Dissertations / Theses on the topic 'Microorganisme extracellulaire'

Les symbioses et le microbiote sont devenus des sujets d’étude prioritaires, souvent explorés grâce à l’organisme modèle Drosophila. Pourtant, nos connaissances des relations naturelles entre drosophiles et symbiotes microbiens, c’est à dire en dehors du laboratoire, sont fragmentaires. Or, comprendre la coévolution entre hôte et symbiotes microbiens nécessite une fine description des effets des partenaires symbiotiques les uns sur les autres. Dans cette thèse, j’ai étudié de façon empirique les interactions entre drosophiles (Drosophila melanogaster et D. suzukii) et symbiotes extracellulaires (bactéries et levures) avec des souches sauvages et dans des conditions qui reproduisaient la nature. Ma thèse a porté sur les trois questions suivantes : (i) comment les drosophiles acquièrent et conservent-elles ces microorganismes tout au long de leur cycle de vie ; (ii) quels sont les effets de ces microorganismes sur le développement de leurs hôtes ; (iii) comment ces microorganismes interagissent-ils entre eux ? Mes travaux ont révélé que levures et bactéries sont plus que des sources de nourriture (i.e. acquisition des ressources) puisqu’elles influencent également comment la larve alloue ses ressources entre traits d’histoire de vie (i.e. plasticité développementale). L’étude des phases d’acquisition et de transmission des symbiotes microbiens par l’insecte en conditions proches de la nature a montré que ceux-ci sont partiellement acquis de l’environnement, conservés entre différents stades de vie, transmis entre générations et lors de l’accouplement. Par ailleurs, j’ai découvert de substantielles interactions entre symbiotes microbiens affectant leur multiplication et leur transmission entre stades de vie de l’hôte. Mon travail révèle une certaine complexité des interactions naturelles entre drosophiles et leurs symbiotes microbiens. Il démontre non seulement que ces interactions sont durables, mais également qu’elles sont composées d’effets imbriqués, simultanés et invisibles dans les systèmes nécessairement simplifiés des laboratoires. Mes travaux apportent également des pistes pour améliorer le contrôle des populations de D. suzukii
Microbiota and symbiotic interactions are priority topics that are often explored using the Drosophila model organism. However, existing knowledge of natural relationships, i.e. in situ, between Drosophila flies and microbial symbionts is fragmented. Understanding coevolution between a host and its microbial symbionts requires a detailed understanding of the co-effects between symbiotic partners. In this PhD, I empirically studied the interactions between Drosophila flies (the model organism D. melanogaster and the pest species D. suzukii) and extracellular microbial symbionts (bacteria and yeasts) using wild strains under near-natural conditions. I investigated three main questions: (i) how do Drosophila flies acquire and transmit their microbial symbionts along their life cycle; (ii) how do these microorganisms affect host development; (iii) how do these microorganisms interact? My work revealed that yeasts and bacteria are not simply sources of nutrition (i.e. resource acquisition) for Drosophila but also influence how fly larva allocate resources between different life history traits (i.e. developmental plasticity). Second, the conducted study on microbial acquisition and transmission phenomena under near-natural conditions showed that symbionts are partially acquired from the environment, conserved through different life stages, and transmitted between generations and through mating. Thirdly, I found substantial interactions between microbial symbionts that affect their multiplication and transmission between host generations. These results reveal natural interactions of some complexity between Drosophila flies and their microbial symbionts. This demonstrates not only that these interactions are durable but also composed of nested effects that are simultaneous and invisible in obligatorily simplified laboratory systems. In addition, this work brings new elements likely to improve population control of the pest D. suzukii

Des bactéries adaptées au froid ont été sélectionnées pour leur capacité à dégrader des polysaccharides naturels. L'influence de la température de croissance, sur la régulation de la production des enzymes exocellulaires, a été étudiée. Nous avons mis en évidence l'existence de cinq profils de régulation qui ne sont corrélés ni a l'origine taxonomique des souches, ni au type d'activité produite. D'autre part, chez une même souche, le profil de production d'une activité donnée peut varier suivant la composition du milieu de culture, notamment en substrat carboné, ainsi qu'en fonction de la présence d'inducteur spécifique. La dépendance des profils de production à l'égard des conditions nutritionnelles, démontre également qu'aucune loi générale ne peut être établie quant à l'influence de la température sur la production des enzymes exocellulaires, chez les bactéries adaptées au froid. Cependant des comportements différents peuvent être observés suivant le groupe thermique des souches. En utilisant la représentation selon Arrhénius, du taux de croissance en fonction de la température, nous avons démontré l'existence d'une température critique de rupture de pente chez toutes les souches psychrotrophes, celle-ci étant absente chez les psychrophiles. Sur la base de ce critère distinctif nous avons établi que, chez les psychrotrophes, la production des enzymes exocellulaires varie de façon discontinue sur l'ensemble de la gamme de températures suboptimales de croissance, ce qui se traduit par l'existence de deux domaines thermiques de production. En revanche chez les psychrophiles la production varie de façon monotone sur la gamme de températures inférieures à l'optimum de croissance. Ces profils pourraient résulter d'adaptation en réponse aux différentes amplitudes de température caractérisant les environnements d'origine des représentants de chacun de ces deux groupes thermiques.

