Dissertationen zum Thema „Rhodopseudomonas“

Rhodopseudomonas palustris est une bactérie pourpre photosynthétique dont le génome, entièrement séquencé, a révélé avec surprise la présence de 6 gènes codant des bactériophytochromes. L'un d'entre eux (RpBphP1) joue un rôle primordial et inhabituel dans la synthèse du photosystème. Chez cette bactérie, trois autres bactériophytochromes (RpBphP2, 3 et 4) sont localisés à proximité d'opérons pucBA codant les polypeptides des antennes collectrices de lumière associées au photosystème. Ce travail de thèse a consisté dans un premier temps à étudier les rôles, les propriétés et les mécanismes d'action de ces 3 bactériophytochromes. Il a pu être ainsi montré que les 2 bactériophytochromes RpBphP2 et 3 agissent de concert dans le contrôle des antennes de types LH4. Cette voie de régulation implique l'action de 3 autres réponses-régulateurs dont la protéine Rpa3018 sensible au potentiel redox. Cette étude a également révélé que, chez certaines souches de Rps. Palustris, la protéine RpBphP4 a perdu sa sensibilité à la lumière mais a acquis en contrepartie une sensibilité au potentiel redox tout en conservant sa capacité à réguler l'expression des antennes de type LH2 via un système à 2 composants. Dans un second temps, l'analyse de la séquence du génome de deux Bradyrhizobium photosynthétiques (ORS278 et BTAi1) a révélé que chaque souche possède un bactériophytochrome spécifique sûrement acquis par transfert horizontal. Les études menées sur ces différents bactériophytochromes ont mis en exergue la diversité de cette famille de senseurs de lumière ainsi que la complexité des voies de signalisation qu'ils initient
Rhodopseudomonas palustris is a photosynthetic purple bacteria which genome was completely sequenced. Six genes encoding putative bactériophytochromes are present in this genome. One of them plays an essential and unusual role in the synthesis of the photosystem. In this bacteria, three other bacteriophytochromes are localized near pucBA operons encoding polypeptides involved in the formation of light harvesting complexes associated with photosystem. First, this PhD work studied roles, properties and mechanisms of action of these three bacteriophytochromes. We showed that RpBphP2 and RpBphP3 act in tandem to regulate the expression of LH4 antennas. This signalling pathway use three reponse-regulators, one of them, Rpa3018 is rdox sensitive. In some strains of Rps. Palustris, a bactériophytochrome, RpBphP4, lost its light sensitivity and acquired a redox sensibility while keeping its ability to regulate the expression of these antennas. In a second time, analysis of the genome sequence of two photosynthetic Bradyrhizobium (ORS278 and BTAi1) revealed, in each strain, the presence of a specific bacteriophytochrome probably acquired by horizontal transfer. The properties of these various bacteriophytochromes led highlighted the variety of this family of light sensors as well as the complexity of the signalling pathways which they introduce

The recent surge in biodiesel production has resulted in a huge surplus of crude glycerol, a by-product of the process to the level of 10% by weight. This is turn has caused the price of glycerol to fall dramatically, and there are now few economically viable channels for using this resource: waste glycerol is usually combusted. Therefore, much interest has arisen in the possibility of making use of glycerol with biotechnology, as this would not only be a more efficient use of resources but also make biodiesel itself more commercially viable. The purple bacterium Rhodopseudomonas palustris is able to metabolize glycerol through photofermentation and thereby produce hydrogen, a commercially useful commodity. R. palustris is of particular interest for this purpose as, in contrast to many other species which have been investigated with a view to fermenting glycerol, it is highly tolerant of crude glycerol. The feedstock requires little purification or dilution to be made suitable for cultivation of R. palustris. Furthermore, the hydrogen gas produced by R. palustris when grown on glycerol is of high purity, and the organism's great metabolic diversity suggests it may be a useful strain for remediation of other waste materials. However, much groundwork is needed to establish R. palustris as a viable chassis organism for biotechnological purposes. This work sets out to establish optimal conditions for cultivating R. palustris in the laboratory, including the design of a suitable batch photobioreactor system. It also determines optimal conditions for electroporation of R. palustris for the purpose of knocking out endogenous genes or introducing heterologous genes. Furthermore, the introduction of heterologous genes is attempted in order to demonstrate the possibility of producing other high-value compounds with R. palustris, and several deletion strains with potential benefits for hydrogen production are created. Finally, several existing deletion strains are investigated to establish their suitability as chassis strains for further genetic manipulation.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2013.
"June 2013." Cataloged from PDF version of thesis.
Includes bibliographical references.
Microbial anaerobic iron oxidation has long been of interest to biologists and geologists, both as a possible mechanism for the creation of banded iron formations before the rise of oxygen, and as a model system for organisms able to accept electrons from an external, inorganic source. Previous work with the purple photoferrotroph Rhodopseudomonas palustris TIE-1 showed that three genes were required for phototrophic growth with Fe(Il): PioA, a decaheme cytochrome, PioB, an outer membrane porin, and PioC, a high potential iron protein (HiPIP). These proteins suggested a model of Fe(II) oxidation that ends with transfer of electrons to the photosynthetic reaction center. The goal of this thesis was to test and extend this model through characterization of the electron transfer proteins PioA and PioC. In the course of our experiments, we discovered that Fe(II) could also delay growth under certain conditions. We then broadened our focus to encompass several facets of the interaction of TIE-1 with Fe(II) under anaerobic conditions: The first portion describes how low amounts of Fe(II) cause a growth delay in TIE-1 cultures growing anaerobically on other substrates - a surprising result for an organism that grows on millimolar concentrations of iron. The cause of this toxicity was found to be dependent on copper, which is toxic to TIE-i at fairly low concentrations. Our results indicate the copper toxicity is synergistically increased by Fe(II) under strictly anaerobic conditions. The second part of this work describes characterization of the HiPIP PioC and a second HiPIP in the TIE-1 genome. The results showed that PioC is capable of reducing the reaction center, as expected, though at a slower rate than is usually found for this kind of interaction. The second HiPIP cannot reduce the reaction center and likely serves an alternate function in the cell unrelated to photosynthesis, possibly involving detoxification of metals. The final section redefines our understanding of the Fe(II) oxidation pathway by putting it in the context of reverse electron transfer, a process that is not well understood in photosynthetic bacteria. Evidence from whole cell experiments using flash induced spectrometry indicated that electrons from Fe(II) may, rather than going to the reaction center, enter the quinone pool through the bc1 complex. This model is significantly different from previous preferred models of phototrophic oxidation, but is similar to the reverse electron transfer system described in acidophilic lithotrophic iron oxidizing bacteria. Taken together, the experiments described in this thesis highlight the complex and interconnected nature of a bacterial cell's interactions with iron under anoxic conditions. It also suggests future avenues of study for phototrophic reverse electron transfer, a poorly understood process that is vital to anoxygenic photoautotrophic growth.
by Lina J. Bird.
Ph.D.

L'une des fonctions essentielles des organismes photosynthétiques est la capture de l'énergie lumineuse par des molécules de chlorophylle spécialisées, ou antennes. Cette énergie électromagnétique est alors transférée vers les centres réactionnels où elle est convertie en énergie chimique, sous forme d'une séparation transmembranaire de charges. Nous avons étudié ces phénomènes chez la bactérie photosynthétique pourpre Rhodopseudomonas sphaeroides par analyse de la fluorescence résolue en temps de l'émission de l'antenne. Nous avons pu comparer les données obtenues sur systèmes globaux à celles mesurées sur complexes-antenne isolés, sur mutants déficients dans certains types d'antennes, et sur centres réactionnels purifiés et totalement dépourvus d'antenne où seuls sont présenta les pigments impliqués dans les réactions primaires de transfert d'électron. Nous obtenons des conclusions sur le type de migration de l'énergie d'excitation vers les centres (chapitre IV), sur le couplage antenne-centre (chapitre II) et sur l'origine de la fluorescence variable (chapitres I et III) en relation avec la stabilisation de l'état de séparation primaire de charges au niveau des centres
The harvesting of light energy is one of the most important function of the photosynthetic organisms. Lt is performed by the chlorophyll antenna molecules. The absorbed energy is transferred very efficiently to the reaction centers where a transmembrane charge separation takes place, converting the energy of photons to chemical free energy. These processes have been studied on the photosynthetic purple bacteria Rhodopseudomonas sphaeroides, by analyzing the time-resolved fluorescence data of the antenna emission. Data obtained on isolated antenna-complex, on mutant lacking one type of antenna and on isolated reaction canters depleted of antenna, were compared to the data obtained on intact systems. Our results are leading to conclusions about the energy migration mechanism in the antenna of purple bacteria (chapter IV), about the antennae to reaction center relationship (chapter II), and about the origin of variable fluorescence (chapter I and III) in relation to the stabilization of the charge separation state of the reaction state

