Depuis la découverte des sources hydrothermales en 1977, un petit nombre d'études ont permis l'isolement et la caractérisation de micro-organismes pouvant résister à de hautes pressions et températures. Parmi ceux-ci, Pyrococcus yayanosii, une archée hyperthermophile de l'ordre des Thermococcales est issue du site Ashadze (dorsale medio-atlantique) à 4100 m de profondeur. Cette espèce représente le premier organisme à la fois hyperthermophile et piézophile strict décrit à ce jour. Elle ne peut en effet se diviser à des pressions inférieures à 20 MPa et sa pression optimale de croissance est de 52 MPa. Afin d'étudier les mécanismes que met en oeuvre cette espèce pour se développer sous hautes pressions, des expériences de transcriptomique (puces à ADN) et protéomique (LC-MS/MS) ont été entreprises à différentes pressions notamment sub- et supraoptimales. La distinction entre les effets « stress » et « adaptations » à la pression a été effectué en comparant les résultats obtenus chez une autre Thermoccocale proche, Pyrococcus furiosus, qui est piézosensible. La détermination des pressions sub et supra optimales a été préalablement effectuée sur une large gamme de pression hydrostatique. Des analyses génomiques ont aussi été effectuées sur les Thermococcales en général et ses deux espèces en particulier et montrent des différences importantes au niveau des voies de biosynthèse des acides aminés ainsi que des transporteurs membranaires. Les analyses transcriptomiques et protéomiquesmontrent que P. yayanosii joue essentiellement sur ses mécanismes de production d'énergie (métabolisme del'hydrogène), de mobilité (chimiotactisme), de traduction (protéines ribosomales) ainsi que sur ses mécanismes de défense (CRISPR/cas). P. furiosus met en place des mécanismes se basant aussi sur la traduction et la mobilité (archaellum). Il semble que ces derniers puissent ainsi être considérés comme des réponses aux stress, alors que la modulation énergétique uniquement présente chez P. yayanosii soit plus un « shift » métabolique permettant à la cellule de s'adapter aux différentes conditions de pression de son environnement. / No

Developing treatments for antibiotic resistant bacterial infections is among the most urgent public health challenges worldwide. Tetracyclines are one of the most important classes of antibiotics, but like other antibiotics classes, have fallen prey to antibiotic resistance. Key small changes in the tetracycline structure can lead to major and distinct pharmaceutically essential improvements. Thus, the development of new synthetic capabilities has repeatedly been the enabling tool for powerful new tetracyclines that combatted tetracycline-resistance. Traditionally, tetracycline antibiotics were accessed through bacterial natural products or semisynthetic analogs derived from these products or their intermediates. More recently, total synthesis provided an additional route as well. Importantly however, key promising antibiotic candidates remained inaccessible through existing synthetic approaches. Heterologous biosynthesis is tackling the production of medicinally important and structurally intriguing natural products and their unnatural analogs in tractable hosts such as Saccharomyces cerevisiae. Recently, the heterologous biosynthesis of several tetracyclines was achieved in Streptomyces lividans through the expression of their respective biosynthetic pathways. In addition, the heterologous biosynthesis of fungal anhydrotetracyclines was shown in S. cerevisiae. This dissertation describes the use of Saccharomyces cerevisiae towards the biosynthesis of target tetracyclines that have promising prospects as antibiotics based on the established structure-activity relationship of tetracyclines but have been previously synthetically inaccessible. Chapter 1 provides an introduction to the pursuit of tetracycline antibiotics using S. cerevisiae. Following an overview of tetracycline drugs, the chapter describes the methods for making tetracyclines and their limitations in accessing the tetracycline analogs targeted in this study. The desirability of making these target analogs as well as key desired properties are then exemplified by natural products, totally synthetic and semisynthetic derivatives. The target tetracycline analogs pursued in this study are then outlined and the considerations in choosing their desired properties are discussed, as well as the reasons for employing S. cerevisiae in their synthesis. Chapter 2 describes the use of Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis, setting the stage for total biosynthesis of tetracyclines in Saccharomyces cerevisiae. Chapter 3 describes the work towards biosynthesis of the target tetracycline analogs using Saccharomyces cerevisiae, utilizing successful expression optimization and gene biomining approaches. Chapter 4 describes the work towards the target tetracycline analogs from fungal anhydrotetracyclines in Saccharomyces cerevisiae. The challenge of enzyme evolution towards unnatural substrates and the complex environment of cells require metabolic engineering efforts to be performed in libraries, as it is currently impossible to predetermine which modifications will prove beneficial. Traditional methods in DNA mutagenesis and increasingly, advances in DNA synthesis, DNA assembly and genome engineering are enabling high throughput strain construction. Thus, there is a need for a general, high-throughput, versatile and readily implemented assay for the detection of target molecule biosynthesis. The development of such an assay is described in Chapter 5. The assay is demonstrated to detect tetracycline derivatives, and differentiate a producer and a nonproducer strain of the fungal anhydrotetracycline TAN-1612. The yeast three hybrid assay for metabolic engineering of tetracycline derivatives described in this chapter could be used in the next steps towards the heterologous biosynthesis of the target tetracycline analogs in S. cerevisiae and beyond.

