This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

This study was undertaken to characterize the role and pathway for assimilation of the intracellular carbon storage compound, poly-beta-hydroxybutyrate (PHB), in Rhizobium (Sinorhizobium) meliloti. Mutants unable to utilize the degradation intermediates, 3-hydroxybutyrate (HB) and/or acetoacetate (AA) were characterized. A mutant unable to utilize HB (Hbu-) while retaining the ability to utilize AA was found to be deficient in 3-hydroxybutyrate dehydrogenase (Bdh) activity. The bdhA mutant showed no symbiotic defects in association with alfalfa plants. However, when co-inoculated with the wild type, the mutant showed significantly reduced competitiveness. A more severe competition defect was observed for a PHB synthesis mutant (phaC). Both these mutants also showed reduced competitiveness when subjected to multiple cycles of subculturing through alternating carbon-rich and carbon-poor media, with the phaC mutant showing a greater loss in competitiveness. The results indicate that the ability to efficiently deposit and utilize cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability. / The gene encoding Bdh (bdhA) was isolated and sequenced. Two transcription start sites, S1 and S2 were identified but no known consensus promoter sequences were identified upstream of either start site. A sigma 54 consensus binding sequence was found to be located between S1 and S2 but no corresponding transcript was detected. Transcriptional bdhA-lacZ fusion studies indicated that gene expression was growth-phase associated. The bdhA gene from Rhizobium sp. NGR234 was also isolated and characterized and found to be highly homologous to the R. meliloti bdhA sequence. Unlike R. meliloti , NGR234 is able to accumulate PHB during symbiosis. An NGR234 bdhA mutant showed symbiotic defects on Leucaena but not on Tephrosia, Macroptilium or Vigna host plants, indicating that the phenotype was host-dependent. / Mutations that suppress the Hbu- phenotype without restoring Bdh activity were identified, indicating the existence of a Bdh-independent pathway for HB utilization. These mutations mapped to the age-1 locus, which causes enhanced growth rate on HB and AA minimal media. Introduction of plasmid-borne multiple copies of a gene encoding acetoacetyl-CoA synthetase (acsA) into the bdhA mutant also results in suppression of the Hbu- phenotype. A possible mechanism of suppression involving direct activation of HB to 3-hydroxybutyryl-CoA, followed by reduction to acetoacetyl-CoA by the NADP-acetoacetyl-CoA reductase (encoded by phaB) was investigated. A strain carrying the triple mutations, age-1::Tn5-Tp, bdhA ::Tn5 and phaB::OSmSp retained the ability to utilize HB, indicating that the bypass mechanism does not involve NADP-acetoacetyl-CoA reductase. / The phaB mutant does not accumulate PHB or utilize HB or AA. Furthermore, colonies of the phaB and phaC mutants exhibit non-mucoid phenotype on yeast extract mannitol agar. The observation that a R. meliloti exoS null mutant is also Hbu- provides further support for a link between PHB and exopolysaccharide synthesis. Since ExoS is a positive regulator of succinoglycan biosynthesis it is hypothesized that regulation of succinoglycan synthesis by ExoS requires PHB synthesis.

This research followed acetate carbon (C) uptake, metabolism, and fate through a typical modern Kraft pulp and paper mill AS system. Freshly collected mill biomass (AS floc suspensions) was placed under conditions representing four key phases of AS biotreatment: (1) initial acetate uptake by aerated starved AS; (2) ongoing acetate uptake; (3) aerobic metabolism of acetate-loaded AS in acetate-stripped effluent; and (4) anaerobic, settled biomass metabolism. Conditions mimicked the mill system as closely as possible. Acetate carbon uptake kinetics and conversion to CO2, growth products, PHB, and secreted metabolites in each of the four phases were measured. The role of PHB synthesis in the initial stripping of acetate from mill effluent and the PHB production potential of this mill AS were also investigated. / Results showed that acetate was rapidly taken up by high-affinity systems in the AS. During the initial exposure of mill-starved AS, acetate greatly stimulated AS-O2 uptake, and was quickly converted to PHB and CO 2. Upon depletion of available effluent acetate, as occurs in the downstream sections of the aeration tank, O2-uptake rates decreased and the acetate-C stored in AS-PHB was slowly released as CO2, and partly used for growth. Under secondary clarifier-like anaerobic conditions, the AS released virtually no CO2. However, substantial amounts of PHB were used for growth under anaerobic conditions and a small proportion of the original acetate C exited the cells as organic acids.

