Depuis 2002 a émergé le concept de « catalyse électromicrobienne ». Cette même année, une équipe du LGC a démontré un phénomène de transfert d’électrons entre un biofilm aérobie marin et une cathode d’acier inoxydable. A partir de ces biofilms a été isolée une souche bactérienne, Algoriphagus yeomjeoni, pouvant former un biofilm électroactif monoespèce. Les objectifs de ce travail ont été de rechercher cette capacité à réduire du dioxygène chez des bactéries marines mais également chez une souche issue de l’industrie agroalimentaire Lactococcus lactis. La première partie de cette étude porte sur l’étude d’Algoriphagus yeomjeoni. Les essais électrochimiques ont eu lieu en eau de mer synthétique, dont on contrôle la composition. Aucune production de courant n’a pu être détectée même en repassant en eau de mer naturelle. La perte d’électroactivité de cette souche nous a amené à la deuxième partie de ce travail qui a été la recherche de nouveaux isolats bactériens électroactifs à partir d’un biofilm formé en milieu marin. La population microbienne de ce biofilm a été étudiée par pyroséquençage. Puis, quatre souches bactériennes ont pu être isolées et identifiées. Ces souches appartenant au genre Bacillus, Roseobacter, Pseudoalteromonas et Marinobacter ont toutes présentées des capacités de réduction du dioxygène à la cathode aussi bien en eau de mer naturelle qu’eau de mer synthétique. Enfin, des essais électrochimiques ont été réalisés avec Lactococcus lactis. Cette souche a présenté des capacités électrochimiques dans un compartiment anodique avec un record de performance de 400 mA.m-2. Et, pour la première fois, Lactococcus lactis a été capable de catalyser une réduction impliquant le dioxygène à une cathode avec une densité de courant maximale de 50 mA.m-2. / Since 2002 emerged the concept of "microbial electro-catalysis". That same year, a team from the Chemical Engineering Laboratory demonstrated a phenomenon of electron transfer between a marine aerobic biofilm and a stainless steel cathode. From these biofilms was isolated a bacterial strain Algoriphagus yeomjeoni which form a mono-species electroactive biofilm. The objectives of this work were to seek the ability to catalyze a reduction of oxygen amongst marine bacteria but also in a strain used in food industry Lactococcus lactis. The first part of this study focuses on the study of Algoriphagus yeomjeoni. Electrochemical tests were conduct in synthetic seawater, whose composition is controlled. No power generation could be detected even by returning in natural seawater. The loss of electroactivity of this strain led us to the second part of the work that has been looking for new electroactive bacterial isolates from a biofilm formed in a marine environment. This microbial population in this biofilm was studied by pyrosequencing. Then, four bacterial strains have been isolated and identified. These strains of the genus Bacillus, Roseobacter, Pseudoalteromonas and Marinobacter have all shown the ability to reduce the oxygen at the cathode in both natural and synthetic seawater. Finally, electrochemical tests were performed with, Lactococcus lactis. This strain showed electrochemical capacity in an anode compartment with a record performance up to 400 mA.m-2. Furthermore, and for the first time, Lactococcus lactis was able to catalyze the oxygen reduction involving a cathode with a maximum current density of 50 mA.m-2.

