Thesis (M.S.)--Oregon State University, 1984. / Typescript (photocopy). Includes bibliographical references (leaves 110-115). Also available online.

Presented as Thesis (Ph. D.)--University of Wisconsin--Madison, 1911. / Cover title. Reprinted from Journal of biological chemistry, vol. X, no 3 (Oct., 1911). Includes bibliographical references.

The biochemical activity of Streptococcus thermophilus and Lactobacillus bulgaricus in a 1:1 ratio (Chr. Hansen's Lab - CH-III) were studied at 43 C in goat and cow milk during the manufacture and storage of yogurt. Determination of pH, titratable acidity, lactose content, proteolytic activity, and enumeration of the starters were performed on samples taken at hourly intervals until the yogurt became set, and after 8, 16, 24 hours, 11 and 22 days of storage at 4 C. In goat milk the number of starter culture organisms showed higher values than those in cow milk. Fermentation was considered to be finished when the pH decreased to 4.7 and 0.7% of lactic acid was produced. Those values were reached after 3 and 4 hours for goat and cow milk respectively. The values for pH in both milks were stable at 4.7 during storage at 4 C. Goat milk and goat milk yogurt had higher levels of free amino acids (12.65 mg/dl and 24.39 mg/dl respectively) than cow milk (10.31 mg/dl) or cow milk yogurt (20.20 mg/dl). During storage at 4 C free amino acids in goat milk decreased by 20%, in cow milk they increased by 70% from values found in fresh yogurt. The proteinase activity in goat milk during elaboration of yogurt was from 2 to 3 hours after incubation, was then stable until 24 hours of storage at 4 C and then decreased. No change in the proteinase activity in cow milk yogurt was seen until 4 hours of incubation for experiments 1 and 2 and 3 hours in experiment 3. During the storage at 4 C, proteinase activity in cow milk yogurt was stable. Polyacrylamide gel electrophoresis of caseins failed to reveal marked changes during the manufacture of yogurt and storage after 22 days at 4 C. However, a scanning gel densitometer showed that the area corresponding to alpha-casein decreased by 22% and for beta-casein 13.32% during the elaboration of goat milk yogurt. After 22 days of storage at 4 C, the alpha-casein fraction decreased an additional 14%, but no change was observed in beta-casein. The area corresponding to alpha- and beta-caseins decreased by 7.16 and 14.48% respectively during yogurt manufacture and an additional 6.74 and 8% during storage of cow milk yogurt. The overall results found in this study suggest that the metabolic activity of S. thermophilus and L. bulgaricus is greater in goat milk than in cow milk during elaboration and storage of yogurt.