Thesis (Ed.D.)--Teachers College, Columbia University, 1994.
Typescript; issued also on microfilm. Sponsor: O. Roger Anderson. Dissertation Committee: Patricia L. Dudley. Includes tables. Includes bibliographical references (leaves 119-129).

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or regarded as undesired yeasts in wine. However, these yeasts have recently come under investigation for their positive contribution towards wine aroma especially when used in sequential or co-inoculated fermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number of enzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases. The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction, as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content of the wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may have an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Although many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no attempts have been made to characterize these enzymes. This study set out to isolate and characterize genes encoding extracellular aspartic proteases from non-Saccharomyces yeasts. An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts, isolated from grape must, was performed. The aspartic protease-encoding genes of two non- Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, were isolated and characterized by in silico analysis. The genes were isolated by employing degenerate and inverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1. The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. The MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular weight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative protein with a predicted molecular weight of 39 kDa. These features are typical of extracellular aspartic proteases. The deduced protein sequences showed less than 40% homology to other yeast extracellular aspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the gene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced in media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was available. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape juice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in the presence of grape juice proteins, which was expected since these proteins are present in the natural habitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 other M. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was also visualized on plates. As far as we know, this is the first report on the genetic characterization of secreted aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the groundwork for further investigations.
AFRIKAANSE OPSOMMING: Nie-Saccharomyces giste is voorheen met wynbederf geassosieer en hul teenwoordigheid in wyn is ongewens. Hierdie giste is onlangs ondersoek vir hulle positiewe bydrae tot wyn aroma, in veral sekwensiële en ko-inokulerings met Saccharomyces cerevisiae. Sommige van die nie-Saccahromyces giste skei ‘n verskeidenhied ensieme af wat moontlik vir die wynmaker van nut kan wees. Een groep van hierdie ensieme is die aspartiese suurproteases. Soos deur sommige navorsers aangetoon word, kan die proteases die vorming van proteïenwaasverlaging, terwyl dit terselfdertyd die assimilerende stikstofinhoud van die wyn vir die gebruik en groei van fermentasie-mikroörganismes verhoog. Die proteases kan moontlik ook ‘n indirekte uitwerking op die aromaprofiel van die wyn hê deur die vrystelling van aminosure wat as aromavoorlopers dien. Alhoewel baie studies gedoen is wat die ekstrasellulêre teenwoordigheid van proteases bevestig in nie-Saccharomyces giste wat van druiwesap/wyn afkoms is, is daar geen dokumentasie oor die genetiese karakterisering van hierdie ensieme beskikbaar nie. Die doel van hierdie studie was om gene wat aspartiese proteases in nie-Saccharomyces giste enkodeer, te isoleer en gedeeltelik te karakteriseer. ‘n Versameling van 308 Saccharomyces en nie-Saccharomyces giste wat uit druiwe sap geïsoleer is, is