Anaerobic bacteria typically degrade many types of aromatic monomers through conversion to benzoyl-CoA which is further degraded by ring reduction, ring cleavage and β-oxidation reactions. Rhodopseudomonas palustris has the ability to grow on different side-chain length aromatic compounds ranging from 3-phenylpropanoic acid to 8-phenyloctanoic acid. A β-oxidation or non β-oxidation metabolic pathway could be responsible for degradation of these compounds. To study the pathways and genes involved, a full cellular proteome analysis using mass spectrometry was carried out and the expression patterns for selected genes were measured by qRT-PCR. Two putative feruloyl-CoA synthetases, Fcs1 and Fcs2, in addition to a long chain fatty acid CoA-ligase (RPA1766) were overexpressed in E. coli as recombinant proteins and the purified enzymes were able to catalyse the formation of CoA thioesters of phenylcarboxylates with different chain lengths. The kinetic properties of Fcs1 enzyme showed a high kcat/Km value for 7-phenyl heptanoic acid. Activity was also recorded with 5-phenyl valeric acid, 6- phenyl hexanoic acid and 8- phenyl octanoic acid, but with lower kcat/Km values, while in contrast Fcs2 only utilised 3-phenyl propionic acid and the related hydroxycinnamic acid. RPA1766 was able to utilize only 8-phenyloctanoic acid with low affinity. These data along with protein structure modelling support the idea that Fcs1 catalyses the first step in a β-oxidation pathway for long chain phenylalkane carboxylates in R. palustris. However, other CoA ligases can contribute, as mutants lacking Fcs1 and Fcs2 genes were able to grow on long chain phenylalkane carboxylic acids. Evidence was obtained that the solute binding proteins of an ABC-transporter previously associated with pimelic acid and long chain fatty acid transportation (RPA3718-RPA3725) could bind certain phenylalkane carboxylates with a Kd value of 1.29 µM for RPA3723 with 7-phenylheptanoic acid.

Rhodopseudomonas palustris is a metabolically versatile phototrophic α-proteobacterium. The organism experiences a wide range of stresses in its environment and during metabolism. The oxidative an pH stresses of four ECF (extracytoplasmic function) σ-factors are investigated. Three of these, σ0550, σ1813, and σ1819 show responses to light-generated singlet oxygen and respiration-generated superoxide reactive oxygen species (ROS). The EcfG homolog, σ4225, shows a high response to superoxide and acid stress. Two proteins, one containing the EcfG regulatory sequence, and an alternative exported catalase, KatE, are presented to be regulated by σ4225. Transcripts of both genes show similar responses to oxidative stress compared to σ4225, indicating it is the EcfG-like σ-factor homolog and controls the global stress response in R. palustris.

0BHG1, a lytic phage specific for Rhodopseudomonas blastica has an icosahedral head of 62 ran diameter and a short, 39 nm long, non- contractile tail with a collar, base plate and short spikes. Caesium chloride density centrifugation of phage normally gave a single phage band corresponding to a density of 1.3850 g cm, but occasionally a second band was observed (1.3838 g cm. No apparent differences were observed between the two phage bands. The nucleic acid of 0BHG1 is double stranded DNA, with a G+C content of 50.6 mol %, and a molecular weight of 48 kb. The capsid proteins are observed ranging in molecular weight from 18,000 to 98,000 and corresponding to approximately 26% of the coding capacity of the genome. 0BHG1 adsorbed to both photo- synthetically and chemohetero-trophically grown Rp. blastica at an identical rate of 1.39 ml'1 min". The phage has no specific cation requirement for adsorption. One-step growth curve studies of 0BHG1 with phage antisera gave a lag period of 80-100 min, a rise period of 100 min and a final burst size of 25 ± 2.5 and 30 + 2.1 for chemohetero-trophically and photoheterotrophically grown cells respectively. Synchronisation of cells for one-step growth curves with potassium cyanide altered the burst size with photosynthetically grown cells. Adsorption of 0BHG1 was specific to the 'old' pole of Rp. blastica. Lysis of Rp. blastica by French pressing gave three distinct bands after differential sucrose density centrifugation corresponding to the cytoplasmic membrane and two cell wall fractions B1 and B2. Both cell wall fractions contained a dominant heat modifiable protein of molecular weight 35,000, and in addition cell wall fraction B1 contained two dominant proteins of Mr 14,500 and 15,000. Amino acid analysis suggests a difference in the amount of cross-linkage of the peptidoglycan between the two cell wall fractions. Comparisons of Rp. blastica with a spontaneous phage resistant mutant Rp. blastica to which 0BHG1 fails to adsorb showed only trace amounts of the Mr 14,500 and 15,000 proteins in the resistant strain. Analysis of lipopolysaccharide from the two strains showed the loss of the neutral sugar 2-0-methyl-6-deoxyhexgse and a reduction in the concentration of galactose in Rp. blastica Evidence suggests that an LPS/protein complex may function as the bacterial phage receptor and is required to inactivate 0BHG1 in vitro.

La degradation du lactate par rhodopseudomonas capsulata a ete etudiee pour differentes conditions de fonctionnement du reacteur : reacteur ferme, reacteur ferme avec ajouts successifs de substrat, reacteur ouvert continu, reacteur ouvert avec recyclage de la biomasse. Dans tous les cas, les concentrations en metabolites formes ont ete mesurees au cours du temps et des bilans matiere realises. La production d'hydrogene, resultat de la degradation du lactate, est optimale pour des cultures en continu diluees, limitees en source azotee. Pour des concentrations bacteriennes elevees, il y a changement de metabolisme : production de propionate et diminution de la production d'hydrogene. Le passage du premier au second metabolisme presente une certaine irreversibilite qui conduit a recommander, pour la conduite de telles cultures, l'usage de techniques de conduite adaptative

L'uranium est un toxique à la fois chimique et radiologique. C'est un élément que l'on retrouve à faible concentration dans l'environnement sauf lors de pollutions causées par les activités humaines. Du fait de sa forte réactivité, l'ion uranyle peut se complexer à de nombreux constituants du sol, qu'ils soient minéraux ou organiques. Ces différentes formes peuvent être ainsi plus ou moins biodisponibles pour les microorganismes et les plantes et peuvent ensuite rentrer dans la chaîne alimentaire de l'homme. La connaissance et la compréhension des mécanismes de transfert et du devenir d'éléments toxiques chimiques et radiologiques dans la biosphère constituent donc un enjeu capital pour permettre une bonne estimation des risques sanitaires et écologiques. La connaissance de la spéciation est capitale pour engager des processus de biorémédiation. Ici, c'est l'effet des microorganismes sur la spéciation de l'uranium dans l'environnement qui nous intéresse. Selon les formes initiales de l'uranium, les bactéries peuvent l'accumuler et/ou le transformer et ainsi modifier sa biodisponibilité. Les modèles utilisés dans cette étude sont Cupriavidus metallidurans CH34, bactérie tellurique résistante aux métaux lourds, Deinococcus radiodurans R1, bactérie connue pour être une des espèces les plus radiorésistantes et Rhodopseudomonas palustris, bactérie pourpre autotrophe capable de dégrader les composés aromatiques en anaérobie. Ces bactéries sont cultivées en présence de deux formes d'uranium retrouvées dans l'environnement : une forme minérale, le carbonate d'uranyle et une forme organique, le citrate d'uranyle. Ces formes ont d'abord été modélisées et les milieux de culture ont été modifiés pour pouvoir travailler avec ces espèces. La capacité des bactéries à résister, à transformer et/ou accumuler l'uranium a été étudiée. On observe une différence entre les concentrations minimales inhibitrices des deux spéciations mais celle-ci est en fait due à une différence de biodisponibilité des phosphates. Aucune accumulation ne semble être mise en évidence aux pH environnementaux par dosage de l'uranium et par observation au microscope électronique à transmission alors qu'une précipitation est observée à pH 1. La spéciation de l'uranium a été étudiée par spectroscopie d'absorption X (EXAFS). La spéciation est bien maitrisée dans les milieux de culture et les précipités observés aux pH très acide sont ces complexes de phosphates d'uranyle.