The present investigation was undertaken to characterize a number of strains of Lactobacillus bulgaricus and Streptococcus thermophilus intended for use by a commercial starter supply company. Thorough characterization of each culture was required in order to combine compatible strains so that their usefulness in Mozzarella cheese manufacture would be maximized. In this regard, cocci were assayed for formate and carbon dioxide production, rods for proteolysis, and both types for salt and phosphate tolerance as well as rate of acid production. In addition, certain combinations of cocci and rods were assayed as mixtures for these characteristics. Analyses of the various strains of lactobacilli and S. thermophilus were performed. Proteolysis, as determined by the Church method, for the rods (L. bulgaricus , L. helveticus and L. lactis ) varied from as low as 11.3 to as high as 34.7 mM when incubated for six hours. Proteolysis analyses for S. thermophilus also revealed a wide range of values from a low of 18.5 to a high of 46.4 mM. However, when strains were incubated for 16 hours, rods were shown to be nearly twice as proteolytic as cocci. When mixed cultures were tested for proteolysis, results were dependent on strain synergism. Values ranged from a low of 5.1 mM to 70.5 mM in mixed cultures. Various strains of S. thermophilus and mixed cultures were assayed for formate production. The S. thermophilus strain values were from a low of 4.2 to as high as 20.3 mg/L. Formate production in mixed cultures varied from traces of formate in one culture to quantities two and a half times that produced by the single S. thermophilus strains tested. Carbon dioxide production for the rods (L. bulgaricus , L. helveticus , and L. lactis ) varied from as low as 0 μl to as high as 376 μl when incubated for six hours at 44 °C. Carbon dioxide production for S. thermophilus ranged from 5 μl to 1259 μl. Also, S. thermophilus strains produced significantly more carbon dioxide than rod cultures, with only three exceptions. All mixtures were weak producers of carbon dioxide. Nine of 19 L. bulgaricus strains were stimulated by 0.1% phosphate ion and one strain showed stimulation at 0.3% phosphate ion. Thirteen of 19 strains were severely inhibited by 0.5% phosphate. Three of 10 L. helveticus strains were stimulated by 0.1% phosphate and another three strains were unaffected. All strains were inhibited by 0.5% phosphate. Two L. lactis strains showed stimulation at 0.1% phosphate, but inhibition at 0.3% and 0.5%. Acid production by strains of S. thermophilus was inhibited in 11 of 13 cases at 0.1% phosphate. The two strains not inhibited were slightly stimulated by 0.1% and 0.3% phosphate and unaffected by 0.5% phosphate. The mixed cultures of L. bulgaricus CR 14/ S. thermophilus 2 and L. bulgaricus Ql S. thermophilus 2 were not inhibited by 0.1% phosphate, but inhibition occurred at higher concentrations. Mixed cultures of L. bulgaricus C, E/ S. thermophilus 7, 12 and L. bulgaricus C, G/ S. thermophilus 4, 12 were stimulated by all three concentrations of phosphate salts tested. Sodium chloride produced toxic effects on the rods at concentrations ranging from 2.5% to 3.0%, and acid production was stimulated 7 of 32 strains by low salt concentrations(0.5%). In general, cocci were more sensitive to NaCl, with 6 of 13 strains showing sensitivity at 0.5%. Sensitivity to salt was a more gradual effect in the cocci as revealed by a gradual reduction in rate of acid production as NaCl concentrations increased. Mixed cultures were more tolerant to NaCl with no inhibition occurring at concentrations of 1.0%. Culture L. bulgaricus C, GIS. thermophilus 4, 7 were stimulated at concentrations through 1.5%. The synergistic properties of the mixed strains increased NaCl tolerance. / Graduation date: 1993