No description available.

Abstract not available

Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field. Factors limiting mycoherbicide efficiency include temperature and humidity. Results from this thesis indicate that solar radiation has both a damaging effect(reduction in germination)limiting efficacy and a photomorphogenic effect(appressorium induction)increasing efficacy. The study has also shown significant interaction between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as third major component of the environment that should be considered when trying to produce mycoherbistats. With the findings presented in this thesis and further research on disease development under different conditions, in combination with the formulation of conidia in suitable UV protectants, a computer simulation modelling the conditions leading to epidemics caused by C.orbiculare, D.avenacea and R.alismatis could be constructed. It may be possible to manipulate fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed. / Doctor of Philosophy (PhD)

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.

Genome plasticity in soil bacteria is predicted to be evolutionarily advantageous, allowing bacteria to sample genetic variation for adaptation to local soil ecology. In the field population of mesorhizobia where the symbiosis island (ICEMlSym[R7A]; an I̲ntegrative C̲onjugative E̲lement) was first identified, individual members were found to have significant chromosomal variation downstream of the phe-tRNA gene or phe-tRNA integrated ICEMlSym[R7A]. However, the nature of this genetic variation and whether it contributed to the adaptation of the indigenous mesorhizobia to their field environment were unknown. This work focused on a nodule isolate, Mesorhizobium sp. strain R88B, a member of the indigenous mesorhizobial population that received ICEMlSym[R7A] from strain R7A. The region downstream of ICEMlSym[R7A] was sequenced, revealing three distinct regions of non-conserved DNA, totalling 34.5 kb. Integrated directly downstream of ICEMlSym[R7A] was IMEMlAdh[R88B], a 24.3-kb novel I̲ntegrative M̲obilisable E̲lement. Using a PCR-based assay, it was shown that the IMEMlAdh[R88B] integrase could excise not only IMEMlAdh[R88B], but also a dual-IMEMlAdh[R88B]/ICEMlSym[R7A] hybrid, indicating the potential mobility of IMEMlAdh[R88B], and a likely evolutionary intermediate of a novel ICE. However, a functional role for MadA, (a putative adhesin and the sole adaptive trait encoded on IMEMlAdh[R88B]) was not discovered. Southern hybridisations with the mesorhizobial population provided evidence for the existence of a novel family of IMEs in the mesorhizobia, which, by diversifying their internal sequences, provide allele-specific variation to the population. The two other regions downstream of IMEMlAdh[R88B] possessed no obvious mobile genetic element structures, and only the region adjacent to the core-chromosome encoded ORFs with putative functions. Mutation of two of these ORFs, fhuD1 and fhuB1, identified their function as two of the four components of a ferrichrome ABC-uptake (Fhu) system. Using genetic screens, the remaining components of this transporter were mapped to two separate loci. Thus, the functional transporter in R88B was a composite of at least two independently-acquired Fhu systems. The genetic screens also revealed that ferrichrome utilisation was dependent on a TonB energy-transduction system encoded downstream of the Fhu ATPase gene, fhuC. Expression studies on the three fhu loci demonstrated that, despite their separate acquisition, their expression was coordinately up-regulated in response to low-iron conditions. Bioinformatics on the predicted promoter regions of the fhu genes identified the binding site of the rhizobial Fur analogue, RirA, which is likely to be responsible for this expression profile. Southern hybridisations of DNA isolated from members of the mesorhizobial population revealed the three fhu loci were not conserved in the mesorhizobial population. The presence of FhuA was the best predictive marker for the trait. It is proposed that multiple rounds of acquisitions and recombinations, both illegitimate and legitimate, formed this transporter, with the constant need for iron offset by the negative selection pressure of FhuA being a target for phage. None of the Fhu-specific genes was present in the sequenced M. loti strain MAFF303099 though flanking sequences were, further emphasizing the role of genome microevolution in forming the Fhu phenotype.

Thesis (Master of veterinary science)--Washington State University, December 2007. / Includes bibliographical references (p. 13-17).

