm se développe dans des conditions statiques, deux populations, sessiles et planctoniques, coexistent et peuvent échanger des cellules. Cependant, l'immigration de cellules planctoniques dans un biofilm n'a, jusqu'à présent, fait l'objet que de peu d'études dans le cas d'un biofilm monoespèce. Chez B. cereus, un pathogène alimentaire, notre équipe a récemment montré que des bactéries planctoniques mobiles peuvent pénétrer en profondeur à l'intérieur d'un biofilm formé en immersion. L'objectif du présent travail était, en partant de ces données, de déterminer le rôle du recrutement dans le développement du biofilm formé en interface air-liquide, et de caractériser ce processus d’un point de vue physiologique et moléculaire. Nous avons montré que la population planctonique est massivement recrutée par le biofilm, mais que, dans nos conditions expérimentales, ce recrutement ne contribue que de façon marginale à la croissance du biofilm. Nous avons mis au point deux dispositifs expérimentaux (en interface air-liquide et en immersion) permettant de quantifier le recrutement, ce qui nous a permis de cribler une banque de mutants, obtenue par mutagenèse aléatoire, pour sélectionner des clones inaptes à être recrutés. Le criblage de 1700 clones a abouti à la sélection d'un gène: Bthur002_62720. La délétion de ce gène par échange allélique affecte fortement la capacité du mutant à être recruté, et la complémentation rétablit le phénotype sauvage. Ce gène code pour une protéine probablement localisée dans l'enveloppe bactérienne. Il est porté par un plasmide, pCT8513, et pourrait être un élément mobile dont l'acquisition augmenterait fortement la capacité de la bactérie réceptrice à être recrutée par un biofilm. Enfin, nous avons mis en évidence le rôle du locus eps dans le recrutement des cellules planctoniques par un biofilm formé en interface air-liquide. Ce locus est homologue du locus epsA-O de Bacillus subtilis, requis chez cette espèce pour la production des exopolysaccharides de la matrice du biofilm. Chez B. cereus, nous avons montré que le locus eps est impliqué dans la formation d’une gaine d’exopolysaccharides faiblement liée à la paroi bactérienne. Cette couche d'exopolysaccharides contribue, avec d'autres exopolysaccharides d'origine inconnue, à la formation de la matrice du biofilm, et joue un rôle important dans l'adhésion de la bactérie sur des surfaces inertes et vivantes. En favorisant l'adhésion de la bactérie sur des surfaces vivantes, le locus eps pourrait faciliter son intégration dans le biofilm. Il pourrait également être impliqué dans le pouvoir pathogène de la bactérie. / When biofilm is developing in static conditions, cell exchanges between sessile and planktonic coexisting population can emerge. Up to now, very few are known about the implication of planktonic cells integration in monospecies biofilm development. in B. cereus, a foodborne pathogen, our team have shown that motility is a key factor for biofilm development and for the deep penetration of motile planktonic bacteria inside a biofilm formed in immersed condition. Based on these data, the purpose of the present work was to determine the role of recruitment in the development of biofilm in air-liquid interface and to characterize this phenomenon physiologically and in a molecular aspect. We showed that a massive planktonic population is integrated in the developing biofilm, however, in our experimental conditions this recruitment contributes only marginally to the biofilm growth. We have developed two recruitment systems (in air-liquid interface and in immersion condition), to quantify recruitment, which has allowed us to screen a library of mutants obtained by random mutagenesis in order to select clones unable to be recruited by a preformed biofilm. Screening of 1700 clones resulted in the selection of a gene: Bthur002_62720. The deletion of this gene by allelic exchange strongly affects the ability of the mutant to be recruited, and complementation restored the wild type phenotype. This gene encodes a protein probably localized in the bacterial envelope. It is carried by a plasmid, pCT8513, and could be a mobile element whose acquisition would greatly increase the ability of the recipient bacterium to be recruited by a biofilm. Finally, we have highlighted the role of the eps locus in the recruitment of planktonic cells in a biofilm formed at air-liquid interface. This locus is homologous to Bacillus subtilis epsA-O locus, required in this species for the production of exopolysaccharides of the biofilm matrix. In B. cereus, we have shown that the eps locus is involved in the formation of an exopolysaccharides sheath weakly bound to the bacterial cell wall. This exopolysaccharides layer contributes with other exopolysaccharides of unknown origin, to the formation of the biofilm matrix, and plays an important role in the adhesion of bacteria on inanimate and living surfaces.By promoting bacterial adhesion on living surfaces; the eps locus could help bacteria integration into the biofilm. It could also be involved in the pathogenicity of bacteria.

