<p> The higher taxonomic groups within Prokaryotes are presently distinguished mainly on the basis of their branching in phylogenetic trees. In most cases, no molecular, biochemical or physiological characteristics are known that are uniquely shared by species from these groups. Comparative genomic analyses are leading to discovery of molecular characteristics that are specific for different groups of Bacteria and Archaea. These markers include conserved inserts and deletions in universal proteins and lineagespecific proteins, which provide novel means for identifying and circumscribing these groups of prokaryotes in clear molecular terms and for understanding their evolution. Because of their taxa specificities, further studies on these newly discovered molecular characteristics should lead to discovery of novel biochemical and physiological characteristics that are unique to different groups of microbes. The focus of my project was phylogenomic studies for two large prokaryotic group: Actinobacteria and Archaea. My goals were to a) identify molecular markers that are specific to Actinobacteria and Archaea at different taxonomic levels, which will help to understand the phylogenetic relationship within these two major groups; b) understand the functional significance of Actinobacteria-specific proteins. By comparative genomics approach, a number of conserved indels in various proteins (viz. Coxl, GluRS, CTPsyn, Gft, GlyRS, TrmD, Gyrase A, SahH and SHMT) have been identified that are specific for all Actinobacteria and additional indels were found to be unique to its major subgroups, such as Corynebacterineae, Bifidobacteriaceae, etc. In parallel, a large number of proteins were discovered to be restricted to Actinobacteria at different phylogenetic depths. These identified conserved indels and proteins for the first time provide useful markers for defining and circumscribing the Actinobacteria phylum or its subgroups in clear molecular terms. Similar comparative genomic studies have been carried out on Archaea and a vast number of proteins have been identified that are unique to Archaea or its various lineages. Lastly, I have performed functional studies on one of the Actinobacteria-specific proteins (ASPl). The structure of ASPl was determined and structural comparison indicates that the function of this protein might be novel since it does not match any known protein with or without known function. </p> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies. / Ph. D.

Open reading frame (ORF) sso0433 from the archaeon Sulfolobus solfataricus encodes a protein kinase, SsoPK5 that exhibits 33% sequence identity to p53 related protein kinase (PRPK) from Homo sapiens and 26% sequence identity to piD261/Bud32 from Saccharomyces cerevisiae. Given this high degree of similarity, the objectives of this thesis were to (a) clone and purify recombinant SsoPK5, (b) examine its commonalities and differences with its eukaryotic homologues, and (c) determine if it was regulated by nucleotides or related compounds. Substantial progress was achieved on each objective. After successful cloning of ORF sso0433 and purification of its protein product, SsoPK5, it was determined that SsoPK5 was cold labile and incubation at 4ºC for an extended period of time rendered SsoPK5 incapable of phosphotransferase activity. When stored at room temperature, SsoPK5 was capable of transferring the γ-phosphate from ATP to casein, reduced carboxyamidomethylated and maleylated (RCM) lysozyme,and p53. SsoPK5 phosphotransferase activity required a divalent metal cofactor; like pid261/Bud32, SsoPK5 preferred Mn²⁺ over the more commonly preferred Mg²⁺. SsoPK5 was shown to phosphorylate itself on threonine and serine residues; one of the specific amino acid residues modified is threonine-151. Recombinant SsoPK5 is activated by ADP-ribose and 5′-AMP. Activation was observed when SsoPK5 was stabilized by ATP or a nonhydrolytic analogue, such as β,γ- methylene adenosine 5′-triphosphate (AMP-PCP). Activation was not a result of phosphoryl transfer nor hydrolytic breakdown of ATP or 5′-AMP. This was deduced by the lack of ³²P radioactivity incorporated into SsoPK5 during pre-incubation with [γ-³²P] ATP for 60 min at 65ºC, and activation by adenosine 5′-O-thiomonophosphate (AMPS), a hydrolysis-resistant analog of AMP. These results may indicate that ADP-ribose acts as a pseudochaperone for SsoPK5 thereby facilitating maximal activity. / Ph. D.