Aromatic compounds are among the most persistent and prevalent toxic pollutants in the environment. Biotransformation and bioremediation processes can convert these compounds into non-toxic compounds and valuable products. The purple phototrophic bacterium Rhodopseudomonas palustris (R. palustris) has the ability to degrade a wide range of aromatic compounds and the identification of several nitroreductases, nitrilases and a nitrile hydratase (NHase) in the genome sequence indicates an ability to degrade nitroaromatic and nitrile compounds respectively. The candidate nitroreductases RPA1711 and RPA4285 were recombinantly produced in E.coli and the purified proteins were assayed for their ability to reduce the nitro-groups of ligands to amino groups by UV-Spectrophotometry. Michaelis-Menten constants (Km) for RPA1711 of 0.21 mM and 0.13 mM were obtained for 2,4 Dinitrotoluene (2,4-DNT) and 2,6-Dinitrotoluene (2,6 DNT) respectively, while RPA4285 had higher Km values with 2,4-DNT of 0.76 mM and with 2,6-DNT of 0.41 mM. On the other hand, nitriles were utilised by nitrilases and a nitrile hydratase and both aromatic and aliphatic nitriles were degraded. The RPA0599, RPA1563 and RPA4166 nitrilases were purified and their activities were determined by the release of ammonia using the continuous coupled assay, phenol-hypochlorite, ophthalaldehyde (OPA) methods. 4-cyanopyridine was a preferred substrate. Furthermore, a NHase of two subunits RPA2805-2806 and NHase with activator (P14K) RPA2805-RPA2806-RPA2807 was also purified and characterised. Benzonitrile was a preferred substrate for the NHase. Exploring the ability of R. palustris to degrade nitroaromatic and nitrile pollutants might help improve bioremediation biotechnology.

This project was aimed to use a purple non-sulphur bacterium, Rhodopseudomonas palustris, as a biocatalyst for hydrogen production, from the waste of biodiesel manufacturing, crude glycerol. The goal of this project was to understand the fundamentals relevant to scaling up the process and developing an off the shelf product. The first objective was to determine the ability of R. palustris to generate hydrogen by non-growing cells in comparison to that by growing cells. Similar average hydrogen production rates and energy conversion were found for both processes but a significant difference in the hydrogen yield was observed. Hydrogen production reached ~ 80 % of the theoretical maximum hydrogen yield by non-growing R. palustris, about eight-fold of that reached by growing R. palustris. The high yield suggested that it is economically appealing to use non-growing R. palustris as the biocatalyst for continuous hydrogen production. To accomplish the proposed scale-up systems, understanding its product formation kinetics is the key. It was found that the hydrogen production rate is not growth-associated and depends solely on the dry cell mass with a non-growth associated coefficient of 2.52 (Leudeking–Piret model dP/dt=2.52 X). Light is vital for hydrogen production by non-growing R. palustris, in terms of light intensity and wavelength range. It was found that excessive or insufficient light intensity may constrain the performance. Only photons of light with appropriate wavelengths can excite cytochrome bacteriochlorophyll complexes II in R. palustris to generate hydrogen. Among white LEDs, infrared LEDs, and incandescent light bulbs, at the same light intensity, infrared LEDs gave the best results in the H2 production rate and energy conversion by non-growing cells, 22.0 % ± 1.5 % higher than that with white LEDs and around 25-30 times of that by incandescent light bulbs. It was found that non-growing R. palustris can be immobilised in alginate beads to give similar H2 production rates as that by cells suspended in media. This preliminary result pointed the direction of developing an off the shelf product of immobilised non-growing R. palustris as a biocatalyst for continuous hydrogen production.

Some purple bacteria species, such as Rhodopseudomonas palustris 2.1.6, produce light harvesting antenna (LH2) with unusual absorption spectra when they are grown under low-light intensities. This ability is often related to the presence of multiple genes encoding the LH2 apoproteins. This thesis describes isolation of pure stable LH2s from Rhodopseudomonas palustris 2.1.6 grown at different light intensities, determination of the polypeptide composition of high- (HL) and low-light (LL) LH2 complexes and characterisation their spectroscopic properties using various optical spectroscopies. The question of whether rings with a heterogeneous apoprotein composition exist has been addressed by single-molecule spectroscopy. For the first time, direct evidence that individual LL LH2 complexes have a heterogeneous αβ-apoprotein composition has been found. Such mixed rings feature Bchl a molecules with both B820-like and B850-like site-energies. This finding was supported by a femtosecond study on the energy transfer reactions and exciton relaxations within both HL and LL LH2 complexes. This thesis also describes attempts to crystallise the HL and LL LH2s. Even though three-dimensional crystals of both HL and LL LH2 complexes only diffracted to low resolution, it was possible to use molecular replacement to obtain structures that suggest both these types of LH2s are nonamers.

A sustainable and low-cost system, namely a photo-bioelectrochemical system (photo-BES), based on the natural blueprint of photosynthetic microorganisms was studied. The aim of this research work is to improve the efficiency of electron transfer of the microorganisms for bioelectricity generation. The first strategy adopted was the evaluation of the exoelectrogenic activity of oxygenic photosynthetic cyanobaterium, Synechococcus elongatus PCC 7942, in biophotovoltaic (BPV) platforms through a comparative performance analysis of bioanode materials. The second approach involved improving the performance of anoxygenic photosynthetic bacterium, Rhodopseudomonas palustris ATCC® 17001™, by varying the ratio of nitrogen to carbon sources (N:C) to maximise both biohydrogen production and exoelectrogenesis for conversion into bioelectricity in photosynthetic microbial fuel cells (photoMFCs). A linear correlation was obtained between average surface roughness/surface area and maximum power density of ITO-coated and graphene/ITO-coated substrates. Graphene/ITO-coated PET bioanodes produced the highest maximum power output of 29±4 μW m-2 in a single chamber BPV device due to improved biofilm formation and improved electrochemical activity. XG Leaf®, also known as graphene paper, helped to bridge the shortcomings of carbon fibres in terms of wettability. The most hydrophilic, 240 μm thick graphene paper, produced the highest maximum power output of 393±20 μW m-2 in a membrane electrode assembly (MEA)-type BPV device, mainly due to reduced electrochemical polarisation. A proof of concept study compared the performance of screen-printed graphene onto a membrane separator against 3D-printed bioanodes coated with carbon nanotubes. One mm thick 3D-printed bioanode was better performing as its structures promoted a much denser biofilm with extensive fibrous extracellular matrix. Using a ratio of N:C=0.20 resulted in higher biohydrogen production and higher exoelectrogenic activity, generating a maximum power output of 361±157 mW m-2 and 2.39±0.13 mW m-2, respectively. This study provided additional insight in improving the electron transfer efficiency, which could be used to further optimise photo-BESs as part of future research and development for sustainable technologies.