Orientadores: Wanderley Dias da Silveira, Eliana Gertrudes de Macedo Lemos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:23:49Z (GMT). No. of bitstreams: 1 Paganelli_FernandaLaroza_M.pdf: 1101767 bytes, checksum: e8882c8b6ec40ea617dd997584b239c3 (MD5) Previous issue date: 2009 / Resumo: Polihidroxialcanoatos (PHAs) são polímeros de hidroxialcarioatos produzidos, e acumulados, intracelularmente, como fonte de carbono e/ou outros materiais energéticos, em vários microorganismos. Freqüentemente, o acúmulo dos PHAs ocorrem em condições abaixo do limite nutricional de elementos como N, P, S, O ou Mg e excesso de carbono, podendo representar até 80% da massa seca total da célula. Mais de 300 diferentes microorganismos podem sintetizar e acumular PHAs. O polihidroxibutirato (PHB) é o mais conhecido dentre os polímeros bacterianos biodegradáveis denominados polihidroxialcanoatos. Por ter propriedades semelhantes ao polipropileno, o PHB pode ser usado na fabricação do plástico biodegradável. Além da busca por maior produção de tal polímero, pouco é conhecido sobre seu papel biológico, em especial nos rizóbios. Estudos revelaram que há variação na capacidade da produção e acúmulo de PHB nessas bactérias quando em simbiose, dependendo da espécie em questão e das condições de cultivo das mesmas, observando-se bactérias incapazes de acumular PHB quando bacterióides, como é o caso do Rhizobium meliloti, ou capazes de produzir e acumular PHB, nessas condições, como é o caso de Rhizobium etli, Bradyrhizobium japonicum e Bradyrhizobium elkanii. Desta forma, este trabalho teve por objetivo a identificação dos genes responsáveis pela síntese de PHB em Bradyrhizobium elkanii, clonagem e expressão dos mesmos em Escherichia coli, uma vez que esta bactéria é bem conhecida como ferramenta molecular e se multiplica rapidamente, podendo atingir uma alta produção do polímero esperado, em um curto período de tempo. Além disso, objetivou-se, também, aumentar a produção de PHB em B. elkanii através de mutações aleatórias (através da inserção do transposon TnphoA), já que esta é uma bactéria naturalmente produtora de PHB. Para isso, os genes phbA, phbB e phbC foram isolados através da técnica de PCR, amplificando-se os genes inteiros. Estes foram clonados em vetores de expressão tipo pET (NOVAGEN), sendo os genes phbA e phbB clonados em "operon " em um mesmo vetor e o gene phbC clonado separadamente. A expressão dos genes foi analisada, bem como sua capacidade de produzir PHB. Os mutantes de B. eíkanii obtidos através da inserção do transposon TnphoA foram analisados com o uso do corante Sudan Black, procurando-se selecionar linhagens maiores produtoras de PHB. A produção dos mutantes selecionados foi, posteriormente, analisada por cromatografia gasosa. Observou-se que a linhagem de E.coli com os três genes clonados teve a capacidade de produzir PHB, porém com baixa eficiência. Já os mutantes aleatórios de B. elkanii apresentação diferentes acúmulos em relação ao selvagem, com destaque para o MUT33 que teve 72% da sua massa seca acumulada na forma de PHB, enquanto o selvagem acumulou 51 % de PHB, nas mesmas condições. / Abstract: Polyhydroxyalkanoates (PHAs) are polymers of hydroxyalkanoate acids, produced and accumulated intracellularly as a source of carbon and energy storage material, in prokaryotic cells. Often, the PHAs accumulation occurs in conditions when the carbon source is in excess but one or several other nutrients are limited, and may represent up to 80% of the cell dry weight. More than 300 different microorganisms can synthesize and accumulate PHAs. The polyhydroxybutyrate (PHB) is the most studied polymer among the bacterial biodegradable polymers (PHAs). By having properties similar to polypropylene, PHB can be used in the manufacture of biodegradable plastic. On the search for greater production of such polymer, little is known about its biological role, especially in the genus Rhizobium. Studies have shown that there is a variation in the PHB capacity production and accumulation when these bacteria are in symbiosis. Depending on the species and cultivation conditions it has been observed either incapacity of PHB production and accumulation, when Bacteroides, as Rhizobium meliloti; or capacity and accumulation of PHB under bacteroides fase, as Rhizobium etli, Bradyrhizobium japonicum and Bradyrhizobium elkanii. Therefore, the present work aimed the identification, cloning and expression, in Escherichia coli, of responsible genes for PHB synthesis in Bradyrhizobium elkanii. Moreover, it was also expected to increase the production of PHB in B. elkanii, through random mutations (insertion of the TnphoA transposon), since B. elkanii is a good natural producer of PHB. For this, the entier phbA, phbB and phbC genes were isolated by PCR. Those genes were cloned in