Les bactéries lactiques, en particulier Lactococcus lactis sont utilisées en industrie agroalimentaire. Ces bactéries sont connues pour avoir une activité réductrice, désignant leur aptitude à abaisser le potentiel redox (Eh) d’un milieu. Le génome de L. lactis MG1363 code plusieurs protéines possédant un motif CXXC potentiellement liées à une activité redox. Pour comprendre le rôle des protéines de surface riches en cystéines, deux approches ont été utilisées. Par l’approche bioinformatique, notre intérêt s'est porté sur deux protéines de surface de fonctions inconnues et à motif CX2CX10CX2C : Llmg_0524 et Llmg_0526. Leurs gènes forment un opéron induit temporairement en début de croissance. Dans les deux protéines, le motif chélate un ion de zinc par les résidus cystéines, formant un complexe très stable. Nos données suggèrent que cet opéron contribue à l'intégrité de la paroi cellulaire et que le zinc participe à la stabilité des protéines. L'identification des protéines à thiols exofaciaux par une approche biochimique indique la présente d’AhpF à la surface de L. lactis. La délétion du gène ahpF entraîne une forte sensibilité du mutant au cumène hydroperoxyde, mais aucune au peroxyde d'hydrogène. Le cumène hydroperoxyde provoque une modification de la proportion en acide gras chez le mutant ahpF, le mécanisme de cyclopropanation contribue à sa survie en réponse à un stress oxydatif. La compréhension des fonctions impliquées dans l'activité réductrice des lactocoques permettra une meilleure maîtrise du Eh dans la fabrication des produits fermentés et un meilleur contrôle des flores pathogènes et d’altérations. Le projet Food-Redox a été financé par l'ANR. / Lactic acid bacteria, particularly Lactococcus lactis are used in dairy industry. These bacteria are known to have a reducing activity, indicating their ability to lower the redox potential (Eh) of a medium. L. lactis MG1363 genome encodes several proteins with a CXXC motif, potentially linked with a redox activity. To understand the role of proteins rich in cysteine located at the surface of L. lactis, two approaches were used, one bioinformatics and biochemical another. For bioinformatic approach, interest was focused on two proteins of unknown function and CX2CX10CX2C motif: Llmg_0524 and Llmg_0526. Their corresponding genes form an operon temporarily induces in early growth phase. In these two proteins, the pattern chelate a zinc ion via its cysteine residues. The zinc-cysteine complexe is very stable, it suggests a probable role in protein stability. Data suggest that this operon contributes to the cell wall integrity. The identification of exofacial thiol proteins by a biochemical approach indicates that AhpF is present at the surface of L. lactis. The ahpF gene deletion causes a strong sensitivity to the cumene hydroperoxide, but no sensibility for hydrogen peroxide. In the mutant ahpF incubation with cumene hydroperoxide modified fatty acid proportion, cyclopropanation mechanism thus contributes to the survival in response to oxidative stress. Understanding the lactococci functions involved in the reduction activity allows a better control of redox potentiel in the fermented food production and thus a better control of foodbornes microorganisms in these products. Food-Redox project is financially supported by the French National Research Agency.

Les bactéries lactiques (BL), communément employées en industrie agroalimentaire, font à présent l’objet d’études visant à les utiliser pour de nouvelles applications telles que le développement de vaccins vivants ou la délivrance de molécules d’intérêt biothérapeutique chez l’hôte. Dans cette optique, différents systèmes de présentation de protéines à la surface des bactéries à Gram positif ont été développés. L’un d’entre eux est basé sur l’activité d’enzymes, les sortases, liant de façon covalente les protéines à la paroi bactérienne. Nous avons utilisé la BL modèle, Lactococcus lactis, afin d’étudier les sortases, jusqu’alors étudiées essentiellement chez les bactéries pathogènes. La sortase A (SrtA) est responsable de l’ancrage d’au moins cinq protéines à motif LPxTG à la surface. Une seconde sortase, de classe C (SrtC), a été identifiée et caractérisée. Nous avons mis en évidence la capacité de L. lactis à produire des pili à sa surface qui sont polymérisés par SrtC et ancrés à la paroi par SrtA. Ces pili résultent de la polymérisation de la piline majeure YhgE qui peut être surplombée par la piline mineure de coiffe YhgD. La production de pili chez L. lactis entraîne un changement de comportement des cellules résultant à des phénotypes particuliers. Nous avons pu l’associer à l’auto-agrégation des cellules en culture liquide, à la formation de biofilms hétérogènes et aériens, et à l’adhésion à la mucine gastrique de porc. Plus précisément, YhgE a été impliquée dans l’auto-agrégation et les biofilms atypiques, et une troisième piline, dont l’appartenance au pilus n’a pas été démontrée, semble aussi impliquée dans la production de biofilms atypiques. / Lactic acid bacteria (LAB), which are commonly used in food industry, are now being studied for their use in new applications such as biotherapeutic molecule delivery vehicules in human host or as live vaccines. Recently, surface protein delivery systems have been developed in Gram positive bacteria and one of them is based on enzymes, the sortases which covalently bind proteins to the cell wall. We used the LAB model, Lactococcus lactis, in order to study the sortases of these non-pathogenic bacteria. This work has functionally characterized the sortase A (SrtA) responsible for cell wall anchoring of at least five LPxTG proteins. A second sortase, from class C (SrtC), has been identified and characterized. We demonstrated the ability of L. lactis to produce pili on its surface that are polymerized by SrtC and cell wall anchored by SrtA. These pili result from polymerization of the YhgE major pilin and can be topped by the YhgD tip minor pilin. Pili production in L. lactis leads a change in cell behavior resulting in individual phenotypes. We were able to associate it with the self-aggregation of cells in liquid cultures, heterogeneous and aerial biofilm formation and bacterial adhesion onto pig gastric mucin. Specifically, YhgE was involved in both self-aggregation and atypical biofilm formation, while a third pilin, whose pilus membership has not been established, was also involved in the production of atypical biofilms.