La bactérie pourpre photosynthétique Rhodopseudomonas palustris présente la particularité de pouvoir se développer en utilisant la photosynthèse ou la respiration et de posséder six bactériophytochromes, ainsi que deux régulateurs de transcription PpsR. Ce travail de thèse a consisté à étudier les rôles des bactériophytochromes et des régulateurs PpsR dans la régulation de la photosynthèse et de la respiration par la lumière et l’oxygène. L’étude de la synthèse du photosystème et de l’activité respiratoire chez Rps. Palustris sauvage, ou inactivée dans des bactériophytochromes ou dans les PpsRs, a révélé (i) que le bactériophytochrome RpBphP1, activé par un éclairement rouge lointain, est capable d’activer la synthèse du photosystème en levant la répression exercée par PpsR2 depuis la microaérobie jusqu’à l’aérobie ; (ii) que la synthèse des antennes LH2 est activée par le régulateur de transcription PpsR1 et le bactériophytochrome RpBphP4, qui, selon les souches de Rps. Palustris, est un senseur d’oxygène ou de lumière et (iii) que RpBphP1, PpsR1 et PpsR2 sont impliqués dans la régulation de la respiration de Rps. Palustris par la lumiére rouge lointain. Les résultats obtenus en combinant des approches in silico, de biologie moléculaire et de biochimie, nous amènent à proposer que le bactériophytochrome RpBphP1, en inhibant l’activation de PpsR2 sur la transcription de sucA – codant pour la sous-unité E1 de l’alpha-cétoglutarate déshydrogénase, une enzyme centrale du cycle de Krebs – est responsable d’une limitation de respiration de 40% sous éclairement rouge lointain par rapport à l’obscurité. Nos résultats indiquent qu’un éclairement rouge lointain, via l’action de RpBphP1 sur PpsR2, a un double effet puisque, d’une part, il active la mise en place du photosystème, et, d’autre part, il limite la respiration. Ce travail a également révélé que les régulateurs PpsR1 et PpsR2 sont à la fois des répresseurs et des activateurs de transcription, qui peuvent agir de concert, ou de façon opposée. Enfin, nous avons montré que les mécanismes moléculaires régissant les régulations métaboliques ayant lieu chez Rps. Palustris ne sont pas tous retrouvés chez une bactérie phylogénétiquement proche de Rps. Palustris, Bradyrhizobium ORS278
The photosynthetic purple bacterium Rhodopseudomonas palustris is able to develop using photosynthesis or respiration. Extraordinarily, it possesses six bacteriophytochromes and two PpsR transcriptional regulators. This work aimed at studying bacteriophytochromes and PpsR roles in the regulation of photosynthesis and respiration by light and oxygen. The study of photosystem synthesis and respiratory activity in Rps. Palustris WT, or inactivated in bacteriophytochromes or in PpsRs showed that (i) the bacteriophytochrome RpBphP1, activated by a far red illumination can trigger photosystem synthesis, by counterbalancing PpsR2 repressive effect, from microaerobic to aerobic conditions ; (ii) that LH2 antennae synthesis is activated by the PpsR1 transcriptional regulator and the bacteriophytochrome RpBphP4, which, depending on the Rps. Palustris strain, can be an oxygen sensor or a light sensor and (iii) that RpBphP1, PpsR1 and PpsR2 are involved in the regulation of Rps. Palustris respiratory activity by far red light. Considering the results obtained combining in silico, molecular biology and biochemistry approaches, we propose that the bacteriophytochrome RpBphP1, by inhibiting PpsR2 activation on the transcription of sucA – encoding the subunit E1 of the alpha-ketoglutarate dehydrogenase, a central enzyme in the Krebs cycle – is responsible for a 40% respiration limitation under far red illumination relative to darkness. Our results showed that a far red illumination, via RpBphP1 action on PpsR2, has a double effect since it activates photosystem synthesis on the one hand, and limits respiration on the other hand. This work also revealed that PpsR1 and PpsR2 regulators are both transcriptional repressors and activators, which can have joint or opposite effects. Finally, we showed that the molecular mechanisms responsible for the metabolic regulations encountered in Rps. Palustris are not all retrieved in the phylogenetically closely related bacterium, Bradyrhizobium ORS278

Nous avons determine le potentiel de demi-reduction et l'orientation par rapport a la membrane de chaque heme du cytochrome tetrahemique du centre reactionnel de trois bacteries photosynthetiques: rhodopseudomonas sulfoviridis mesophile et thermophile et roseobacter denitrificans. Les resultats obtenus montrent qu'il existe une forte similitude entre le cytochrome des souches de rhodopseudomonas sulfoviridis (h#1: +390 mv, 75 ; h#2: +310 mv, 56 ; l#1: +72 mv, 58 ; l#2: -40 mv, 72,5) et celui de rhodopseudomonas viridis deja bien caracterise (h#1: +380 mv, 75 ; h#2: +310 mv, 55 ; l#1: +20 mv, 60 ; l#2: h#2: -60 mv, 73,5) alors que celui de roseobacter denitrificans est different (h#1: +290 mv, 39 ; +240 mv, 55 ; l#1: +90 mv, 40 ; l#2: +90 mv, 90). L'etude des cinetiques rapides des transferts d'electrons entre les hemes de ce cytochrome et le donneur du centre reactionnel p nous ont permis de conclure que l'arrangement des hemes au sein du cytochrome des deux souches de rhodopseudomonas sulfoviridis est le meme que celui de rhodopseudomonas viridis (l#2-h#2-l#1-h#1-p) alors que celui de roseobacter denitrificans en differe au moins par la position des hemes de haut potentiel (l#2-h#1-l#1-h#2-p). Les hemes de bas potentiel du cytochrome du centre reactionnel de rhodopseudomonas viridis, sulfoviridis et de roseobacter denitrificans ne participent pas a un transfert cyclique d'electrons vers le centre reactionnel meme en conditions fortement reductrices. L'heme de haut potentiel h#2 est couple au cytochrome bc#1 par l'intermediaire d'un cytochrome soluble (c#2 chez rhodopseudomonas viridis et sulfoviridis et c#5#5#1 chez roseobacter denitrificans). Ce cytochrome forme, a l'etat reduit, un complexe electrostatique stable avec le cytochrome du centre reactionnel de rhodopseudomonas viridis et sulfoviridis ce qui n'est pas le cas pour roseobacter denitrificans

Rhodopseudomonas palustris est une bactérie pourpre photosynthétique dont le génome, entièrement séquencé, a révélé avec surprise la présence de 6 gènes codant des bactériophytochromes. L'un d'entre eux (RpBphP1) joue un rôle primordial et inhabituel dans la synthèse du photosystème. Chez cette bactérie, trois autres bactériophytochromes (RpBphP2, 3 et 4) sont localisés à proximité d'opérons pucBA codant les polypeptides des antennes collectrices de lumière associées au photosystème. Ce travail de thèse a consisté dans un premier temps à étudier les rôles, les propriétés et les mécanismes d'action de ces 3 bactériophytochromes. Il a pu être ainsi montré que les 2 bactériophytochromes RpBphP2 et 3 agissent de concert dans le contrôle des antennes de types LH4. Cette voie de régulation implique l'action de 3 autres réponses-régulateurs dont la protéine Rpa3018 sensible au potentiel redox. Cette étude a également révélé que, chez certaines souches de Rps. palustris, la protéine RpBphP4 a perdu sa sensibilité à la lumière mais a acquis en contrepartie une sensibilité au potentiel redox tout en conservant sa capacité à réguler l'expression des antennes de type LH2 via un système à 2 composants. Dans un second temps, l'analyse de la séquence du génome de deux Bradyrhizobium photosynthétiques (ORS278 et BTAi1) a révélé que chaque souche possède un bactériophytochrome spécifique sûrement acquis par transfert horizontal. Les études menées sur ces différents bactériophytochromes ont mis en exergue la diversité de cette famille de senseurs de lumière ainsi que la complexité des voies de signalisation qu'ils initient.

碩士
臺灣大學
分子與細胞生物學研究所
98
Rhodopseudomonas palustris (R. palustris) is a purple nonsulfur anoxygenic phototrophic bacterium that belongs to the alpha-proteobacteria. It is a common soil and water bacterium that makes its living by converting sunlight to cellular energy and by absorbing atmospheric carbon dioxide and converting to biomass. This microbe can also degrade and recycle components of the woody tissues of plants. Because of its intimate involvement in carbon management and recycling, R. palustris was selected by the DOE Carbon Management Program for genome sequencing. R. palustris exhibits the ability to grow under photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic conditions, and can switch between the four different modes of metabolism for survival by protein phosphorylation. Here, we analyzed the phosphoproteome of R. palustris in photoheterotrophic condition with a shotgun approach and identified 80 phosphopeptides from 73 phosphoproteins and 74 phosphopeptides from 68 phosphoproteins at chemohetrotrophic and photoheterotrophic condition, respectively. Our results revealed that the threonine phosphorylated peptide “GGMpTSHAAVVAR” from pyruvate phosphate dikinase (PPDK, RPA1051) in photoheterotrophic condition is elevated more than 2 folds than in chemoheterotrophic condition. PPDK performs a light-dependent activity and plays an important role in carbon fixation in C4 and CAM plants. However, the function of phosphorylated PPDK is still unknown in prokaryotic cell. Here, we showed that PPDK enzyme activity is higher in photoheterotrophic than in chemoheterotrophic. In our in vitro point mutation experiment revealed that threonine phosphorylation site played an important role in regulating PPDK activity. We suggested that light could stimulate threonine phosphorylation of PPDK and might enhance its activity which could regulate the switch mechanism of R. palustris in photoheterotrophic and chemoheterotrophic conditions.

