Contamination of perchlorate and chlorate in nature is primarily the result of various industrial processes. The microbial respiration of these oxyanions of chlorine plays a major role in reducing the society’s impact on the environment. The focus with this thesis is to investigate the oxygen-dependent regulation of key components involved in the chlorate respiration in the gram‑negative bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of the enzymes chlorate reductase and chlorite dismutase and results in the end products molecular oxygen and chloride ion. Up‑regulation of chlorite dismutase activity in the absence of oxygen is demonstrated to occur at the transcriptional level, with the participation of the transcriptional fumarate and nitrate reduction regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated at the transcriptional level with the possible involvement of additional regulating mechanisms as well. Interestingly, the corresponding chlorate reductase operon was found to be part of a polycistronic mRNA which also comprises the gene for a cytochrome c and a putative transcriptional regulator protein.

No description available.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Northern Australia and Southeast Asia. Melioidosis can present with acute, chronic and latent infections and can relapse several months or years after initial presentation. Currently not much is known about the ways in which B. pseudomallei can persist within the host, although it has been speculated that the ability to survive within an anaerobic environment will play some role. B. pseudomallei is able to survive anaerobically for extended periods of time but little is known about the molecular basis of anaerobic respiration in this pathogenic species. Bioinformatic analysis was used to determine the respiratory flexibility of both B. pseudomallei and B. thailandensis, identifying multiple genes required for aerobic and anaerobic respiration, and molybdopterin biosynthesis. Using B. thailandensis as a model organism a transposon mutant library was created in order to identify genes required for anaerobic respiration. From this library one transposon mutant was identified to have disrupted moeA, a gene required for the molybdopterin biosynthetic pathway. This B. thailandensis transposon mutant (CA01) was unable to respire anaerobically on nitrate, exhibiting a significant reduction in nitrate reductase activity, altered motility and biofilm formation, but did not affect virulence in Galleria mellonella. It was hypothesised that the reduction in nitrate reductase activity was contributing to the phenotypes exhibited by the B. thailandensis moeA transposon mutant. To determine whether this was true an in-frame narG deletion mutant was created in B. pseudomallei. Deletion of B. pseudomallei narG (ΔnarG) resulted in a significant reduction in nitrate reductase activity, anaerobic growth, motility and altered persister cell formation, and but did not affect virulence in G. mellonella or intracellular survival within J774A.1 murine macrophages. This study has highlighted the importance of anaerobic respiration in the survival of B. thailandensis and B. pseudomallei.

Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of the present work was to evaluate alternative methods to conventional (CA) and dynamic controlled atmosphere (DCA), in order to maintain the quality of 'Royal Gala' apples, particularly the physical disorders occurrence. So, we evaluated the effect of initial low oxygen stress in maintaining the quality of 'Royal Gala' apples stored in ultralow oxygen conditions (less than 0.8 kPa) and also the effect of low oxygen conditioning through the gradual decreasing of oxygen in the storage chambers, and comparison of these techniques by applying 1-MCP. Two experiments were established, one in 2010 and another in 2011. At the first, the treatments were as follows: [1] CA with 1.2 kPa O2 + 2.0 kPa CO2; [2] 1.0 kPa O2 + 2.0 kPa CO2; [3] 0.8 kPa O2 + 1.0 kPa CO2; [4] 0.6 kPa O2 + 1.0 kPa CO2 e [5] 0.5 kPa O2 + 1.0 kPa CO2. For each CA treatment fruits were divided into two lots, one of them received 1-MCP application and another don´t. Then the fruits of each were again divided into two lots, and a portion was subjected to an initial low oxygen stress (0.3 kPa for seven days) and the other was enclosed in CA chambers. For the second experiment were used the following treatments: [1] CA with 1.0 kPa O2 immediately installed; [2] 1.0 kPa O2 and conditioning for seven days; [3] 0.8 kPa O2 and conditioning for seven days; [4] 0.7 kPa O2 and conditioning for seven days; [5] 0.7 kPa O2 and conditioning for 14 days; [6] 0.7 kPa O2 and conditioning