expression vectors such pET (NOVAGEN), where phbA and phbB genes were cloned in operon, in a single vector, whereas phbC gene was cloned separately, in another vector. The expression of those genes was analyzed, as well as its ability to produce PHB. Mutants B. elkanii, obtained by insertion of the TnphoA transposon, were analyzed using the dye Sudan Black, in order to select different strains that might produce higher quantities of PHB. The production of PHB by mutants was then analyzed by gas chromatography. It was observed that the E. coli with the three cloned genes had the ability to produce PHB, but with low efficiency. The B. elkanii random mutants show different accumulation compared to the wild, especially MUT33 that had 72% of its dry mass accumulated in the form of PHB, while the wild 51% of accumulated PHB under the same conditions. / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

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Estratégias para minimizar o efeito do balanço energético negativo (BEN) em vacas leiteiras vêm sendo estudadas constantemente no passar dos anos, pois o período de transição é caracterizado por grande estresse metabólico. O objetivo deste estudo foi avaliar parâmetros metabólicos, produtivos e reprodutivos em vacas da raça Holandesa após a aplicação de drench no pós-parto imediato e 24 h após, como alternativa para minimizar o BEN e proporcionar melhor desempenho produtivo e reprodutivo aos animais. Foram utilizadas 129 vacas, 64 no grupo 1 (1ª e 2ª lactação) e 65 no grupo 2 (≥ 3ª lactação), distribuídas em: verão (n= 63) e inverno (n= 66) e região da Serra Gaúcha (Fazenda A, n= 48) e Planato Médio (Fazenda B, n= 81). Foram coletadas amostras de sangue para obtenção de soro e plasma e determinação de β-hidroxibutirato (BHB) com sistema Abbot Precision Xtra. Foi medido o escore de condição corporal e a temperatura corporal, e realizado acompanhamento de controle leiteiro para avaliar produção e composição de leite. Foi realizada ultrassonografia ovariana para avaliação de ciclicidade ovárica e primeira ovulação pós-parto. O tratamento drench provocou uma tendência a diminuir os valores de BHB sérico no período experimental, sendo que, no período de verão essa diminuição foi significativa na Fazenda A. Na Fazenda B, nas semanas 3 e 4, o tratamento com drench aumentou significativamente a produção de leite nos grupos 1 e 2. A exceção foi no grupo 1 no período de verão, onde o efeito foi negativo. Também na Fazenda B, o tratamento com drench teve efeito positivo no percentual de gordura nas vacas do grupo 1 e efeito negativo nas vacas do grupo 2. Parâmetros reprodutivos não tiveram interações significativas. Em conclusão, o tratamento com drench preveniu a ocorrência de cetose subclínica, aumentou a produção leiteira no inverno e no verão em vacas de 3 ou mais lactações e também aumentou o percentual de gordura no leite em vacas de uma ou duas lactações. / Strategies to minimize the effect of negative energy balance (NEB) in dairy cows are being studied constantly over the years, since the transition period is characterized by excessive metabolic stress. The present study had the objective to evaluate metabolic, productive and reproductive parameters in dairy cows after application of drench in the immediate postpartum period and 24 hours after as an alternative to minimize NEB providing better productive and reproductive performance of animals. 129 cows, 64 were used in group 1 (1st and 2nd lactation ) and 65 in group 2 ( ≥ 3rd lactation ), distributed in: summer ( n = 63 ) and winter ( n = 66 ) and Serra Gaucha (Farm A, n = 48) and Planato Médio (Farm B, n = 81 ) . Blood samples were collected to obtain serum and plasma and determination of β-hydroxybutyrate (BHB) with the Abbott Precision Xtra system . Body condition score and body temperature, were also obtained by milk information system to evaluate production and milk composition was measured. Ovarian ultrasonography was performed for evaluation of cyclicity and days to first ovulation postpartum. The drench treatment had a trend towards significance in the reduction of mean serum BHB during the experimental period, whereas in the summer period this decrease was significant in farm A. On farm B at weeks 3 and 4, drench significantly increased milk production in groups 1 and 2. Except for group 1 during the summer, where the effect was negative. Also on farm B, drench had a positive effect on fat percentage in cows of group 1 and negative effect on cows in group 2. Reproductive parameters showed no significant interactions. In conclusion, drench prevented the occurrence of subclinical ketosis, increased milk production during winter and during summer in cows of third or more lactation and also increased of milk fat percentage in cows of first and second lactations.