碩士
國立臺灣大學
分子與細胞生物學研究所
96
Rhodopseudomonas palustris (R. palustris), a purple nonsulfur bacteria, is among the most metabolically versatile bacteria and ubiquitous in soil and water. The microbe could use sunlight, organic or inorganic compound for cellular energy, and use atmosphere carbon dioxide, or green plant-derived compound for cell material. It also could produce hydrogen gas by biological nitrogen fixation. The microbe could adjust and reweave itself in response to changes in light, carbon, nitrogen and electron sources. The ability of R. palustris to use carbon dioxide depends on two metabolic states: photoautotrophic and chemoautotrophic state. In this study, we used microarray for transcriptomic study, and two-dimensional polyacrylamide gel electrophoresis and mass spectrometry for proteomic study, respectively. Our results showed that 1357, 2512, 1629, 168 genes were differentially expressed over two-fold in photoautotrophic verus chemoautotrophic conditions, chemoautotrophic versus chemoheterotrophic conditions, photoautotrophic versus photoheterotrophic conditions, and chemoheterotrophic versus photoheterotrophic conditions, respectively. We identified 38 proteins differentially expressed over two-fold between two of the four major metabolic states. Some proteins, such as 60kDa chaperonin 2, ABC transporter related proteins, phosphoenolpyruvate carboxykinase, catalase, and phosphoglycerate dehydrogenase, could differentially expressed in three of these four major metabolic states. Interestingly, ABC transporter pathway was related to switch of autotroph and heterotroph. With computational prediction of plausible protein-protein interactions among these 38 proteins, we found 60kDa chaperonin 2, transcription terminator rho, acyl-CoA dehydrogenase, S-adenosylmethionine synthetase, alcohol dehydrogenase were the hub proteins in the predicted interaction network. These findings help us understand the regulatory mechanism of R. palustris.

碩士
中華醫事科技大學
生物科技研究所
97
The nitrogen cycle of aquatic ecosystem plays an important role, especially inaquatic farms. The effluent of nitrogen source is an invisible killer that could harm the aqua products. In the seawater or fresh water systems, the concentration of Ammonium(NH4+) and Nitrous acid(NO2-) higher than 2mg/L may cause the pathological change of fish. Therefore,this research was designed to study the use of Photosynthetic bacteria (PSB) to biomanipulate the concentration of inorganic nitrogen in the aquatic system. It might be helpful to the aquatic farms and aquatic environments if the increase of nitrogen concentrate is controlled efficiently. Rhodopseudomonas sphaeroides was used in this study and Microbial Pesticides methods were applied to control the content of nitrogen source in aquatic experiments. The results show that the amount of bacteria was saturated after 120 hours culture of R. sphaeroides. The saturated bacterial concentration reached to 109no./ml. In the process of culture, the pH value becomes weak alkalescence after 24 hour culture. The correlation between the amount of R.sphaeroides and the absorbance value of optical density at 660 nm is R2=0.9941. The Abs(OD660) values were utilized to measure the quantities of R. sphaeroides(Klaas,1982). Then, R. sphaeroides (108 no./ml) were added in five different concentrations of Nitrous acid in water to study the removal of Nitrous acid after 24 hours. The results show that all Nitrogen acid can be removed by R. sphaeroides completely. Next, R. sphaeroides (108 no./ml) were added in five different concentrations of Ammonium to study the removal rate of Ammonium. The removal rate of Ammonium is about 58% after 120 hours. Third, among three levels of bacterium concentration, the concentration of 109no. /ml is the best for removing Nitrous acid. Fourth, R. sphaeroides can stabilize the pH value in the water of aquaculture pond, but it has no effect on the removal of Nitrogen.

碩士
中國文化大學
生物科技研究所
88
Purple nonsulfur bacteria have been known to have potential use in agriculture and environment protection. They have been reported to enhance the soil fertility, stimulate flower bud diffraction and fruit maturation, and improve wastewater treatment and malodor removing and produce of single cell protein and amino acid for animal feed. In this study we screen and isolated purple nonsulfur bacteria from waste of local hog farm and investigate the results of co-culture with Bacillus megaterinm for the purple nonsulfur bacteria. In these studies the experiment on growth rate of purple nonsulfur bacteria are conducted with various cultural and nutrient conditions. The reduction of acetylene to ethylene is used to estimate the activity of nitrogen fixation. Comparison of the amount of ethylene production is conducted before and after the co-culture experiments for the investigation of our nitrogen fixation experiment. Our results show that the purple nonsulfur bacteria from local manure identify to be Rhodopseudomonas palustris and can grow under both aerobic and anaerobic condition with light and without light illumination for more than ten months. Cultures of Rhodopseudomonas palustris are red in anaerobically grown condition and yellowish white in aerobically grown condition. Under the 3000 lux illumination the generation time of Rhodopseudomonas palustris shorten and the pigment of bacteriochlorophyll a of each cell lessen as light intensity increase. The activity of nitrogen fixation of Rhodopseudomonas palustris and Bacillus megaterium are insignificant, however a 10-15 times increase while both bacteria culture together. The activity of nitrogen fixation of Rhodopseudomonas palustris demonstrates to be enhanced by co-culture with Bacillus megaterium.

碩士
國立臺灣大學
分子與細胞生物學研究所
97
Transketolase is a thiamin diphosphate (ThDP)-dependent enzyme that functions as a key catalyst in the oxidative pentose phosphate pathway of virtually all organisms. It also plays an important role in the Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle of photosynthetic organisms. In Rhodopseudomonas palustris, a purple, non-sulfur photobacterium, there are two isoforms of transketolase, CbbT1 and CbbT2, encoded by transketolase genes, cbbT1 (rpa4643) and cbbT2 (rpa0945), respectively. In this study, we overexpressed cbbT1 and cbbT2 under photoautotrophic conditions, in which CO2 was the only carbon source. We integrated oligo microarray analysis, real-time quantitative PCR (qPCR), bioluminescent ATP assay and absorbance spectra to study the effects of overexpressed transketolase. From our microarray data, overexpressed cbbT1 had more effects on R. palustris than overexpressed cbbT2. In the T1 strain, an increase of the peak amplitudes in the absorption spectrum and increased expression of related photosynthetic genes were observed. On the other hand, we measured the glycolytic ATP synthetic activity by bioluminescent ATP assay and observed an increased amount of ATP in the T2 strain. Moreover, immunogold labeling of HA-tag fusion tranketolase reveals that CbbT1 is mainly located in the intracytoplasmic membrane systems, but CbbT2 in the cytosol and the ICM structures are not abundant in the T2 strain compared with the T1 strain. In this study, we suggest that the two isoforms of transketolase are likely involved in different carbon metabolic pathways and have distinct effects on bacterial physiology.

Mass Spectrometry of proteins and biologically relevant molecules is an area in which a growing interest in being expressed. However, the field is still in its infancy with respect to the compilation of proteomes of both sub-cellular fractions and whole cells. A two step approach has been used to evaluate the suitability of the mass spectrometry technique on the purple non-sulphur bacteria, Rhodopsuedomonas palustris. I first analyzed the mass spectrometry of isolated chromatophores, vesicles formed by invaginations of the bacterial inner membrane, to evaluate our approach. I searched for proteins expected to be located in these structures, identified proteins that may be associated with the chromatophores and searched for potentially novel photosynthetically related hypothetical proteins. Subsequently, I investigated the complete proteome of the bacteria under a number of different environmental conditions and used a mutant strain of this bacterium. From the preliminary results, I created a new approach for display of the proteomics data obtained by my collaborators. This allows for the rapid examination of qualitative and quantitative aspects of proteins by colour-coded display of grouped peptides.