for 28 days; [7] 0.5 kPa O2 and conditioning for seven days; [8] 0.5 kPa O2 and conditioning for 14 days; [9] 0.5 kPa O2 and conditioning for 28 days; [10] 0.7 kPa O2 with conditioning and low oxygen stress; [11] 1.0 kPa O2 immediately installed, plus 1-MCP and [12] DCA with chlorophyll fluorescence. For all treatments the partial pressure of CO2 was 1.2 kPa. The initial low oxygen stress increases the occurrence of physiological disorders, and shouldn t recommended for use immediately after harvest, however, after one low O2 adaptation period of the fruit, this procedure helps to maintaining quality during storage in ultralow oxygen conditions. The gradual reduction of O2 for approximately one month, allows storage in ultralow oxygen conditions and maintaining fruit quality better than the DCA. 1-MCP application offers low efficiency when the O2 concentration in the storage chambers are extremely low, or when the fruits presented an advanced maturity stage. / O objetivo do presente trabalho foi avaliar métodos alternativos à atmosfera controlada convencional (AC) e dinâmica (ACD), na manutenção da qualidade de maçãs Royal Gala , em especial a ocorrência de distúrbios fisiológicos. Para tanto, avaliou-se o efeito do estresse inicial com baixo O2 na manutenção da qualidade de maçãs armazenadas em condições ultrabaixas de oxigênio (menor que 0,8 kPa) e também o efeito do condicionamento ao baixo O2, por meio da redução gradativa do oxigênio nas câmaras de armazenamento, além de comparação destas técnicas com a aplicação de 1-MCP. Foram instalados dois experimento, um no ano de 2010 e outro em 2011. No primeiro, os tratamentos foram os seguintes: [1] AC com 1,2 kPa O2 + 2,0 kPa CO2; [2] 1,0 kPa O2 + 2,0 kPa CO2; [3] 0,8 kPa O2 + 1,0 kPa CO2; [4] 0,6 kPa O2 + 1,0 kPa CO2 e [5] 0,5 kPa O2 + 1,0 kPa CO2. Para cada condição de AC os frutos foram divididos em duas subamostras, sendo que uma recebeu aplicação de 1-MCP e outra não. Em seguida, os frutos foram novamente divididos em dois lotes, sendo que uma parte foi submetida a um estresse inicial por baixo O2 (sete dias em 0,3 kPa) e a outra foi acondicionada nas minicâmaras de AC. Para o segundo experimento utilizou-se os seguintes tratamentos: [1] AC com 1,0 kPa O2 e instalação imediata; [2] 1,0 kPa O2 e condicionamento de sete dias; [3] 0,8 kPa O2 e condicionamento de sete dias; [4] 0,7 kPa O2 e condicionamento de sete dias; [5] 0,7 kPa O2 e condicionamento de 14 dias; [6] 0,7 kPa O2 e condicionamento de 28 dias; [7] 0,5 kPa O2 e condicionamento de sete dias; [8] 0,5 kPa O2 e condicionamento de 14 dias; [9] 0,5 kPa O2 e condicionamento de 28 dias; [10] 0,7 kPa O2 com condicionamento e estresse por baixo O2; [11] 1,0 kPa O2 com instalação imediata, mais 1-MCP e [12] ACD com fluorescência de clorofilas. Para todos os tratamentos a pressão parcial de CO2 foi de 1,2 kPa. O estresse inicial por baixo O2 aumenta a ocorrência de distúrbios fisiológicos, não sendo recomendada a sua utilização logo após a colheita, no entanto, após um período de adaptação dos frutos ao baixo O2, mantém a qualidade durante o armazenamento em condições ultrabaixas de oxigênio. A redução gradativa de O2 durante aproximadamente um mês, permite o armazenamento em condições ultrabaixas de O2, com resultados superiores a ACD na manutenção na qualidade dos frutos. A aplicação de 1-MCP apresenta pouca eficiência quando as concentrações de O2 nas câmaras de armazenamento são extremamente baixas, ou quando os frutos apresentam um estádio de maturação avançado.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of this work was to evaluate the effect of post harvest ethanol or acetaldehyde application and to compare them with consolidated storage techniques on the quality maintenance of Royal Gala‟ apples. Also, we aimed to evaluate the effect of controlled atmosphere condition with ultra low oxygen partial pressures on the post storage quality maintenance of Royal Gala‟ apples. To reach such aims, three experiments were performed during 2008 and 2009. In the first experiment, carried out in 2008, the following treatments were evaluated: [1] control; [2] application of 0.5mL of ethanol month-1 kg-1; [3] application of 0.25mL acetaldehyde month-1 kg-1; [4] application of 625 ηL L-1 1-MCP; and [5] ethylene scrubbing (<0.02μL L-1). All fruits from the evaluated treatments were stored in CA with 1.0kPa O2 and 2.0kPa CO2. In the second experiment, carried out in 2009, the treatments were constituted in the following way: [1] control; [2] low relative air humidity (LRH); [3] LRH and application of 0.3mL of ethanol month-1 kg-1; and [4] application of 0.3mL of ethanol month-1 kg-1. All treatments were stored in CA with 