<p>Denna rapport är en sammanställning av de 100 undersökta enskilda brunnarna i Landskrona kommun vid årsskiftet 05/06. Det utfördes analyser på mikrobiologisk-, nitrat-, nitrit- och även kemiska bekämpningsmedelspåverkan. Syftet med denna rapport är att bearbeta och presentera analysresultaten av de 100 enskilda brunnarna i Landskrona kommun och försöka dra slutsatser kring dessa. Alla analysresultaten sammanställdes i tabeller och så småningom kundes vissa slutsatser dras kring varför resultatet såg ut som det gjorde. Resultatet visade att endast åtta av de 100 brunnarna var otjänliga gällande mikrobiologisk påverkan. Denna parameter är väldigt varierande beroende mycket på yttre omständigheter men det kan ge en indikation på att brunnen inte är tillräckligt tät och därför kan ytvatten tränga in i brunnen. När det gäller nitritpåverkan blev bara en brunn otjänlig. Men när det gällde nitrat var det istället 17 % av brunnarna som visade sig vara otjänliga. Detta är ändå ett relativt bra resultat beroende på hur landskapet ser ut i Skåne med stor påverkan från enskilda avlopp, jordbruk och gödsling. Sist men inte minst analyserades kemiska bekämpningsmedel och gav ett oroande resultat. Hela 35 % av brunnarna var otjänliga pga. kemiska bekämpningsmedel. Detta resultat är inget ovanligt utan kan genom danska undersökningar bara bekräftas. Kemiska bekämpningsmedel kan sprida sig långa sträckor och inte bara vid den plats där ämnena använts eller spillts. De slutsatser som kan dras genom denna undersökning är att grävda brunnar i ytliga jordlager löper störst risk att i framtiden bli otjänliga då dessa utsetts för störst påverkan av föroreningar och ytvatten. Brunnar placerade på gårdsplan är speciellt utsatta och är starkt påverkade av både mikroorganismer och kemiska bekämpningsmedel. När det gäller kemiska bekämpningsmedel är resultaten från Landskrona liknande de från både Danmark och övriga Skåne.</p> / <p>The problem with bad drinking water in wells are a wide problem in an landscape such as Skåne. The purpose of this report is to work up all the data from the different analysis regarding drinking water quality from wells in Landskrona municipality. The parameters that were analysed were microorganisms, the content of nitrate and nitrite and also the concentration of pesticides. Many reports in the field of the subject were collected to get a good picture and a better knowledge of the different problems that lie ahead. This resulted in various conclusions but some of the conclusions was confirmed from other examinations. That wells in the ground are more exposed than wells in mountain. The concentration of pesticides were very high in many of the wells wich shows that this will be a great problem for us to deal with in the future. Examinations in both Denmark and remaining parts of Sweden also show high concentration of pesticides. This only confirm that the problem isn’t just a local here in the municipality.</p>

This thesis focused on using microcosms to better understand the aerobic cometabolic processes of TCE and cis-DCE transformation that occurred during a Cometabolic Air Sparging (CAS) demonstration at McClellan Air Force Base. The microcosms were created with groundwater and aquifer materials from the demonstration site. Concentrations of compounds in the microcosms were maintained to mimic conditions where the demonstration was performed. Propane was used as the primary substrate to stimulate indigenous propane-utilizers present in the McClellan subsurface. The microcosms were used to test the potential of the propane-utilizers to transform the CAHs of interest, and determine their nutrient requirements while transforming these compounds. Vadose zone microcosms were also created and used to compare the cometabolic processes and nutrient requirements of the propane-utilizers under these different conditions. After the addition of propane a ten-day lag period was observed before the propane-utilizers were stimulated in all the microcosms. The presence of CAHs and excess nitrogen did not have any effect on the lag period required to stimulate these microorganisms. Microcosms that received nitrogen amendments maintained effective transformation of TCE and c-DCE with successive additions. The rate of c-DCE transformation was observed to be faster than TCE transformation. Complete removal of the CAHs occurred in these microcosms. No other nutrients, such as phosphorous, were observed to cause any nutrient limitations. However, the microcosms that only had limited amounts of nitrogen present were only able to maintain transformation ability for a short time. Propane utilization rates gradually decreased with each addition, and CAH transformation eventually ceased. This was also observed during the CAS field demonstration after successive additions of propane. Ammonia gas was added to the sparge gas in the field and propane utilization and CAH transformation resumed. Ammonia gas was added to the nitrogen-limited microcosms, and like the field demonstration, propane utilization and CAH transformation resumed. Nitrogen was found to be a critical nutrient for effective cometabolism of CAHs. Nitrogen supplied either as ammonia or nitrate was required for the propane-utilizers to maintain effective rates of propane utilization and CAH transformation ability. By comparing different sets of microcosms under different conditions, estimates were made to the amount of nitrogen required by the propane-utilizers with and without CAHs transformed. The transformation of CAHs significantly increased the propane-utilizers requirements for nitrogen. A 2.0-3.8-fold increase in was observed for nitrogen consumption when CAHs were transformed, possibly resulting from toxic effects caused by the transformations. The sparge gas used at the CAS demonstration also contained ethylene at a low concentration (1% vol/vol). The microcosm experiments with this concentration of ethylene were found not to have any negative effects on CAH transformation. The propane-utilizers were also able to maintain propane utilization and CAH transformation at high CAH concentrations. The vadose zone microcosms showed that propane utilization in the vadose zone was an order of magnitude lower than what was observed in the saturated microcosms. Also bioavailable nitrogen was required to maintain propane utilization rates. However, higher CAH concentrations were found to inhibit the stimulation of the propane-utilizers under these conditions. / Graduation date: 2002