碩士
國立臺灣海洋大學
水產養殖學系
97
The influence of different light sources on the growth and carotenoid content of photosynthetic bacteria (Rhodopseudomonas palustris) was studied. For the experiment set up, 27 250ml flasks were used and 25ml (4.4×104 cell/ml ) photosynthetic bacteria solution per flask was added. Light intensity was set to 2000±100 lux. Eight light sources were used: incandescent lamp (IL), halogen lamp (HL), fluorescence lamp (FL), light-emitting diode (LED): LED white (LW), LED yellow (LY), LED red (LR), LED blue (LB), LED green (LG). Dark (DK) served as control. Each of the nine treatments had triplicates. Two trials were conducted to culture the bacteria for 96 and 144 hours. In the first trial, the effect of light sources on bacteria growth was: LB＞IL＞HL＞FL≧(LW＝DK＝LG＝LR)≧LY and on carotenoid content: LB＞IL≧LY≧(HL＝LR＝LG) ≧LW≧DK≧FL. In the second trial, light resource effect on growth was: (LB＝IL)＞FL＞LW≧HL≧LR≧(LG＝LY＝DK). Bacteria biomass production per unit electric power consumption were: LB＞LW＞LY＞IL＞LG＞HL＞FL＞LR. Production of photosynthetic bacteria by LB can save up to 75% of electricity cost than by IL. This study provides practical application of illumination for the production of photosynthetic bacteria.

碩士
國立臺灣大學
生化科技學系
104
Rhodopseudomonas palustris NTUIOB-PS3, a phototrophic purple non-sulfur bacterium, was originally isolated from paddy field in Taiwan. It can not only improve plant growth, but also reduce the nitrate accumulation in plants. The aim of this study was to investigate the mechanism of its plant growth promotion effect. We would like to elucidate the effects of rhizosphere bacteria on plant physiology through evaluating the nitrogen uptake efficiency, activity of nitrogen assimilation associative enzymes, gene expression of nitrate transporters, and CO2 assimilation rate of plants. When hydroponic solution was supplemented with the PS3 inoculant, plant growth (fresh weight of shoots and roots) of Chinese cabbage was greater than that without inoculation (control group). Since the gene expression of the nitrate transporter BjNRT1.1 in the roots and total amounts of N in the plants were dramatically elevated, we deduced that PS3 could stimulate the N uptake efficiency (NupE) of plants. The N uptake efficiency was around 40% increase in comparison with that in the control group. On the other hand, the plants inoculated with PS3 showed higher photosynthetic rate in the early stage of plants, then the leave sizes and total C amounts in shoot were increased nearly 25 % and 70%, respectively. Taken together, we deduced that PS3 improves both N and C metabolic efficiencies of plants through plant-microbe interaction, as a result of higher biomass accumulation.

碩士
國立中興大學
環境工程學系所
102
Environmental concerns, energy shortage, and consequently increasing energy costs result in the need to produce sustainable and renewable fuels. As Hydrogen has high energy density (142 MJ/Kg), a sustainable platform of hydrogen utilization can be established if it can be generated sustainably and used safely, as well as being stored and transported efficiently. There are various ways to produce hydrogen, among which photosynthetic hydrogen production by purple non-sufur photosynthetic bacteria (PNSB) is one of the favorable methods for biohydrogen production because of its high substrate conversion efficiency and its capability of utilizing abundant substrates. The most serious problem of photohydrogen production is that hydrogen production is inhibited by high ammonium concentration. The hydrogen production center of PNSB, the nitrogenase, is regulated by NifA protein. High ammonium concentration will inhibit NifA protein activities, and hence inhibit the transcription of nitrogenase gene and its expression. As a result, no hydrogen can be produced. The mutant of Rhodopseudomonas palustris WP3-5, Rhodopseudomonas palustris NifAnQ was constructed by molecule biotechnology. This mutant can tolerate high ammonium concentration. In this study, this mutant was employed to treat dark-fermentation effluent as substrate to recover hydrogen energy. The dark-fermentation effluent is rich in organic acid including acetate (1~6 g/L), lactate(1 g/L), butyrate(0.4~0.6 g/L), and high ammonium concentration(135 mg/L). This study treats the effluent as continuous culture condition’s substrate. To stabilize the pH value in the reactor, a continuous monitoring system was set up to prevent non-toxic form from transforming to toxic form of ammonium. At a high level of ammonium concentration with acetate and butyrate being carbon sources, the mutant photobioreactor operated continuously at HRT = 12 hr ; the maximum hydrogen concentration and hydrogen production rate are 74% and 172 mL-H2/day. At a high level of ammonium concentration with lactate being carbon source, it was proven that the wild type photobioreactor could not generate hydrogen. This study proved that mutant photobioreactor can operate under continuous culture condition with high ammonium concentration synthetic wastewater, and it can generate hydrogen continuously over 57 days. This demonstrates that the mutant photobioreactor has the capacity to treat effluent with high ammonium concentration to recover most of hydrogen energy.