Estratégias para minimizar o efeito do balanço energético negativo (BEN) em vacas leiteiras vêm sendo estudadas constantemente no passar dos anos, pois o período de transição é caracterizado por grande estresse metabólico. O objetivo deste estudo foi avaliar parâmetros metabólicos, produtivos e reprodutivos em vacas da raça Holandesa após a aplicação de drench no pós-parto imediato e 24 h após, como alternativa para minimizar o BEN e proporcionar melhor desempenho produtivo e reprodutivo aos animais. Foram utilizadas 129 vacas, 64 no grupo 1 (1ª e 2ª lactação) e 65 no grupo 2 (≥ 3ª lactação), distribuídas em: verão (n= 63) e inverno (n= 66) e região da Serra Gaúcha (Fazenda A, n= 48) e Planato Médio (Fazenda B, n= 81). Foram coletadas amostras de sangue para obtenção de soro e plasma e determinação de β-hidroxibutirato (BHB) com sistema Abbot Precision Xtra. Foi medido o escore de condição corporal e a temperatura corporal, e realizado acompanhamento de controle leiteiro para avaliar produção e composição de leite. Foi realizada ultrassonografia ovariana para avaliação de ciclicidade ovárica e primeira ovulação pós-parto. O tratamento drench provocou uma tendência a diminuir os valores de BHB sérico no período experimental, sendo que, no período de verão essa diminuição foi significativa na Fazenda A. Na Fazenda B, nas semanas 3 e 4, o tratamento com drench aumentou significativamente a produção de leite nos grupos 1 e 2. A exceção foi no grupo 1 no período de verão, onde o efeito foi negativo. Também na Fazenda B, o tratamento com drench teve efeito positivo no percentual de gordura nas vacas do grupo 1 e efeito negativo nas vacas do grupo 2. Parâmetros reprodutivos não tiveram interações significativas. Em conclusão, o tratamento com drench preveniu a ocorrência de cetose subclínica, aumentou a produção leiteira no inverno e no verão em vacas de 3 ou mais lactações e também aumentou o percentual de gordura no leite em vacas de uma ou duas lactações. / Strategies to minimize the effect of negative energy balance (NEB) in dairy cows are being studied constantly over the years, since the transition period is characterized by excessive metabolic stress. The present study had the objective to evaluate metabolic, productive and reproductive parameters in dairy cows after application of drench in the immediate postpartum period and 24 hours after as an alternative to minimize NEB providing better productive and reproductive performance of animals. 129 cows, 64 were used in group 1 (1st and 2nd lactation ) and 65 in group 2 ( ≥ 3rd lactation ), distributed in: summer ( n = 63 ) and winter ( n = 66 ) and Serra Gaucha (Farm A, n = 48) and Planato Médio (Farm B, n = 81 ) . Blood samples were collected to obtain serum and plasma and determination of β-hydroxybutyrate (BHB) with the Abbott Precision Xtra system . Body condition score and body temperature, were also obtained by milk information system to evaluate production and milk composition was measured. Ovarian ultrasonography was performed for evaluation of cyclicity and days to first ovulation postpartum. The drench treatment had a trend towards significance in the reduction of mean serum BHB during the experimental period, whereas in the summer period this decrease was significant in farm A. On farm B at weeks 3 and 4, drench significantly increased milk production in groups 1 and 2. Except for group 1 during the summer, where the effect was negative. Also on farm B, drench had a positive effect on fat percentage in cows of group 1 and negative effect on cows in group 2. Reproductive parameters showed no significant interactions. In conclusion, drench prevented the occurrence of subclinical ketosis, increased milk production during winter and during summer in cows of third or more lactation and also increased of milk fat percentage in cows of first and second lactations.

