Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33Å MgADP heterotropic interaction.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-10-05T12:37:57Z No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) / Made available in DSpace on 2016-10-05T12:37:57Z (GMT). No. of bitstreams: 1 2011 - Simone de Souza Fernandes.pdf: 745580 bytes, checksum: 7836d760ce3ea36d3e5602714715cd42 (MD5) Previous issue date: 2011-05-27 / Yogurt is a fermented milk resulting from a mutual interaction between the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. For yoghurt quality assurance, the cell numbers of each microorganism, should not be less than 1x107per gram, therefore a relative ratio lactobacilli and streptococci should be 1:1, although a 1:2 ratio is also accepted. During storage of yoghurt exposed for sale, post-acidification may occur, changing the ratio of the two microorganisms. The objectives of this research were to evaluate yoghurts of four different manufacturers named A, B, C and D, in relation to the number and balance between streptococii and lactobacilli during the storage and its relation with acidity and pH. In this way, yoghurts with up to 20 days of manufacturing (band I) and more than 20 days (band II) were evaluated. Better recovery of L. delbruckii subsp. bulgaricus and S. thermophilus was observed with LB and M17 media respectively, which were used to determine the relative ratio of the two microorganisms. An imbalance in the lactobacilli numbers which were lower than that of streptococci was verified and considered inadequate, in two out of four commercial brands. In yoghurts from the manufacturer A, there was a significant reduction in the number of lactobacilli from band I to band II leading to an increase in the relative ratio of streptococii to lactobacilli. There was no significant difference in the counts of streptococci or bacilli from one band to another with the other three brands of yoghurts. The acidity of yoghurts from manufacturer D revealed significantly higher (P< 0.05) than the others, although it did not result in an increased pH reduction. All samples attended the legislation in relation to total lactic acid bacteria counts, acidity, pH and mould and yeast count / Iogurte ? um leite fermentado resultante de uma intera??o microbiana mutualista entre as bact?rias l?cticas Streptococcus thermophilus e Lactobacillus delbrueckii subsp. bulgaricus. Para que a qualidade do iogurte seja garantida o n?mero de c?lulas destes dois microrganismos individualmente, n?o deve ser inferior a 1x107 por grama, portanto uma propor??o relativa de lactobacilos e estreptococos de 1:1 sendo a propor??o 1:2 tamb?m aceita. Durante o armazenamento dos iogurtes expostos para venda podem ocorrer p?s-acidifica??o e modifica??o na propor??o dos dois microrganismos. Os objetivos desta pesquisa foram avaliar iogurtes de quatro diferentes fabricantes as quais foram denominados A, B, C, D quanto ao n?mero e equil?brio entre as duas bact?rias l?cticas durante o armazenamento, e ao mesmo tempo determinar as caracter?sticas f?sico-quimicas (pH e acidez) e contagem de bolores e leveduras. Para tanto, adotou-se duas faixas de avalia??o, faixa I (at? 20 dias de fabrica??o) e faixa II (ap?s 20 dias). Foi observada melhor recupera??o L. delbruckii subsp. bulgaricus e S. thermophilus, respectivamente com os meios LB e M17 que foram usados para determina??o da propor??o relativa dos dois microrganismos. Foi verificado um desequil?brio no n?mero de lactobacilos que foi inferior ao de cocos e, considerado inadequado, em duas das quatro marcas comerciais. Nos iogurtes da marca A houve redu??o significativa no n?mero de lactobacilos da faixa I para faixa II levando a um aumento na propor??o de cocos relativa ao de bacilos. Nos iogurtes dos tr?s outros fabricantes n?o houve diferen?a significativa nas contagens de cocos ou de lactobacilos de uma faixa para outra. Tamb?m n?o foi observada diferen?a significativa de acidez e pH relacionados ao tempo de prazo comercial nas faixas I e II nas quatro marcas de iogurte analisadas. A acidez dos iogurtes do fabricante D foi significativamente mais elevada (P<0,05) que a dos demais, embora n?o tenha resultado em maior redu??o de pH nestes produtos. Todas as amostras analisadas estavam em conformidade com a legisla??o vigente no que se refere ao m?nimo exigido de bact?rias l?ticas totais que ? de 1x107 UFC/mL. Tamb?m estavam em conformidade com rela??o ? acidez, pH e contagem de bolores e leveduras. Palavras-chave: leite

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).

Yogurt is a characteristic fermented dairy product of Turkey and Bulgaria and its popularity has been increasing all over the world. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) are used together as starter culture in production of yogurt. The objective of this study was to isolate and characterize yogurt cultures from traditionally produced yogurts (i.e. produced without using commercial starter cultures) and to search the genotypic diversity within traditional S. thermophilus isolates. Yogurt cultures were isolated from traditionally produced yogurts collected from different regions of Turkey and identified biochemically. Acidification ability of the isolates was examined and the cultures giving best acidifying rates were further subjected to a selection in terms of their acetaldehyde production ability. Then, phage resistance and proteolytic activity of chosen isolates were tested. Finally, twenty-five L. bulgaricus and twenty-two S. thermophilus isolates were selected as cultures having best technological properties. Furthermore, subtyping studies were carried out to indicate strain diversity among isolates. S. thermophilus was selected as target organism for subtyping in this study. Clustered regularly interspaced short palindromic repeats (CRISPR) loci are highly polymorphic genetic regions, which are composed of partially palindromic direct repeats interspaced by sequences called spacers. In order to characterize S. thermophilus isolates genotypically, CRISPR1 locus of the isolates were analyzed. Additionally, nineteen isolates selected after CRISPR1 analysis were characterized using multilocus sequence typing (MLST). This provided to compare CRISPR1 analysis with MLST as a typing method. According to CRISPR1 analysis S. thermophilus isolates were grouped into 6 main clusters with a total of 15 sub-clusters. MLST results demonstrated an evolutionary relationship among these strains compatible with that derived from the CRISPR1 analysis.

Zvýšená konzumace fermentovaných mléčných výrobků v posledních letech vede k zavádění nových výrobků na trh. Funkční a probiotické produkty jsou v dnešní době také hojně rozšířeny. Cílem diplomové práce bylo posoudit mikrobiální kvalitu komerčních jogurtů a nefarmakologických probiotických výrobků. Bylo zkoumáno 24 vzorků jogurtů a 8 vzorků probiotických mléčných výrobků. Pozornornost byla zaměřena zvláště na kvantifikaci mikroorganismů v jednotlivých výrobcích a na sledování viability buněk. Většina mléčných produktů obsahovala počty odpovídající minimální požadované hodnotě 1x107cfu/ml. Nicméně byly objeveny produkty, které neobsahovaly živé bakterie nebo obsahovaly jen jejich menší množství. To platí zejména pro běžné jogurty. Probiotické mléčné výrobky v zásadě obsahovaly větší množství bakterií než jogurty, protože je u nich navíc požadováno 1x106cfu/ml minimálního množství specifických bakterií jiných než jogurtových startovacích kultur. Isoláty z fermentovaných mléčných výrobků byly identifikovány jako Streptococcus thermophilus and Lactobacillus spp. Isoláty byly poté rozlišeny na jednotlivé kmeny pomocí RAPD použitím tří různých primerů. Byla zjištěna velká různorodost mezi kmeny rodu Lactobacillus spp. používaných různými podniky.