The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively. BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety. The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target. In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann’s reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann’s reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above. We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB. The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme. Our structural analysis revealed that despite its low sequence identity of 28%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD
Die vorliegende Arbeit befasst sich mit der strukturellen und biochemischen Charakterisierung der beiden unterschiedlichen bakteriellen Enzyme BadI von Rhodopseudomonas palustris und MenD von Staphylococcus aureus. Die 2-Ketocyclohexancarboxyl-CoA-Hydrolase BadI ist eines der Schlüsselenzyme des anaeroben Abbaus aromatischer Verbindungen. Der Abbau aromatischer Verbindungen ist essentiell für die Aufrechterhaltung des biogeochemischen Kohlenstoffkreislaufs und der biologischen Beseitigung von Xenobiotika, welche in höheren Konzentrationen eine Gefahr für den menschlichen Organismus darstellen können. Wegen des inerten Charakters aromatischer Verbindungen sind Enzyme, welche deren Abbau katalysieren, von besonderem Interesse für industrielle Anwendungen. BadI ist eines der Schlüsselenzyme für den anaeroben Abbau aromatischer Verbindungen zu aliphatischen Gruppen. Das Hauptaugenmerk dieses Projekts lag auf der Aufklärung des Reaktionsmechanismus, welcher von BadI katalysiert wird. BadI gehört zur Überfamilie der Crotonasen und zeigt hohe Sequenzhomologie mit der zugehörigen Dihydroxynaphthoat-Synthase MenB. Durch die Hydrolyse einer C-C Bindung katalysiert BadI den Schnitt des zyklischen Rings von 2-Ketocyclohexancarboxyl-CoA, welcher zur Bildung der aliphatischen Verbindung Pimelyl-CoA führt. MenB, andererseits, katalysiert die Kondensationsreaktion von O-Succinylbenzyl-CoA zu Dihydronaphthoyl-CoA. Ein umfassender Aminosäuresequenzvergleich zwischen BadI und MenB zeigt, dass die Reste des aktiven Zentrums von MenB aus Mycobacterium tuberculosis (mtMenB) in BadI von R. palustris konserviert sind. MenB ist Teil des Menaquinon Biosynthesewegs und ein potentielles Wirkstoffziel gegen M. tuberculosis, da kein humanes Homolog existiert. Wegen der ausgeprägten Homologie zwischen MenB und BadI und der Tatsache, dass bisher keine MenB-Inhibitor Komplex Strukturen gelöst werden konnten, erweiterten wir unser Interesse auf BadI, da es als Model für mtMenB als Wirkstoffziel dienen könnte. Darüber hinaus besitzt BadI einige einzigartige mechanistische Charakteristika. Wie zuvor erwähnt, hydrolysiert es das Substrate durch eine reverse Dieckmanns Reaktion in Gegensatz zu seinem ähnlichsten Homolog MenB, das einen Ringschluss durch eine Dieckmanns Reaktion katalysiert. Dennoch scheinen die Reste des aktiven Zentrums streng konserviert zu sein. Daher entschieden wir die strukturelle Charakterisierung von BadI anzugehen um Gemeinsamkeiten und Unterschiede zwischen BadI und MenB aufzuzeigen und einen Einblick zu erhalten, wie sie die gegenläufigen Reaktionen durchführen. Wir lösten die ersten Strukturen von BadI in seiner Apo-Form und einer Substrat-Mimik gebundenen Form. Die Kristallstrukturen von BadI zeigten die gleiche Gesamtfaltung wie andere Mitglieder der Crotonase Familie. Allerdings gibt es in BadI kein Anzeichen für Domain-Swapping, wie es in MenB beobachtet wurde. Das Fehlen des Domain-Swappings ist bemerkenswert, da die vertauschte C-terminale helikale Domäne in MenB ein Tyrosin enthält, welches essentiell für die Katalyse ist und auch in BadI konserviert vorliegt. Der Vergleich des aktiven Zentrums zeigt, dass der C-Terminus von BadI so auf seinen Kern/Hauptteil faltet, dass das konservierte Tyrosin an der gleichen Stelle positioniert ist wie in MenB und mit dem Liganden interagieren kann. Die Struktur von BadI bestätigt auch die Rolle eines Serin- und eines Aspartatrests für die Ligandenbindung und bekräftigt damit, dass das konservierte aktive Zentrum an der enzymatischen Reaktion teilnimmt. Die Strukturen zeigen auch eine bemerkenswerte Verschiebung des aktiven Aspartats, welches zwei Hauptkonformationen einnimmt. Strukturelle Analysen zeigten auch die Nähe des Serinrests zu einem Wasser- und Chlormolekül, sowie einem Kohlenstoffrest, an dessen Stelle der Carbonylrest des eigentlichen Substrats läge. Die biochemische Charakterisierung von BadI in enzymkinetischen Untersuchungen bestätigte dass die vorgeschlagenen Reste des aktiven Zentrums an der Substratbindung beteiligt sind. Jedoch ist die Rolle der verschiedenen Reste sehr verschieden, wobei dem Serin eine herausragende Rolle zugedacht wird. Die hier dargestellte Arbeit bestätigt die Mitwirkung des mutmaßlichen aktiven Zentrums und zeigt, dass die Reste des Aktiven Zentrums von BadI eine unterschiedliche Rolle, im Vergleich zu ihrem ähnlichsten Homolog MenB, spielen. MenD, eine SEPHCHC (2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carbonsäure) Synthase, ist an der Menaquinonbiosynthese von S. aureus beteiligt. Obwohl S. aureus gewöhnlich als Kommensale betrachtet wird, kann es als bemerkenswertes Pathogen auftreten, wenn es die Epithelwand durchbricht und eine Vielzahl an Erkrankungen, von einfachen Hautinfektionen bis zu lebensbedrohlichen Zustanden, verursachen. Sogenannte „Small colony variants“ (SCVs), eine langsam wachsende, kleinzellige Subpopulation der Bakterien wurde mit persistenten, rezidivierenden und antibiotika-resistenten Infektionen assoziiert. Diese Varianten weisen einen Mangel von Thiamin, Menaquinon und Hämin auf. Menaquinon ist ein essentieller Bestandteil der Elektronentransport-Kette in grampositiven Organismen. Daher sind Enzyme dieses Stoffwechselwegs attraktive Wirkstoffziele gegen Krankheitserreger wie M. tuberculosis oder Bacillus subtilis. MenD, das Enzym, welches den ersten irreversiblen Schritt des Menaquinon-Biosynthesewegs katalysiert, wurde mit dem SCV Phänotyp von S. aureus in Verbindung gebracht. In dieser Arbeit werden die biochemischen und strukturellen Eigenschaften dieses wichtigen Enzyms untersucht. Unsere strukturelle Untersuchung zeigte, dass trotz einer niedrigen Sequenzidentität von 28%, die Gesamtfaltung von S. aureus MenD (saMenD) mit derjenigen von Escherichia coli MenD (ecMenD), trotz einiger signifikanter Abweichungen, übereinstimmt. Größere strukturelle Unterschiede können nahe des aktives Zentrums des Proteins beobachtet werden, vor allem in der C-terminalen Helix und einer Schleife nahe dem aktiven Zentrum. Die Schleife enthält kritische Reste für die Kofaktorbindung und liegt nur in der ecMenD-ThDP Komplexstruktur definiert vor, während die in der Apo-Form und der Substrat-gebundenen Struktur von ecMenD ungeordnet ist. In unserer saMenD Struktur zeigt sich die Schleife erstmals komplett geordnet in der Apo-Form und stellt eine neue Konformation der Kofaktor-Bindeschleife dar. Die Schleife nimmt eine ungewöhnlich offene Konformation an und die konservierten Reste, welche für die Kofaktorbindung verantwortlich sind, sind zu weit entfernt, um in dieser Position einen produktiven Komplex mit dem Kofaktor zu bilden. Zudem haben biochemische Studien in Verbindung mit den strukturellen Daten zur Identifizierung der Substratbindetasche und der an der Bindung und Katalyse beteiligten Aminosäuren beigetragen. In der vorliegenden Arbeit wurden die biochemischen und strukturellen Charakteristika von saMenD erfolgreich aufgeklärt

碩士
國立中興大學
環境工程學系所
97
Nowadays, the aggravation of greenhouse effect by combustion of non-renewable sources of energy such as fossil fuel, coal, oil, and nature gas has become a global issue. However, hydrogen can be an alternative and sustainable energy source due to the properties of clean and high energy yield（122 kJ/g）, which is an environmentally friendly technology in the future. Biohydrogen, comparing with the other technologies of hydrogen production, has the advantages of organic waste recycling and cost- effectiveness. Therefore, the aim of this study focuses on the combination of three different types of microorganisms and trying to enhance the efficiency of hydrogen production. This study was divided into two parts. Firstly, after anaerobic fermentation of leftovers, the possibility of using effluent as substrate for phototrophic hydrogen production was evaluated. Secondly, the feasibility of biohydrogen production from co-culture of purple nonsulfur bacterium, Rhodopseudomonas palustris WP3-5, and heterocyst-forming filamentous cyanobacteria, Anabaena sp. CH1, was estimated. In the first part, the inoculum of anaerobic fermentation was isolated from the sludge from wastewater treatment plant via heat treatment and BMP medium enrichment. The results show that the maximum cumulative volume of biohydrogen from semi-continuous anaerobic fermentation system is 248 ml, which is utilizing the leftovers as substrates. The results also indicate that the effluent contains large amount of volatile fatty acid but little amount of ammonium（5 mg/L）. Because the ammonium concentration in effluent is lower than the inhibition threshold（17 mg/L）of Rhodopseudomonas plaustris WP3-5 during biohydrogen production, phototrophic biohydrogen system could be successfully compatible with anaerobic fermentation system as two-stage biohydrogen production system. In the second part, the results from co-cultured system show that the maximum cumulative volume of biohydrogen reaches 140.83 ml in the WP3-5/CH1 mixed ratio of 2/1, which increases 2.48 fold comparing with the sum of cumulative volume（56.77 ml）from individual biohydrogen systems. Acetate, the metabolite of fructose via heterotrophic metabolism of Anabaena sp. CH1, could be taken by Rhodopseudomonas plaustris WP3-5 as energy source to produce biohydrogen. Therefore, the results from this study indicate that the co-culture biohydrogen system combining those two phototrophic bacteria is exactly feasible and the efficiency of biohydrogen production by co-cultured system is better than that of the single culture system.