gesif vir ensiematiese aktiwiteit deur plaattoetse uit te voer. Twee gene wat aspartiese protease enkodeer, is geïsoleer van twee nie-Saccharomyces giste. Dit hetpositief gedurende die aktiwiteitstoetse getoets en is deur in silico–analise gekarakteriseer. Die gene is deur die uitvoering van gedegenereerde en inverse PKR geïdentifiseer. Een geen is vanaf Metschnikowia pulcherrima IWBT Y1123 geïsoleer en is MpAPr1 genoem, terwyl die ander van Candida apicola IWBT Y1384 geïsoleer en CaAPr1 genoem is. Die MpAPr1-geen is 1137 bp lank en enkodeer ‘n proteïen wat uit 378 aminosure bestaan met ‘n voorspelde molekulêre massa van 40.1 kDa. Daar teenoor is die CaAPr1-geen 1101 bp lank en enkodeer vir ‘n proteïen wat uit 367 aminosure met ‘n molekulêre massa van 39 kDa bestaan. Hierdie eienskappe is kenmerkend van aspartiese protease. Die afgeleide proteïenvolgorde het minder as 40% homologie met ander ekstrasellulêre aspartiese proteases vertoon, wat dui op die nuwigheid van hierdie ensieme. Die MpAPr1-geen is heterologies in S. cerevisiae YHUM272 uitgedruk en dit het bevestig dat die geen inderdaad ‘n ekstrasellulêre aspartiese protease enkodeer. Die MpAPr1-geen is uitgedruk in media wat alleenlik proteïen as stikstofbron bevat het, terwyl dit onderdruk is in gevalle waar ‘n verkose stikstofbron beskikbaar was. Die geen is uitgedruk in die teenwoordigheid van kaseïen, BSA en proteïene afkomstig vanaf druiwesap en in die teenwoordigheid van ammoniumsulfaat onderdruk. Die hoogste uitdrukking was in die teenwoordigheid van druifproteïene. Hierdie proteïene is teenwoordig in die natuurlike habitat van die gis en is dus dalk ‘n bekende stikstofbron vir die gis. ‘n Genetiese sifting het die teenwoordigheid van die MpAPr1-geen in 12 ander M. pulcherrima–rasse, wat ook van wynkundige oorsprong is, bevestig. Die aspartiese protease-aktiwiteit van die 12 rasse is ook op agarplate waargeneem. Na ons wete, is dit die eerste verslag oor die genetiese karakterisering van afgeskeide aspartiese proteases van nie- Saccharomyces giste van wynkundige oorsprong en verskaf die grondslag vir verdere ondersoek.

L’objectif de ces travaux a été d’étudier le rôle des exopolysaccharides dans la formation des biofilms de Listeria monocytogenes et dans leur résistance face à des procédures de nettoyage rencontrées en entreprise dans des circuits fermés. Nous avons montré que l’exopolysaccharide majeur présent dans la matrice extracellulaire des biofilms de L. monocytogenes était de l’acide téichoïque identique à l’acide téichoïque pariétal. Nous avons identifié que sur 93 souches de sérotype 1/2a étudiées la moitié présentaient une mutation sur le gène lmo2550 ce qui pouvait entraîner la non-ramification de l’acide téichoïque avec le résidu N-Acétylglucosamine (GlcNAc). Des mutants de la souche de référence EGD-e inactivés pour les gènes lmo2549 ou lmo2550 intervenant dans la ramification de l’acide teichoïque par le GlcNAc ainsi que des mutants inactivés pour les gènes lmo2537 ou tagO1tagO2 intervenant dans la voie de biosynthèse des acides téichoïques ont été étudiés. Cela nous a permis de montrer que l’absence de GlcNAc sur les acides téichoïques avait modifié les propriétés de surface de L. monocytogenes, diminué l’adhésion à l’acier inoxydable et modifié l’architecture des biofilms de 48h. Par ailleurs, ils étaient plus sensibles à une procédure de nettoyage en circuit fermé avec un détachement plus important, une modification de l’architecture des biofilms et la présence uniquement de cellules viables non cultivables et mortes après passage d’un flux de soude. Le but de ces travaux était de mieux comprendre le rôle de la matrice extracellulaire dans la formation des biofilms de L. monocytogenes pour ensuite trouver le meilleur moyen de l’éradiquer dans l’environnement industriel