Mobile group II introns are retroelements that insert site-specifically into DNA target sites by a process called retrohoming. Retrohoming is mediated by a ribonucleoprotein particle (RNP) that contains both the intron RNA and the intronencoded protein (IEP). My dissertation focuses on two mobile group II introns: Lactococcus lactis Ll.LtrB and Escherichia coli EcI5, which belong to structural subclasses IIA and CL/IIB1, respectively. Previous studies showed that the Ll.LtrB IEP, denoted LtrA protein, is pole localized in E. coli. First, I found that active LtrA protein is associated with E. coli membrane fractions, suggesting that LtrA pole localization might reflect association with a membrane receptor. Second, I found that EcI5 is highly active in retrohoming in E. coli and obtained a comprehensive view of its DNA target site recognition by selection experiments. I found that EcI5 recognizes DNA target sequences by using both the IEP and base pairing of the intron RNA, with the IEP having different target specificity than for other mobile group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate at ten different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Finally, I developed methods for gene targeting in the frog Xenopus laevis by using Ll.LtrB RNPs for site-specific DNA modification in isolated sperm nuclei, followed by in vitro fertilization to generate genetically modified animals. The site-specific integrations were efficient enough to detect in fifty sperm nuclei for a multiple copy target site, the Tx1 transposon, and several hundred sperm nuclei for protein-encoding genes. Based on these results, I obtained transgenic tadpoles with sitespecific Tx1 integrations by simple screening. To facilitate screening for embryos with targeted integrations in protein-encoding genes, I constructed an intron carrying a GFPRAM (Retrotransposition-Activated Marker). By using this GFP-RAM with introns containing randomized sequences that base pair with the target DNA, I obtained tadpoles with intron integrations at different genomic locations, including protein-encoding genes. The methods for using group II introns for targeted sperm DNA modification in X. laevis may be applicable to other animals. / text

Submitted by Suzana Kotzent (su_kotzent@hotmail.com) on 2017-03-17T13:27:02Z No. of bitstreams: 1 Dissertação final 170317 Suzana Kotzent.pdf: 1627152 bytes, checksum: fb879e427474125c781d95d8fcc0faa5 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-23T16:43:06Z (GMT) No. of bitstreams: 1 kotzent_s_me_jabo_par.pdf: 1254030 bytes, checksum: 86a63f13335a097462b3bb9d50cce954 (MD5) / Made available in DSpace on 2017-03-23T16:43:06Z (GMT). No. of bitstreams: 1 kotzent_s_me_jabo_par.pdf: 1254030 bytes, checksum: 86a63f13335a097462b3bb9d50cce954 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os probióticos são microorganismos vivos que afetam de forma benéfica o hospedeiro ou o ambiente. Na aquicultura podem ser usados tanto na água como na ração, mas seu uso na alimentação é destacado como uma das principais medidas profiláticas. As doenças bacterianas são consideradas um dos principais entraves no crescimento da aquicultura, e assim, há a necessidade urgente no desenvolvimento de probióticos. O tambaqui Colossoma macropomum é a espécie nativa mais produzida no Brasil, e apesar de sua importância econômica, não há estudos que estabeleçam os microorganismos com potencial probiótico para esta espécie de peixe. Neste trabalho foi possível identificar e caracterizar bactérias autóctones com potencial probiótico para o tambaqui a partir de testes de: caracterização morfológica, catalase, tolerância à bile, antagonismo frente à patógenos, sensibilidade a antimicrobianos e sequenciamento do gene 16S rRNA. As cepas selecionadas foram: Enterococcus faecalis, Enterococcus hirae, Lactococcus lactis, Pediococcus pentosaceus, Staphylococcus hominis e Staphylococcus saprophyticus. Todas as cepas foram tolerantes aos ácidos biliares do tambaqui e capazes de inibir o crescimento dos patógenos Enterococcus casseliflavus, Lactococcus garvieae e Aeromonas hydrophila. Todas as cepas foram parcialmente resistentes contra sete antibióticos. Como as cepas de S. saprophyticus e