碩士
中興大學
環境工程學系所
99
Hydrogen is a clean energy that generates only water upon burning and it releases high amount of energy (122 kJ/g) by combustion. Photo-biological hydrogen production by purple non-sulfur photosynthetic bacteria is one of promising methods. Purple non-sulfur photosynthetic bacteria can produce hydrogen by nitrogenase under photo-heterotrophic condition. In recent years, many researchers make efforts to improve its efficiency of biological hydrogen production. Poly-β-hydroxybutyrate (PHB) is accumulated as carbon and energy storage material under unbalanced growth. The synthesis of PHB may compete with hydrogen production for energy and reducing power. In addition, maximum hydrogen production rate usually takes place during early stationary phase. Therefore, energy distribution between cell growth, hydrogen production and PHB accumulation is an important issue. Besides, dark fermentation effluent containing various volatile organic acids is suitable for purple non-sulfur photosynthetic bacteria to produce hydrogen. To treat wastewater and recycle clean energy efficiently by biological method are also essential. In this research, Rhodopseudomonas palustris WP3-5 and its PHB synthase-deficient mutant Rps. palustris M23 were used in batch experiments to explore energy distribution between cell growth, hydrogen production and PHB accumulation under different culture conditions. This study also tried to isolate purple non-sulfur photosynthetic bacteria from environment, and tested their ability to produce hydrogen using different volatile organic acids. Then, hydrogen production and treatment efficiency in co-culture system with Rps. palustris WP3-5 were evaluated and continuous photosynthetic hydrogen production was performed. From the results, Rps. palustris WP3-5 accumulated PHB in growth phase, and PHB could be utilized as another carbon and energy source when its content reached maximum value. Microorganism ceased growing when nitrogen source was exhausted, and used excess energy to produce more hydrogen in stationary phase. Rps. palustris WP3-5 could not synthesize PHB under low concentration of carbon source because substrate was degraded rapidly, and there were no difference of hydrogen production between wild-type strain and mutant Rps. palustris M23. When concentration of carbon source was three times higher, cumulated hydrogen volume and substrate conversion efficiency of Rps. palustris WP3-5 were better than Rps. palustris M23. This result coincided with other experiments in different culture conditions. But when using malate as carbon source, Rps. palustris WP3-5 had lower substrate degrading rate and cumulated hydrogen volume, and PHB was not accumulated. Different species of purple non-sulfur photosynthetic bacteria had different substrate assimilation pathway when using same carbon source and accumulated PHB content might be different. Rps. palustris WP3-5 used most energy to produce hydrogen in stationary phase and PHB content was below 10% cell dry weight, accounting for less than 5% of the substrate electrons utilized. This portion of energy might partially redistribute to synthesize soluble microbial product (SMP). Therefore, the competition relationship between hydrogen production and PHB accumulation was insignificant. In second part, several purple non-sulfur photosynthetic bacteria were isolated, and there were no obvious difference in utilizing volatile organic acids to produce hydrogen between these strains. Hydrogen production was not improved and competition for energy might exist in co-culture system. Additionally, new designed photo-bioreactor was used for Rps. palustris WP3-5 to produce hydrogen continuously when using tungsten filament lamp as light source. Operating hydraulic retention time from 2 day to 3 day, gas production rate was highest in initial period, up to 317.6 mL gas/L culture-day. MLSS was always between 220 and 360 mg/L, and MLSS wasn’t enhanced by changing operational strategies. Thinking of light transmission efficiency and problem of light shielding effect caused by high biomass concentration, experimental results was not as expected, and this photo-bioreactor might not favorable for continuous hydrogen production by purple non-sulfur photosynthetic bacteria.

碩士
國立臺灣海洋大學
水產養殖學系
100
Photosynthetic bacteria have a wide application, is rich in carotenoids, and can be used as probiotics in aquaculture. Since light source can affect the growth of photosynthetic bacteria and their carotenoid composition, this study used 8 light sources: light-emitting diode (LED) green (LG), blue (LB), red (LR), white (LW), yellow (LY), halogen light (HL), incandescent light (IL), and fluorescence (FL) to find out their effects on the growth and carotenoid content of 3 photosynthetic bacteria (Rhodobacter capsulatus, Rhodopseudomonas palustris, Rhodobacter sphaeroides). Three experiments were conducted: Experiment I and II were after low (4.1 x 105 cell/ml) and high (4.2 x 106 cell/ml) concentration of inoculation of 3 photosynthetic bacteria, respectively, the effects of various light sources on growth of photosynthetic bacteria. Experiment III was the effects of various light sources on the carotenoid content of 3 photosynthetic bacteria. Experiment I indicated that for the bacteria growth the sequences were: (LB＝IL)＞(LW＝HL＝FL)＞LR＞(LG＝LY)＞DK, for the total carotenoid content the sequences were: LB＞IL＞(LW＝HL＝FL)＞(LG＝LR＝LY)＞DK, and for the bacteria growth and total carotenoid content were: R. capsulatus＞R. palustris＞R. sphaeroides. Since R. capsulatus had the highest wave length absorbance in 400~500 nm and the wave length of LB was 460~470 nm, the culture of R. capsulatus under LB resulted in the highest production. Experiment II indicated that for the bacteria growth the sequences were: (LB＝IL)＞FL＞LW＞(LG＝LR)＞LY＞DK. Cultured under LB the highest bacteria growth was 1.0 x 107cell/ml and 0.9 x 107cell/ml for Experiment I and II, respectively. Although headed with higher inoculation concentration, Experiment II had lower growth, which was attributed to nutrient deficiency and self-shadow effect. In Experiment III, light source effects on the production of carotenoid were as the following: β-carotene: LB＞IL＞LW＞(HL＝FL)＞(LG＝LR＝LY)＞DK, zeaxanthin: LB＞LW＞(LY＝IL＝FL)＞(HL＝LR＝DK＝LG), lutein: (LB＝LW)＞LR＞LG＞(LY＝DK＝HL＝IL＝FL), and canthaxanthin: IL＞(LY＝HL＝FL)＞DK＞(LG＝LB＝LR＝LY). LB was in the best absorbance range of β-carotene, zeaxanthin and lutein, but not for canthaxanthin.

博士
國立臺灣大學
分子與細胞生物學研究所
101
Protein phosphorylation is one of the most important post-translational modifications in cells, including prokaryotes and eukaryotes. Systematic study of phosphoproteome in cells is becoming a crucial determinant for understanding the regulation of cell physiology. In the study, we applied the phosphoproteomic analyses to investigate the molecular regulation in both prokaryotic and eukaryotic systems. An efficient approach, hydroxy acid-modified metal oxide chromatography (HAMMOC), was used for phosphopeptide enrichment. In the prokaryotic phosphoproteome study, the regulation of different metabolic states in Rhodopseudomonas palustris (R. palustris) was studied. R. palustris is a purple nonsulfur anoxygenic phototrophic bacterium with metabolic versatility and is able to grow under photoheterotrophic and chemoheterotrophic states. It also has the potential for bioenergy production and biodegradation. This study is the first to identify the phosphoproteome of R. palustris including 100 phosphopeptides from 54 phosphoproteins and 74 phosphopeptides from 42 phosphoproteins in chemoheterotrophic and photoheterotrophic growth conditions, respectively. In the identified phosphoproteome, phosphorylation at the threonine residue, Thr487, of pyruvate phosphate dikinase (PPDK, RPA1051) was found to participate in the regulation of carbon metabolism. Here, we show that PPDK enzyme activity is higher in photoheterotrophic growth, with Thr487 phosphorylation as a possible mediator. Under the same photoheterotrophic conditions, R. palustris with overexpressed wild-type PPDK showed an enhanced accumulation of total lipids than those with mutant PPDK (T487V) form. This study reveals the role of the PPDK in the production of biodiesel material, lipid content, with threonyl-phosphorylation as one of the possible regulatory events during photoheterotrophic growth in R. palustris. In the eukaryotic phosphoproteomic study, the response pathways of ATP synthase inhibitor in lung cancer cells were investigated. ATP synthase is a multimeric protein complex that catalyzes the synthesis of ATP. For a long time, animal ATP synthase was believed to be found only in mitochondria; however, in recent studies, ATP synthase was also found on the extracellular surface of some cell types including cancer cells, named as ectopic ATP synthase. ATP synthase inhibitor is a potential drug candidate for fighting cancer by blocking the ectopic ATP synthase without obvious damages to mitochondrial function. In this study, we performed quantitative phosphoproteomics to elucidate the molecular response to ectopic ATP synthase inhibition in both cell and xenograft systems. A total of 2834 phosphopeptides covering 836 phosphoproteins and 862 phosphopeptides containing 423 phosphoproteins were identified in cells and xenograft phosphoproteome, respectively. The gene ontology analysis showed that ATP synthase inhibitor treatment had the impacts on phosphorylated proteins involved in protein folding, cell cycle, and cytoskeleton. Clustering and network analysis of time-dependent phosphorylation profiles further revealed that inhibiting ectopic ATP synthase could immediately down-regulate the phosphorylation of heat shock protein 90 (HSP90AB1), and reduce the function of mitogen-activated protein kinase 1 (MAPK1) in the late-response by dual de-phosphorylation of a TEY activation motif, which is involved in the MAPKs/ERKs cascade associated with cancer progression. We next found that the dephosphorylated HSP90-S255A with alanine substitution of the identified phosphorylated residue Ser255 was unable to mediate the phosphorylation of MAPK1 and the activity of MAPKs/ERKs signaling. These results imply that inhibiting ecto-ATP synthase might suppress the cancer growth through the de-phosphorylation of HSP90-regulated MAPKs/ERKs signaling. This study provides the new insight into the development of novel therapeutic strategies that exploit response pathways on lung tumor suppression.