S. thermophilus est une bactérie lactique largement utilisé dans l'industrie laitière et, comme toute bactérie, doit s'adapter à des environnements variés tels que le lait, le yaourt et même le tractus digestif, après que le produit ait été ingéré. Les systèmes à deux composants (TCS) constituent un des mécanismes essentiels qu'utilisent les bactéries pour percevoir et s'adapter à des changements environnementaux. D'un point de vue structural, les TCS sont constitués de deux composants: un « senseur » ou protéine histidine kinase (HK) qui s'auto-phosphoryle en réponse à un stimulus puis transfère son groupement phosphate au « response regulator » (RR), le deuxième composant. Celui-ci se comporte alors le plus souvent comme un régulateur transcriptionnel permettant une réponse physiologique adaptée. Afin de mieux comprendre ces phénomènes de régulation impliqués dans la réponse aux changements environnementaux, nous avons étudié la contribution de chacun des 8 TCS de Streptococcus thermophilus LMD-9 à son adaptation dans le lait. Ainsi, des études transcriptionnelles effectuées sur des cultures en lait montrent que tous les RR sont exprimés, à des niveaux et profils d'expression différents. Nous avons noté en coculture avec Lactobacillus bulgaricus, le partenaire de Streptococcus thermophilus dans le yaourt, une induction de l'expression de 4 RR qui atteint, pour rr02 et rr09, un facteur 6. Nous avons construit par ailleurs des mutants négatifs pour 7 des 8 RR de S. thermophilus et montré l'essentialité de RR05, un orthologue de YycF chez B. subtilis ou de or de WalR chez S. aureus. Pour les 7 autres mutants RR, l'absence d'un seul gène rr n'impacte pas suffisamment la croissance du streptocoque en lait. Enfin, la détermination du régulon du TCS06 par des études post-génomiques a permis de montrer que ce système est impliqué dans la résistance à la bacitracine en modulant entre autres la voie de biosynthèse du polysaccharide à rhamnose (RGP). / The lactic acid bacterium Streptococcus thermophilus is widely used in the dairy industry and, as a food bacterium, has to cope with changing environments such as milk, yogurt as well as the digestive tract, after the product has been ingested. Two-component systems (TCS), typically composed of a sensor kinase (HK) that detects a stimulus and of a response regulator (RR) which acts as a transcriptional regulator, are among the most prevalent means for bacteria to adapt to changing environments via fine-tune gene expression. To get a more comprehensive view of the role of all two-component systems in S. thermophilus physiology, we have investigated the contribution of each S. thermophilus LMD-9 TCS to its fitness and adaptation to milk. Transcriptomic studies (RT-qPCR) and construction of negative mutants of the rr genes were performed for LMD-9 S. thermophilus strain. We have shown that all LMD-9 response regulators were expressed in milk, at different levels and with different profiles of expression during growth. In mixed culture with Lactobacillus bulgaricus, the S. thermophilus partner in yoghurt, the expression of four LMD-9 rr increased; for two of them, rr02 and rr09, the increase reached a factor 6. These results indicate that Lb. bulgaricus induces regulatory changes in S. thermophilus and that S. thermophilus is able to adapt to these changes by probable fine tuning regulations. We constructed negative mutants for 7 out of 8 LMD-9 RRs and we showed that RR05 -an ortholog of B. subtilis YycF or S. aureus WalR- was essential for the optimum growth of S. thermophilus. For the 7 other RR, the absence of a single rr gene was not sufficient to notably impact the growth of LMD-9 in milk. The determination of the TCS06 regulon by post-genomics shows that TCS06 is involved in bacitracin resistance through the modulation of the rhamnose polysaccharide pathway.

No description available.