E. faecalis apresentaram menores valores no teste de antagonismo e por estas bactérias serem relatadas como agentes zoonóticos, concluímos este estudo selecionando quatro potenciais cepas: E. hirae, L. lactis, P. pentosaceus, S. hominis. Este é o primeiro estudo a referir o potencial uso probiótico de cepas autóctones para o tambaqui. / Probiotics are living microorganisms that beneficially affect the host or the environment. In aquaculture it can be used in both water and feed, but its use in feed is highlighted as one of the main prophylactic measures. Bacterial diseases are considered to be one of the major obstacles to the growth of aquaculture, and thus, there is an urgent need for the development of probiotics. The tambaqui Colossoma macropomum is the most produced native species in Brazil, and despite its economic importance, there are no studies that establish the microorganisms with probiotic potential for this fish species. In this study it was possible to identify and characterize autochthones bacteria with probiotic potential for tambaqui from tests of: morphological characterization, catalase, bile tolerance, antagonism of pathogens, antimicrobial susceptibility and 16S rRNA gene sequencing. The selected strains were: Enterococcus faecalis, Enterococcus hirae, Lactococcus lactis, Pediococcus pentosaceus, Staphylococcus hominis and Staphylococcus saprophyticus. All strains were tolerant to acids bile from tambaqui and capable of inhibiting the growth of Enterococcus casseliflavus, Lactococcus garvieae and Aeromonas hydrophila pathogens. All strains were partially resistant against seven antibiotics. Since the strains of S. saprophyticus and E. faecalis presented lower values in the test of antagonism and because these bacteria were reported as zoonotic agents, we conclude this study selecting four potential strains: E. hirae, L. lactis, P. pentosaceus, S. hominis. This is the first study to mention the potential probiotic use of autochthonous strains for tambaqui.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 136-145).

Techn. University, Diss., 2005--München.

Thèse (Ph.D)--Université Laval, 2003. / Titre de l'écran-titre (visionné le 22 mars 2004). Bibliogr.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-11-06T10:14:45Z No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) / Made available in DSpace on 2017-11-06T10:14:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1239459 bytes, checksum: e1a7cd2486c370dcab988719055849df (MD5) Previous issue date: 2017-06-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização de cepas fermentadoras de Lactococcus lactis subsp. lactis produtoras de nisina em alimentos fermentados tem vantagem para a indústria pois permite um controle adicional de contaminantes, como o patógeno de origem alimentar Listeria monocytogenes. No entanto, as interações microbianas devem ser avaliadas para garantir a produção da bacteriocina no alimento e o efeito na população do patógeno. L. monocytogenes tem a capacidade de resistir a diversas condições de estresse encontradas no alimento e durante o processamento, expressando diferentes genes como o fator siga alternativo (sigB), a enzima glutamato descarboxilase (gadD), a chaperona GroEL e o transportador de glicina betaína (gbu). A exposição a uma condição de estresse em nível subletal é capaz de conferir uma maior resistência a L. monocytogenes de sobreviver a outras situações. Nesse contexto, o objetivo deste trabalho foi avaliar a expressão de genes de estresse de L. monocytogenes em interação com L. lactis subsp. lactis produtor de nisina em meio de cultura e leite. A produção de nisina em caldo BHI e leite foi avaliada por sua detecção no sobrenadante do meio de crescimento e pela expressão do gene nisK. A expressão dos genes de estresse de L. monocytogenes sigB, gadD2, groEL e gbu foi avaliada relativamente a cultura pura e na interação com L. lactis subsp. lactis. A expressão relativa dos genes de estresse de L. monocytogenes foi variável. No entanto, a expressão dos genes sigB, groEL e gbu foi inferior no tempo de 24 h durante a interação, em relação a cultura pura, o que pode indicar uma menor capacidade de sobrevivência aos estresses quando a bactéria se encontra