Estratégias para minimizar o efeito do balanço energético negativo (BEN) em vacas leiteiras vêm sendo estudadas constantemente no passar dos anos, pois o período de transição é caracterizado por grande estresse metabólico. O objetivo deste estudo foi avaliar parâmetros metabólicos, produtivos e reprodutivos em vacas da raça Holandesa após a aplicação de drench no pós-parto imediato e 24 h após, como alternativa para minimizar o BEN e proporcionar melhor desempenho produtivo e reprodutivo aos animais. Foram utilizadas 129 vacas, 64 no grupo 1 (1ª e 2ª lactação) e 65 no grupo 2 (≥ 3ª lactação), distribuídas em: verão (n= 63) e inverno (n= 66) e região da Serra Gaúcha (Fazenda A, n= 48) e Planato Médio (Fazenda B, n= 81). Foram coletadas amostras de sangue para obtenção de soro e plasma e determinação de β-hidroxibutirato (BHB) com sistema Abbot Precision Xtra. Foi medido o escore de condição corporal e a temperatura corporal, e realizado acompanhamento de controle leiteiro para avaliar produção e composição de leite. Foi realizada ultrassonografia ovariana para avaliação de ciclicidade ovárica e primeira ovulação pós-parto. O tratamento drench provocou uma tendência a diminuir os valores de BHB sérico no período experimental, sendo que, no período de verão essa diminuição foi significativa na Fazenda A. Na Fazenda B, nas semanas 3 e 4, o tratamento com drench aumentou significativamente a produção de leite nos grupos 1 e 2. A exceção foi no grupo 1 no período de verão, onde o efeito foi negativo. Também na Fazenda B, o tratamento com drench teve efeito positivo no percentual de gordura nas vacas do grupo 1 e efeito negativo nas vacas do grupo 2. Parâmetros reprodutivos não tiveram interações significativas. Em conclusão, o tratamento com drench preveniu a ocorrência de cetose subclínica, aumentou a produção leiteira no inverno e no verão em vacas de 3 ou mais lactações e também aumentou o percentual de gordura no leite em vacas de uma ou duas lactações. / Strategies to minimize the effect of negative energy balance (NEB) in dairy cows are being studied constantly over the years, since the transition period is characterized by excessive metabolic stress. The present study had the objective to evaluate metabolic, productive and reproductive parameters in dairy cows after application of drench in the immediate postpartum period and 24 hours after as an alternative to minimize NEB providing better productive and reproductive performance of animals. 129 cows, 64 were used in group 1 (1st and 2nd lactation ) and 65 in group 2 ( ≥ 3rd lactation ), distributed in: summer ( n = 63 ) and winter ( n = 66 ) and Serra Gaucha (Farm A, n = 48) and Planato Médio (Farm B, n = 81 ) . Blood samples were collected to obtain serum and plasma and determination of β-hydroxybutyrate (BHB) with the Abbott Precision Xtra system . Body condition score and body temperature, were also obtained by milk information system to evaluate production and milk composition was measured. Ovarian ultrasonography was performed for evaluation of cyclicity and days to first ovulation postpartum. The drench treatment had a trend towards significance in the reduction of mean serum BHB during the experimental period, whereas in the summer period this decrease was significant in farm A. On farm B at weeks 3 and 4, drench significantly increased milk production in groups 1 and 2. Except for group 1 during the summer, where the effect was negative. Also on farm B, drench had a positive effect on fat percentage in cows of group 1 and negative effect on cows in group 2. Reproductive parameters showed no significant interactions. In conclusion, drench prevented the occurrence of subclinical ketosis, increased milk production during winter and during summer in cows of third or more lactation and also increased of milk fat percentage in cows of first and second lactations.