The aim of this work was to study the role of exopolysaccharides in the formation of Listeria monocytogenes biofilm and their resistance to cleaning procedure in industry. We showed that the major exopolysaccharide present in the extracellular matrix of L. monocytogenes biofilm was teichoic acid, which was identical at the structure to the parietal teichoic acid. We identified that 50% of 93 strains of 1/2a serotype studied had a mutation in the lmo2550 gene that could lead to non-branching of the teichoic acid with the N-Acetylglucosamine residue (GlcNAc). We therefore examined mutants of EGD-e reference strain which had inactivated lmo2549 or lmo2550 or lmo2537 or tagO1tagO2 genes allowing highlighting the absence of the GlcNAc residue on teichoic acids. The mutation of these genes had changed the L. monocytogenes surface properties, decreased the adhesion to stainless steel and modified the architecture of 48h-biofilms. Furthermore, they were more susceptible to a circuit cleaning procedure with an important detachment, a change in the architecture of the biofilm and the presence of non-cultivable but viable cells and dead cells after passage of the caustic soda flow versus the wild-type EGD-e strain. The aim of this work was to better understand the role of the extracellular matrix in the formation of L. monocytogenes biofilm and then find the procedure to eradicate it in the industrial environment

Bioflocculation happens naturally and microorganisms aggregate into flocs during wastewater treatment. It is critical to understand the mechanisms of bioflocculation and its impact on the following solid/liquid separation process since seperation by settling is one of the key aspects that determine the efficiency and the overall economy of activated sludge systems. Bioflocculation occurs via extracellular polymeric substances (EPS) and cations by creating a matrix to hold various floc components together so the cations become an important part of the floc structure. The main objective of this study is to investigate the effects of monovalent cations specifically potassium and sodium (K and Na) on the bioflocculation, settleability and dewaterability of activated sludge. The particular aim is to grow the mixed culture microorganisms in the presence of specific cation so that the effect of cation on the stimulation of EPS production can be seen. In order to achieve this aim, semi-continuous reactors were separately operated at concentrations of 5, 10, and 20 meq/L of each cation with mixed culture bacteria and fed with synthetic feed medium representing influent to the activated sludge systems. Also, a control reactor at low cation dose was operated for each reactor set. The effective volume of the reactors was 2 L with 8 days of sludge residence time (SRT) and pH was kept at 7.7±
0.3. The activated sludge reactors were operated until the reactors reached steady state and then related analyses were conducted. It was found that addition of potassium and sodium ions at increasing concentrations resulted in increase in total polymer concentration. However, potassium ions promoted the synthesis of both polysaccharide and protein type polymers whereas sodium ions tended to stimulate production of protein type polymers and had an affinity to bind more protein within the floc structure. Sodium sludges had lower hydrophobicity and higher surface charges, so sodium ions led to deterioration in flocculation of sludges. Addition of both these ions decreased the dewaterability, sodium ions had more detrimental effect on dewaterability of sludges compared to potassium ions. The examination of data related to settleability showed that potassium ions led to no drastic deterioration in settling characteristics of the activated sludge but the addition of sodium ions deteriorated the settleability. In addition, it was seen that while the addition of potassium ions to the feed led to a decrease in viscosity, increase in sodium concentration correlated with an increase in viscosity. Finally, the comparison of chemical oxygen demand (COD) removal efficiency of these cations showed that sodium is more efficient in COD removal.