1.2kPa O2 associated with 2.5kPa CO2. In the two experiments the fruits were stored at 0.5°C. In the third experiment, carried out in 2008, five CA conditions were evaluated: [1] 1.0kPa O2 and 2.0kPa CO2; [2] 0.8kPa O2 and 1.5kPa CO2; [3] 0.8kPa O2 and 1.0kPa CO2; [4] 0.6kPa O2 and 1.5kPa CO2; and [5] 0.6kPa O2 and 1.0kPa CO2. All conditions were stored in 0.0°C, 0.5°C and 1.0°C resulting in a factorial experiment. The application of 0.5mL ethanol month-1 kg-1 or 0.25mL acetaldehyde month-1 kg-1 cause increase in mealiness, flesh browning and rottenness incidence and decrease flesh firmness, resulting in decrease in the quality maintenance when compared with the application of 1-MCP and ethylene scrubbing. However, 0.3mL ethanol month-1 kg-1, performed on second experiment, resulted in higher flesh firmness retention, lower ACC oxidase activity and lower ethylene production. The lower relative air humidity is not efficient in maintain apple quality, however, it potentiated the effect of the application of 0.3mL ethanol month-1 kg-1. Royal Gala‟ apples stored in CA with O2 partial pressures below to 0.8kPa show lesser quality after storage. Temperature of 1.0ºC, compared with 0.0°C and 0.5°C, provided highest apple quality maintenance after eight months of storage and more seven days in shelf life at 20ºC. The best ULO condition for fruits stored at 1.0ºC is 1.0kPa O2 with 2.0kPa CO2. The interaction between ULO and storage temperature showed a necessity to increase O2 partial pressures when the storage temperature was increased. This behavior were verified especially when ACC oxidase activity and flesh browning incidence were analyzed. / O objetivo deste trabalho foi avaliar o efeito da aplicação pós-colheita de etanol e aldeído acético e comparar com tecnologias de armazenamento comprovadamente eficientes sobre a manutenção da qualidade de maçãs Royal Gala‟. Além disso, objetivou-se avaliar o efeito de condições de atmosfera controlada (AC) com pressões parciais ultrabaixas de oxigênio sobre a manutenção da qualidade de maçãs, Royal Gala‟ após o armazenamento. Para atingir tais objetivos foram conduzidos três experimentos no decorrer dos anos de 2008 e 2009. No primeiro experimento, realizado em 2008, foram testados os seguintes tratamentos: [1] controle; [2] aplicação de 0,5mL de etanol mês-1 kg-1; [3] aplicação de 0,25mL de aldeído acético mês-1 kg-1; [4] aplicação de 625 ηL L-1 de 1-MCP; e [5] absorção de etileno (<0,02μL L-1). Todos os frutos dos tratamentos avaliados foram armazenados em AC na condição de 1,0kPa O2 e 2,0kPa CO2. No segundo experimento, conduzido em 2009, os tratamentos avaliados foram constituídos da seguinte forma: [1] controle; [2] baixa umidade relativa do ar (BUR); [3] BUR com aplicação de etanol na dosagem de 0,3mL mês-1 kg-1; e [4] aplicação de etanol na dosagem de 0,3mL mês-1 kg-1. Todos os frutos dos tratamentos foram armazenados em AC com 1,2kPa O2 + 2,5kPa CO2. Nos dois experimentos os frutos foram armazenados na temperatura de 0,5°C. No terceiro experimento, conduzido em 2008, foram avaliadas cinco condições de AC: [1] 1,0kPa O2 e 2,0kPa CO2; [2] 0,8kPa O2 e 1,5kPa CO2; [3] 0,8kPa O2 e 1,0kPa CO2; [4] 0,6kPa O2 e 1,5kPa CO2; e [5] 0,6kPa O2 e 1,0kPa CO2, combinadas com temperaturas de 0,0°C, 0,5°C e 1,0°C, resultando em um experimento fatorial. A aplicação de 0,5mL de etanol mês-1 kg-1 ou 0,25mL de aldeído acético mês-1 kg-1, provocou aumento da incidência de polpa farinácea, degenerescência de polpa e podridões, assim como diminuição da firmeza de polpa, resultando em menor manutenção da qualidade comparada com a aplicação de 1-MCP e absorção de etileno. Entretanto, a dose de 0,3mL de etanol mês-1 kg-1, do segundo experimento, resultou na manutenção de maior firmeza de polpa, menor atividade da ACC oxidase e menor produção de etileno. A redução da umidade relativa do ar (BUR) não apresentou eficiência na manutenção da qualidade, porém, potencializou o efeito da aplicação de 0,3mL de etanol mês-1 kg-1. Maçãs Royal Gala‟ armazenadas em AC sob pressões parciais de O2 abaixo de 0,8kPa apresentaram inferior qualidade após o armazenamento. A temperatura de 1,0ºC, comparado a 0,0°C e 0,5°C, manteve a qualidade superior após oito meses de armazenamento e mais sete dias de exposição à temperatura de 20ºC. A melhor condição de ULO para frutos armazenados na temperatura de 1,0ºC é de 1,0kPa O2 combinado com 2,0kPa de CO2. A interação entre ULO e temperatura de armazenamento evidenciou a necessidade de elevação das pressões parciais de O2 quando a temperatura de armazenamento foi aumentada. Este comportamento foi verificado quando analisadas atividade da ACC oxidase e incidência de degenerescência de polpa.