Notre modèle d'étude, Mycoplasma mycoides subsp. mycoides (Mmm), est l'agent de la péripneumonie contagieuse bovine (PPCB). Cette maladie fut un des plus grands fléaux de l'élevage bovin au XIXème siècle et sévit encore largement en Afrique. A cause de son impact sur l'économie, elle a bénéficié de grandes attentions dans les pays industrialisés, notamment lors des épisodes de résurgence européens dans les années 1980-90. Inscrite sur les listes de l'OIE, la PPCB est à déclaration obligatoire, ce qui entraine de sévères mesures de restriction sur le commerce des animaux vivants. Plusieurs espèces de mycoplasmes sont phylogénétiquement proches de Mmm. Pour mieux comprendre la pathogénie de cette maladie, il est nécessaire d'analyser les facteurs qui peuvent concourir à la virulence de l'agent pathogène incriminé. Dans ce contexte, nous nous sommes intéressés aux exopolysaccharides (EPS) de Mmm, candidats clés dans la virulence de cette bactérie. Ils ont été caractérisés chimiquement par HPLC et RMN, puis comparés aux polysaccharides capsulaires (CPS) dans le cadre d'une étude sur des variants intraclonaux de Mmm. Ces expériences ont été rendues possibles grâce à l'élaboration d'un protocole offrant une production optimale des EPS pour les analyses et un affranchissement des contaminants du milieu lors de leur isolement à partir des surnageants de culture. Ce protocole a permis, par ailleurs, d'élargir l'étude de caractérisation chimique des EPS au groupe « mycoides » auquel appartient Mmm. La production d'anticorps monoclonaux anti-polysaccharides a offert la possibilité d'étudier les communautés antigéniques au sein du groupe et de caractériser la localisation du polymère sécrété (CPS et/ou EPS). Les génomes séquencés et annotés disponibles ont fait l'objet d'études in silico sur les voies de biosynthèse potentiellement impliquées dans la production des polysaccharides. Les résultats montrent que dans le cas de Mmm, les variants d'un même clone présentent des différences de production de polysaccharides se traduisant par un phénotype opaque/translucide en culture sur milieu solide pouvant être associé à un « switch ON/OFF » du gène qui code un transporteur de glucose. Mmm sécréte du galactane (polymère de β-D-(1-6) galactofuranose) identique au composé associé à la membrane. Au sein du groupe « mycoides », deux espèces sécrètent un β-D-(1-2) glucane dont la structure linéaire est originale. L'analyse des voies de biosynthèse des génomes concernés concorde avec les deux produits mis en évidence. De nombreux gènes impliqués semblent résulter de transferts latéraux venant de mycoplasmes éloignés phylogénétiquement mais qui partagent le même habitat / Our model, Mycoplasma mycoides subsp. mycoides (Mmm), is the agent of contagious bovine pleuropneumonia (CBPP). CBPP is a severe contagious disease currently present in Africa. CBPP is classed as a notifiable disease by the OIE, which implies severe restrictive measures in in the trade of live animals. Because of its economic importance, CBPP has received much attention in indusdrialized country, notabily when the disease had reemerged in Europe in 1980’s and 90’s. To better understand the pathogenesis of this disease, it is necessary to analyze the factors that may contribute to the virulence of this agent. In this context, we were interested in Mmm exopolysaccharides (EPS), potential key molecules in the virulence of this bacterium. First, Mmm EPS were chemically characterized by HPLC and NMR and compared to the capsular polysaccharides (CPS) in a study on intraclonal Mmm variants. To conduct these experiments, a suitable methodology was developed to produce and to purify EPS from culture supernatants by avoiding technical difficulties due to the complexity of the mycoplasma growth medium. Then, this protocol allowed extending the study to the chemical characterization of EPS from mycoplasmas belonging to the Mycoplasma mycoides cluster (MMC) as Mmm. The production of monoclonal antibodies recognizing Mmm and Mccp polysaccharides was used to study the antigenic communities within this group of mycoplasmas and to characterize the location of the secreted polymer (CPS and/or EPS). The sequenced and annotated genomes were used to manage in silico studies of biosynthetic pathways potentially involved in the production of polysaccharides. The results showed that Mmm intraclonal variants resulting in an opaque / translucent phenotype on solid culture may be associated with an “ON/OFF switch" of the gene encoding a glucose transporter and, in turn associated to the production of either CPS or EPS. Mmm secretes a β-(1-6) galactofuranose polymer identical to that of the membrane associated compound, excepted it has no lipid anchor. Within MMC, there are two species that secrete a β -(1-2) glucan, the linear structure of which is original. The analyses of the biosynthetic pathways of the different genomes were consistent with the structure of the related secreted products. Many genes could appear to originate from phylogenetically distant mycoplasmas that share the same habitat