Trypanosoma brucei est un parasite protozoaire responsable de la trypanosomiase humaine africaine. Il présente un cycle de vie complexe alternant entre des hôtes mammifères et un vecteur insecte, la mouche tsé-tsé. Au cours de ce cycle, il rencontre des environnements radicalement distincts auxquels il s’adapte en régulant son métabolisme. Nous avons étudié le métabolisme intermédiaire et énergétique de la forme procyclique évoluant dans le tractus digestif de l’insecte vecteur. Dans cet environnement dépourvu de glucose, la néoglucogenèse est cruciale pour la croissance et la survie des parasites car elle permet la synthèse d’hexoses phosphates et en particulier du glucose 6-phosphate qui alimente plusieurs voies de biosynthèse essentielles. Nos travaux confirment ce flux néoglucogénique alimenté par la proline mais aussi par le glycérol. Nous montrons que le glycérol est une source de carbone efficacement métabolisée et préférentiellement utilisée par la forme procyclique à défaut de la proline et même du glucose pour alimenter son métabolisme intermédiaire. Cette situation qu in’a jamais été décrite auparavant met en évidence la répression du glycérol sur le métabolisme du glucose. Nous montrons également que l’enzyme fructose 1,6-biphosphatase(FBPase), spécifique de la néoglucogenèse, n’est pas essentielle à la survie du parasite en conditions dépourvues de glucose indiquant qu’il existe une alternative à cette enzyme.Toutefois, FBPase joue un rôle important dans la virulence de T. brucei dans l’insecte.De plus, nous avons mis en évidence une autre stratégie d’adaptation de T. brucei basée sur des réarrangements génomiques qui peuvent mener à la synthèse de gènes chimères
Trypanosoma brucei is a protozoan parasite responsible for human African trypanosomiasis. His complex life cycle alternates between mammalian hosts and the insect vector, the tsetsefly. During this cycle, the parasite encounters dissimilar environments and adapts to the sechanging conditions by regulating his metabolism. We have studied intermediate and energetic metabolism of the procyclic form living in the midgut of the insect vector. In this glucose-depleted environment, gluconeogenesis is crucial for growth and viability of the parasites. Indeed, it allows the synthesis of hexoses phosphates and in particular glucose 6-phosphate which feeds several essential biosynthetic pathways. Our work has confirmed the existence of a gluconeogenic flux fed by proline and glycerol. We have shown that glycerol is an efficiently metabolized carbon source and is preferentially used by the procyclic form rather than proline or even glucose. This situation never described before highlights glycerol repression on glucose metabolism. We have also showed that the enzyme fructose 1,6-biphosphatase (FBPase), specific of the gluconeogenesis, is not essential for the viability ofthe parasite in glucose-depleted conditions, suggesting that there is an alternative to this enzyme. However, FBPase plays an important role for virulence of T. brucei in the insect. Moreover, we have showed another adaptation strategy developed by T. brucei which is basedo n genomic rearrangements leading to the synthesis of chimeric genes

This thesis presents studies that examine microbial extracellular electron transfer that an emphasis characterizing how environmental conditions influence electron flux between microbes and a solid-phase electron donor or acceptor. I used bioelectrochemical systems (BESs), fluorescence and electron microscopy, chemical measurements, 16S rRNA analysis, and qRT-PCR to study these relationships among chemical, physical and biological parameters and processes.
Engineering and Applied Sciences

The presence of nitrates (NO3-) in groundwater is a worldwide concern. The high energy demand and environmental impact of available technologies requires investigating new technologies. This thesis was focused on investigating the usage of bioelectrochemical systems (BES) for treating nitrate-polluted groundwater. BES uses microorganisms able to catalyze oxidation/reduction processes by delivering/obtaining electrons from an electrode. In this thesis, microorganisms able to use the electrode as an electron donor (biocathode) to reduce nitrates into dinitrogen gas (inert) were investigated. As a result, a process could be patented, in which BES are able to treat nitrates at high denitrification rates (up to 700 gN•m-3NCC•d-1), with a competitive energy demand (0.68•10-2 – 1.27•10-2 kWh•gN-1treated), without sludge generation nor chemical dosing. Moreover, the microorganisms were electrochemically characterized, and the key subcommunities of the process were elucidated. In summary, bioelectrochemical systems have the potential for becoming a competitive alternative for the treatment of nitrate-polluted groundwater
La presència de nitrats (NO3-) en aigües subterrànies és una preocupació global. L’alt cost energètic i ambiental de les tecnologies actuals requereixen la investigació de noves estratègies. Aquesta tesi ha investigat la utilització de sistemes bioelectroquímics (BES) pel tractament d’aigües subterrànies contaminades per nitrats. Les BES es basen en microorganismes capaços de realitzar oxidacions/reduccions tot alliberant/captant electrons d’un elèctrode. Aquesta tesi ha investigat l’ús de bactèries capaçes d’utilitzar l’elèctrode com a donador d’electrons (biocàtode) per reduir el nitrat a dinitrogen gas (compost inert). Com a resultat, s’ha patentat un procés que permet desnitrificar a altes velocitats (700 gN•m-3NCC•d-1), a un cost energètic competitiu (0.68•10-2 – 1.27•10-2 kWh•gN-1tractat), sense generar fangs ni addicionar substàncies químiques. També s’ha caracteritzat electroquímicament els microorganismes i s’ha elucidat les subcomunitats microbianes responsables de la desnitrificació. En definitiva, aquesta tesi demostra que els sistemes bioelectroquímics poden esdevenir una alternativa competitiva pel tractament d’aigües subterrànies contaminades per nitrats