em interação com L. lactis subsp. lactis produtor de nisina. / The use of nisin producing fermentative strains of Lactococcus lactis subsp. lactis in fermented foods has an advantage for the industry because it allows an additional control of contaminants, such as the food-borne pathogen Listeria monocytogenes. However, microbial interactions should be evaluated to ensure bacteriocin production in the food and the effect on the pathogen population. L. monocytogenes has the ability to withstand the various stress conditions encountered in food and during processing, expressing different genes such as the alternative sigma factor (sigB), the glutamate decarboxylase enzyme (gadD), the GroEL chaperone and the glycine betaine transporter (gbu). The exposure to a stress condition at the sublethal level is able to confer a greater resistance to L. monocytogenes to survive other situations. In this context, the aim of this work was to evaluate the expression of L. monocytogenes stress genes in interaction with nisin producing L. lactis subsp. lactis in culture medium and milk. The production of nisin in BHI broth and milk was evaluated by its detection in the supernatant of the growth medium and by the expression of the nisK gene. Expression of sigB, gadD2, groEL and gbu stress genes of L. monocytogenes was evaluated for pure culture and for the interaction with L. lactis subsp. lactis. The relative expression of the L. monocytogenes stress genes was variable. However, expression of the sigB, groEL and gbu genes was lower at 24 h during interaction than in pure culture, which may indicate a lower ability to survive stress when the bacterium is interacting with nisin producing L. lactis subsp. lactis.

The proteolytic system of Lactococcus lactis starter cultures influences both flavor and the characteristic body and texture of cheese. The ability to further understand and control how different components of this proteolytic system work together to hydrolyze milk proteins would be of immense importance to the dairy industry. The goal of this research was to characterize Lactococcus lactis subsp. lactis starter bacteria with varying prt operon compositions by proteinase specificity, aminopeptidase and lipase activities, growth, and influence on cheese flavor. By using a cheese slurry system, a statistical model to predict milk protein hydrolysis patterns was developed. Lactococcus lactis subsp. lactis C20 has five plasmids of 55 (pJK550), 48 (pJK480), 43 (pJK430), 3.7 (pJK037), and 2.1 (pJK021) kilo bases. Two of these plasmids (pJK550 and pJK430) are necessary for full proteolytic capability, i.e., clotting milk in 16 h at 20°C. Plasmid pJK550 codes for a proteinase that catalyses the first step in casein degradation. Plasmid pJK430 codes for an oligopeptide transport system, which further transports peptides across the membrane for bacterial metabolism. Strains were constructed containing twelve different combinations of proteolytic phenotypes, such as Lac+PrtP+Opp+, Lac+PrtP+Opp-, Lac+PrtP-Opp+, Lac+Prt-Opp-, Lac-PrtP+Opp+, Lac-PrtP+Opp-, Lac-Prt-Opp+, and Lac-Prt-Opp-. The proteinase specificities of these strains toward milk proteins were dependent on the genotypes present. Genetically all strains showed a P1-type proteinase. Enzymatically C20 had group g proteinase specificity, whereas the rest of the strains containing the proteinase gene showed mixed group specificity. a a-Casein was only slightly hydrolyzed by all strains. B-Casein had a variable pattern, as did mixed casein and milk. K-Casein hydrolysis showed similar degradation patterns in all strains except CB06, which varied in its profile from the other strains. Sensory evaluation showed that culture had a significant effect on rancidity but not on acidity or bitterness. It also showed that the proteolytic system was associated with lipase activity in these strains. A statistical prediction model was developed that allowed strains to be classified according to their amino acid hydrolysis patterns. Mixed casein solution proved to be the best substrate for this analysis. Relationships among strains were seen more easily with canonical analysis and distance tables than by looking only at amino acid hydrolysis patterns.