Rhodobacter capsulatus is a versatile organism capable of growing on different growth conditions including photofermentation in the presence of carbon source, aerobic respiration, anaerobic respiration in the presence of an external electron acceptor such as DMSO. The photofermentative growth of R.capsulatus results in hydrogen production which stands out as an environmentally harmless method to produce hydrogen and accepted as one of the most promising process. Due to the serious problems such as as global climate change and environmental pollution caused by the fossil fuels, there is an increasing requirement for a clean and sustainable energy source. Furtherrmore, the ability of R.capsulatus to fix nitrogen, to use solar energy makes it a model to study various aspects of its metabolism. Thus the goal of this study is to increase the potential in biohydrogen production with the photofermentative bacteria and to investigate the proteins playing roles in different growth modes of the bacteria. In the present study, protein profiles of Rhodobacter capsulatus grown on respiratory, anaerobic respiratory and photofermentative growth modes were obtained. LC-MS/MS system is used to analyze the proteome as a high throughput technique. Physiological analysis such as HPLC for the analysis of the carbon source consumption, GC and analysis of pigments were carried out to state the environmental conditions. As a result, total of 460 proteins were identified with 17 proteins being unique to particular growth condition. Ratios of the proteins in different growth conditions were compared and important proteins were highlighted.

La respiration anaérobie peut être un trait essentiel dans le mode de vie, la colonisation de l'environnement et la survie. Jusqu'à présent, la seule respiration anaérobie confirmée chez Agrobacterium spp. est la dénitrification. De façon intéressante, cette voie est inégalement répandue chez les agrobactéries. Ces observations m'ont amené à mon hypothèse, à savoir la respiration anaérobie et notamment la dénitrification pourraient expliquer la coexistence d'agrobactéries et leur distribution dans des niches spécifiques de la rhizosphère. Ma thèse visait à explorer les stratégies de respiration anaérobie d’Agrobacterium spp. et de les relier à l'adaptation de niche écologiques différentes. Les objectifs de ma thèse étaient (1) de caractériser tous les gènes impliqués dans la dénitrification chez A. fabrum C58 in vitro, (2) d'explorer les gènes de la dénitrification nécessaires à la colonisation des racines du maïs et (3) de découvrir de nouvelles respirations anaérobies pendant la colonisation racinaire du maïs (Figure 16). Réaliser des mutants et les tester dans des conditions particulières est le moyen classique de déterminer l'implication d'un gène dans une voie spécifique. Cependant, cette méthode implique une vision à priori et des connaissances solides sur les gènes cibles et ne peut pas être appliquée à toutes les situations. Nous avons alors dû développer une méthode plus adaptée pour identifier les gènes essentiels impliqués dans la croissance dans des conditions anaérobies spécifiques. - Gènes de dénitrification chez A. fabrum C58 in vitro. Pour compléter la voie de dénitrification chez A. fabrum C58 et identifier tous les gènes et régulateurs impliqués dans la dénitrification, nous avons adopté deux stratégies : Premièrement, une vision à priori pour (1) identifier la nitrate réductase impliquée dans la première étape de la dénitrification et (2) valider le rôle d'un ARN non codant dans le contrôle de la dénitrification. Pour ce faire, nous avons construit un mutant napA de A. fabrum C58 et un mutant de l'ARN non codant NopR et nous avons évalué leur croissance et leur capacité à produire du N2O dans des conditions anoxiques. Deuxièmement, pour identifier tous les gènes impliqués dans la dénitrification, nous avons construit une banque de transposons mutants de C58 et testé sa croissance dans des conditions de dénitrification in vitro en présence de nitrate ou de nitrite. - Rôle des gènes de la denitrification de A. fabrum C58 dans la colonisation racinaire du maïs. Il est bien connu que le séquençage de transposons (Tn-Seq) est une méthode très puissante pour déterminer les gènes nécessaires à la croissance bactérienne en présence de leur hôte. Pour déterminer les gènes de dénitrification impliqués dans la colonisation des racines en anoxie, nous avons utilisé la banque construite chez C58 et l’avons