Le principal objectif de cette thèse était de mieux comprendre l’importance des Substances Polymériques Extracellulaires (SPE) dans la structuration et la formation des biofilms benthiques ; tout en s’inscrivant dans une étude plus globale des mécanismes écologiques impliqués dans le fonctionnement des vasières intertidales. La mise au point des dosages biochimiques a été effectuée sur le mucilage de l’algue Chaetomorpha aerea et a permis en parallèle de purifier un polysaccharide sulfaté riche en galactose, présentant une activité bactéricide sélective contre la souche Staphylococcus aureus (ATCC 25923). Les études biochimiques et écologiques menées sur les SPE extraits de la vasière charentaise ont ensuite permis de quantifier leur dynamique de production et leur composition, en fonction des conditions environnementales. La présence de désoxy-sucres et d’acides uroniques au sein des SPE capsulaires a laissé supposer que ces fractions jouaient un rôle important dans la formation et le devenir du biofilm microphytobenthique. La dernière partie des travaux a permis de caractériser les propriétés acide/base de Lewis et hydrophile/hydrophobe de la surface de la micro-algue Navicula jeffreyi, impliquée dans la formation de biofilms benthiques, par des méthodes classiques d’analyse. L’utilisation d’une nouvelle méthode, la Chromatographie Gazeuse Inverse (CGI), a permis d’obtenir des résultats intéressants et relativement similaires, confirmant le caractère prometteur de la CGI pour l’étude des propriétés de surface des micro-organismes
The main goal of this thesis was to better understand the importance of Extracellular Polymeric Substances (EPS) in the structuring and formation of benthic biofilms; while considering a global conception of the ecological mechanisms involved in the functioning of intertidal mudflats. The development of the biochemical assays was done on the mucilage of the macroalgae Chaetomorpha aerea and allowed purifying a polysaccharide rich in galactose, showing a selective bactericidal activity against Staphylococcus aureus (ATCC 25923). Then, the biochemical and ecological studies concerning the EPS extracted from the local mudflat allowed studying their dynamic of production and composition in relation to environmental conditions. The presence of deoxy sugars and uronic acids in the bound EPS highlighted their important roles during the formation and the life of microphytobenthic biofilms. The last part of the work was used to characterize the acid/base of Lewis and hydrophilic/hydrophobic surface properties of the microalgae Navicula jeffreyi, involved in the formation of benthic biofilms, by using classical analysis methods. The use of a new method, named Inverse Gas Chromatography (IGC), allowed getting interesting and relatively similar results, confirming the potential of the method to study the surface properties of microorganisms

碩士
元智大學
生物科技與工程研究所
107
Precious metals such as gold (Au) and palladium (Pd) are widely used in industrial and biomedical applications. Recovery of these precious metals has become an area of particular interest. Biosorption of these rare metals is of the promising approach for recovery of these metals from industrial wastes. However, these metal ions have also been recognized to possess antimicrobial properties. Microorganisms able to tolerate high concentrations of these metals may facilitate the application of biosorption of these precious metals We evaluate several heavy metal resistant strains including Pseudomonas aeruginosa EJ01 previously isolated from our laboratory for their ability to tolerate Pd when grown in general complex medium such as Luria-Bertani (LB) medium. These strains could grow in LB medium containing Pd ranging from 180 to 270 ppm. In addition to these heavy metal resistant strains, we also isolated two palladium resistant microorganisms from soil contaminated with high concentrations of heavy metals. The two isolates were able to tolerate Pd up to 360 ppm. Analysis of 16S rRNA gene sequences led to the identification of these two isolates as Ochrobactrum sp. and Microbacterium sp. and were designed as Ochrobactrum sp. strain 71 and Microbacterium sp. strain 73, respectively. It was found that about 25% of the total palladium concentration could be adsorbed when the microorganisms grown in a palladium-containing medium by ion inductively coupled plasma atomic emission spectrometry (NIEA W311.53C). According to the literature, the adsorption capacity of microorganisms for noble metals is mostly derived from the function of extracellular polysaccharides. Therefore, we also examined the extracellular polysaccharides extracted from Ochrobactrum sp. and Microbacterium sp. for palladium adsorption. The polysaccharide showed a significant adsorption effect on palladium, about 10-15%. Finally, the extracellular polysaccharides of the two strains were subjected to saccharide composition.