Il est reconnu, depuis des années, que la flore intestinale par son équilibre complexe et dynamique joue un rôle essentiel dans la santé humaine. Une des stratégies les plus prometteuses pour la maintenir ou l’améliorer consiste à moduler le microbiome par l’utilisation de bactéries probiotiques ou de sucres prébiotiques. C’est dans ce contexte que s’inscrivent les études structurales et fonctionnelles d’α glucosidases bactériennes développées dans cette thèse. Ces enzymes hydrolysant les liaisons α-(1,4) glucosidiques sont classées, selon la base de données CAZy, dans les familles de glycoside hydrolases (GH) 4, 13, 31, 63, 97 et 122. Ces travaux de thèse, centrés sur trois α-glucosidases issues de Lactobacillus bulgaricus (11842aglu, GH31), Lactococcus lactis (1403aglu, GH13) et Shewanella sp. ANA-3 (SHWaglu, GH97), exposent la mise au point de leurs protocoles de surexpression et de purification. Ils présentent également des études bioinformatiques de 11842aglu et de 1403aglu, ainsi qu’une caractérisation enzymatique préliminaire de cette dernière. Une analyse structurale et fonctionnelle approfondie de SHWaglu a aussi été réalisée. La résolution, par cristallographie aux rayons X, des structures de SHWaglu seule, en complexe avec différents ligands et de mutants, a participé à enrichir les connaissances, jusqu’à présent peu étendues, sur les enzymes de la famille GH97. Ainsi, un motif structural conservé au sein de cette famille a notamment été mis en évidence. Par ailleurs, ces informations structurales combinées aux études enzymatiques ont permis de révéler des déterminants moléculaires de l’activité de cette α-glucosidase et, par conséquent, d’établir les relations structure-fonction-activité de cette enzyme. Ainsi, l’ensemble des données obtenues, couplé à des études d’ingénierie protéique, contribue à ouvrir de nouvelles perspectives industrielles, notamment en suggérant d’optimiser ou de conférer des activités enzymatiques modifiées dans certaines cibles de choix afin de leur faire synthétiser des sucres de type prébiotiques / It is now generally accepted that the gut flora with its complex and dynamic nature plays a vital role in human health. One of the most promising strategies for maintaining or improving health is to modulate the microbiome by the use of probiotics and prebiotics as food supplements. The structure/function/activity relationship studies of bacterial α-glucosidases described in this thesis have been performed within this context. These α-(1,4)-glucosidic bond hydrolyzing enzymes are classified, according to the CAZy database, into glycoside hydrolases families (GH) 4, 13, 31, 63, 97 and 122. This thesis work, has focused on three α-glucosidases from Lactobacillus bulgaricus (11842aglu, GH31), Lactococcus lactis (1403aglu, GH13) and Shewanella sp. ANA-3 (SHWaglu, GH97), and the development of their overexpression and purification protocols. It also presents a bioinformatics studies of 11842aglu and 1403aglu, as well as preliminary enzymatic characterization of the latter. As for SHWaglu, detailed structural and functional studies have been carried out. The crystal structures of SHWaglu in its native state, in complex with different ligands as well as site directed mutants have contributed to increase our knowledge on enzymes from the GH97 family which to date remains relatively limited. Notably, a conserved structural motif in this family has been identified. Overall, the structural- and enzymatic studies and analyses have revealed molecular-and structural determinants governing the activity and broad substrate specificity of this α-glucosidase which is adapted to cold temperatures. Apart from the insight gained from a fundamental research point of view, data described within this work, coupled with protein engineering studies may contribute to open up new industrial perspectives, in particular by suggesting optimized or altered enzyme activities in some attractive enzyme targets with the aim of synthesizing prebiotic compounds

S. thermophilus est une bactérie lactique largement utilisé dans l'industrie laitière et, comme toute bactérie, doit s'adapter à des environnements variés tels que le lait, le yaourt et même le tractus digestif, après que le produit ait été ingéré. Les systèmes à deux composants (TCS) constituent un des mécanismes essentiels qu'utilisent les bactéries pour percevoir et s'adapter à des changements environnementaux. D'un point de vue structural, les TCS sont constitués de deux composants: un " senseur " ou protéine histidine kinase (HK) qui s'auto-phosphoryle en réponse à un stimulus puis transfère son groupement phosphate au " response regulator " (RR), le deuxième composant. Celui-ci se comporte alors le plus souvent comme un régulateur transcriptionnel permettant une réponse physiologique adaptée. Afin de mieux comprendre ces phénomènes de régulation impliqués dans la réponse aux changements environnementaux, nous avons étudié la contribution de chacun des 8 TCS de Streptococcus thermophilus LMD-9 à son adaptation dans le lait. Ainsi, des études transcriptionnelles effectuées sur des cultures en lait montrent que tous les RR sont exprimés, à des niveaux et profils d'expression différents. Nous avons noté en coculture avec Lactobacillus bulgaricus, le partenaire de Streptococcus thermophilus dans le yaourt, une induction de l'expression de 4 RR qui atteint, pour rr02 et rr09, un facteur 6. Nous avons construit par ailleurs des mutants négatifs pour 7 des 8 RR de S. thermophilus et montré l'essentialité de RR05, un orthologue de YycF chez B. subtilis ou de or de WalR chez S. aureus. Pour les 7 autres mutants RR, l'absence d'un seul gène rr n'impacte pas suffisamment la croissance du streptocoque en lait. Enfin, la détermination du régulon du TCS06 par des études post-génomiques a permis de montrer que ce système est impliqué dans la résistance à la bacitracine en modulant entre autres la voie de biosynthèse du polysaccharide à rhamnose (RGP).