inoculée sur les plants de maï cultivées sur un sol fertile et cultivées dans des conditions inondées mimant des conditions anaérobies. Le séquençage des cellules d ‘A. fabrum C58 récupérées mettra en évidence les gènes impliqués dans la colonisation anaérobie de cette niche spécifique. - Découverte de nouvelles voies de respiration anaérobie chez A. fabrum C58. Pour découvrir de nouvelles voies de respiration anaérobie, nous avons mis en place des tests de croissance de C58 dans des conditions anoxiques en présence de sources de C et de N en tant qu'accepteurs terminaux d'électrons. De façon interéssante, en cultivant des souches WT et mutée dans le gène napA au contact de la racine de maïs dans des conditions anoxiques (chapitre 1), nous avons montré une croissance des deux souches. Ce résultats suggère que les exsudats de racine servent d'accepteurs d'électrons terminaux pour la croissance anaérobie de C58. Pour déterminer quels composés exsudés du maïs peuvent servir de TEA, les principaux métabolites ont été identifiés par HPLC et certains ont été testés en tant que TEA dans des conditions anoxiques / Anaerobic respiration may be an essential trait in lifestyle, environment colonization and survival. Until now, the only confirmed anaerobic respiration in Agrobacterium spp. is denitrification. Interestingly, this pathway is unequally widespread among Agrobacteria. These observations led me to my hypothesis which is anaerobic respiration and notably denitrification could explain the coexistence of Agrobacteria and their distribution in specific niches in the rhizosphere. My thesis was undertaken to explore the anaerobic respiration strategies of Agrobacterium spp. and to relate them to niche adaptation. The objectives of my thesis were to (1) characterize all the genes involved in denitrification in A. fabrum C58 in vitro, (2) explore the genes of denitrification that are needed during maize root colonization and (3) discover new anaerobic respirations that occur during maize root colonization (Figure 16). Mutational analysis is the classic way to determine the involvement of a gene in specific pathway. However, this method implies an a priori view and solid knowledge on target genes and cannot be applied for every situation. We have to develop a more adapted method to identify essential genes involved in growth in specific anaerobic conditions. - Denitrification genes in A. fabrum C58 in vitro. To complete denitrification pathway in A. fabrum C58 and identify all the genes and regulators involved in the denitrification function, we adopted two strategies: Firstly, an a priori view to (1) identify the nitrate reductase involved in the first step of denitrification and (2) validate the role of a non-coding RNA in denitrification control. To do so, we constructed a mutant of napA of A. fabrum C58 and a mutant of the non-coding RNA NopR and we evaluated their growth and capacity to produce N2O under anoxic conditions. Secondly, to identify all the genes involved in denitrification, we constructed a mutant transposon library of C58 and tested its growth under denitrification conditions in vitro in the presence of either nitrate or nitrite. - Role of A. fabrum C58 denitrifying genes in the root colonization of maize. It is well known that Transposon-sequencing (Tn-Seq) is a very powerful method to determine genes required for bacterial growth in the presence of their host. To determine denitrifying genes involved in root colonization under anaerobic conditions, we used the library constructed in C58 and performed in planta assays. The mutant library was inoculated on maize plants grown on fertile-ground and cultured under flooded conditions miming anaerobic conditions. Sequencing the recovered A. fabrum C58 cells will evidence the genes involved in this anaerobically specific niche colonization. - Discovery of new anaerobic respiration pathways in A. fabrum C58. To discover new anaerobic respiration pathways, we set-up growth assays of C58 under anoxic conditions in the presence C and N sources as terminal electrons acceptors. Interestingly, by culturing WT and NapA-deficient strains in contact with maize root under anoxic conditions (Chapter 1), we showed growth of both strains, suggesting that root exudates serve as terminal electrons acceptors for anaerobic growth of C58. To determine which maize exuded compounds can serve as TEAs, primary metabolites were identified by HPLC and some were tested as TEAs under the set-up conditions