Microorganisms may evade killing by neutrophils (PMNs) by altering signal transduction and hence phagosome maturation. Secreted, active matrix metalloproteinases (MMPs) appear to be required for PMN killing of pseudomonas microorganisms, via an MMP and complement-dependent, but otherwise unknown mechanism. This also depends on the absence of the inhibitor of MMPs, tissue inhibitor of metalloproteinases-1 (TIMP-1). By altering their particular complement opsonin and hence the PMN complement receptor bound, microorganism may evade killing, as not all PMN complement receptors trigger phagosome maturation and hence killing of microorganisms. C1 inhibitor of the classical complement cascade, required for the exposure of C1q and further assembly of complement factors on the bacterial surface and hence binding to specific PMN receptors, is MMP sensitive. MMP secretion may, therefore, not only facilitate the killing of microorganisms, but inappropriate secretion, induced by pathogens, may prevent complement assembly and killing via complement-mediated pathways. It was, therefore, decided to assess MMP-9 and TIMP-1 secretion in the presence of C1q-opsonized polystyrene beads and subsequently upon stimulation with pseudomonas organisms, and explore the relationship between secretion of PMN MMPs (specifically MMP-9) and TIMP-1 and phagocytic uptake and maturation of the PMN phagosome into a killing body. MMP-9 and TIMP-1 secretion was seen to occur at low levels under most conditions. However, in the presence of serum, and hence complement, MMP-9 secretion was found to be upregulated during uptake of C1q-coated beads. MMP-9 possibly inactivates C1 inhibitor at this stage, causing local tissue swelling (normally associated with the inactivation of C1-inhibitor), entry of various white blood cells and further complement into the area of infection, assisting in the extracellular killing of microorganisms. MMP secretion may simultaneously down-regulate the activation of further PMNs via inactivation of C1q assembly and hence phagocytic uptake and activation of PMNs. Unlike MMP-9, secretion of TIMP-1 was not upregulated by C1q receptor binding, implying that any secreted MMP-9 may, therefore, be in excess and hence uninhibited by TIMP-1. A distinct regulatory mechanism seems to be responsible for the release of TIMP-1, though TIMP-1 secretion was upregulated by extracellular calcium levels, partially contradicting previous findings which suggested that TIMP-1 was not calcium regulated. It seems unlikely that extracellular calcium levels would be the only mechanism by which TIMP-1 is regulated, however, and further surface receptor mediated agonists should be explored. Levels of MMP-9 and TIMP-1 secretion in the presence of pseudomonas microorganisms now need to be assessed to see whether these secretion patterns are altered to favour the evasion of opsonization by C1q. Uptake of C1q-opsonized beads was also increased by the presence of serum, possibly due to presence of complement. MMP-9 and TIMP-1 secretion patterns still need to be correlated with phagosomal uptake and killing of microorganisms, before their role in killing of microorganisms becomes fully evident.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.