The goal of my dissertation research was to investigate the structure and function of ammonia-oxidizing microbial communities in temperate forest soils. Accomplishing this goal required a hybrid approach: I used modern molecular biology techniques alongside soil biogeochemical measurements and framed my research using ecological theory largely developed in plant systems. All of my field work was done at Coweeta Hydrologic Laboratory, a Forest Service Station and Long Term Ecological Research Site near Otto, NC. Watershed-level land use manipulations have been performed at Coweeta since the 1930s, including clear-cutting, fertilizing, liming, burning, grazing by cattle, and replanting entire watersheds in white pine. While these treatments were originally imposed to assess the effects of land use on water yield, they have resulted in changes in soil characteristics as well. Working at Coweeta has therefore allowed me to sample ammonia-oxidizer communities across a gradient of soil variables, such as pH and nitrogen (N) availability, within the geographically-constrained area of the Coweeta Basin. First, I used amplicon-based pyrosequencing to independently assess the diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) at several sites within Coweeta. I found that AOA and AOB diversity were a function of both resource availability (i.e. N availability) and environmental harshness (i.e. soil pH) in line with general ecological theory developed for plant systems by Tilman and Grime, respectively. Next, I tested whether AOA and AOB were substrate or nutrient limited in this system by adding either N or a nutrient solution containing both potassium and phosphorus to soil incubations and assessing the growth response of AOA and AOB using quantitative polymerase chain reaction (qPCR). I found strong evidence for substrate limitation by AOB and a marginally-significant positive effect of nutrient addition on growth of AOA. Another intriguing finding from this study was that both AOA and AOB grew during unamended soil incubations. Unamended (buried-bag) incubations have been used to estimate in situ rates of nitrification for over 50 years. By measuring the growth of AOA and AOB alongside nitrification during buried-bag incubations, I discovered that AOA are the dominant ammonia-oxidizers in temperate forest soils. However, I found that AOA are much less efficient at using the energy from ammonia oxidation to create biomass than AOB in the forest soils I sampled. Overall, I found that temperate forest soils contain low abundances of AOA and AOB, with relatively low diversity in both groups. This is especially true for the diversity of AOA, where a single taxon dominated the community at every site. Soil pH and N availability seem to be major selective forces for forest soil ammonia oxidizers, though other nutrients such as potassium and phosphorus may regulate the activity of AOA as well. AOA are most-likely the dominant ammonia oxidizers in temperate forest systems, though this may change with increased disturbance. In a broader sense, I found that ecological theory developed for plant communities was applicable to chemoautotrophic microbes despite the large differences in life history between these groups of organisms. / Ph. D.

Les Archaea planctoniques contribuent de façon significative aux grands cycles biogéochimiques dans les écosystèmes aquatiques, néanmoins la structure des communautés actives ainsi que leurs variations saisonnières sont encore largement méconnues. En outre, la découverte de l’implication des Archaea dans le cycle de l’azote (Ammonia Oxidizing Archaea ou AOA), plus particulièrement dans le processus de nitrification a considérablement modifié la perception d’un processus autotrophe réalisé uniquement par des bactéries (Ammonia Oxidizing Bacteria ou AOB). Dans les écosystèmes marins, la large distribution des AOA suggère que ces microorganismes joueraient un rôle prépondérant dans le cycle de l’azote néanmoins, ces observations ne sont pas généralisables à l’ensemble des écosystèmes aquatiques en raison de leur grande diversité et/ou d'un manque d'informations et d’études sur certains d'entre eux. Ainsi, les objectifs de ce projet étaient i) d’étudier la structure spatiale et temporelle des communautés d’Archaea actives dans des écosystèmes aquatiques contrastés en termes d’apports anthropiques et/ou de gradients de salinité (lac, estuaire, milieu côtier) ; ii) de déterminer la contribution relative des Archaea au processus d’oxydation de l’ammonium, en comparaison avec celle des bactéries ; et iii) de mieux comprendre les paramètres environnementaux qui pourraient déterminer l’établissement des communautés d’AOA ou d’AOB. / Aquatic Archaea are important players among microbial plankton and significantly contribute to biogeochemical cycles, especially nitrogen, but details regarding their community structure and seasonal activity and dynamics remain largely unexplored. In marine ecosystems, the widespread distribution of Ammonia Oxidizing Archaea (AOA) suggests that they probably play a major role in nutrients cycling. However, we cannot generalize these observations to all aquatic ecosystems because of their high diversity and/or a lack of information and studies on these organisms for some of these ecosystems. More precisely, lacustrine and coastal ecosystems were less studied while they are potentially subjected to strong anthropogenic impacts. Moreover, notable differences in terms of diversity and activity between marine and freshwater communities can be expected, considering the specific environmental parameters of each ecosystem. The objectives of this thesis were: i) to study the archaeal community structure across a temporal scale and assess the diversity of archaeal communities and AOA in diverse aquatic ecosystems along anthropogenic and/or salinity gradient (lacustrine, estuarine and coastal ecosystems); ii) to determine their relative contribution in ammonia oxidation, compared to Ammonia Oxidizing Bacteria (AOB) by looking at their spatial and temporal distribution and activity, and iii) to explore more precisely the environmental parameters that could drive AOA and/or AOB establishment.

Trotz der zunehmenden Bedeutung, die der Biogasprozess in der Reihe der regenerativen Energiequellen seit einigen Jahren einnimmt, sind dessen mikrobiologische Prozesse häufig nur zum Teil verstanden und die Zusammensetzung der mikrobiellen Biozönose oft unbekannt. Um jedoch die Biogasgewinnung möglichst effektiv zu gestalten, ist es essentiell, die physikochemischen Bedingungen im Reaktor an die Mikroorganismen anzupassen. Daher war es ein Ziel dieser Arbeit, die mikrobielle Gemeinschaft in verschiedenen Biogasanlagen abhängig vom eingesetzten Substrat zu charakterisieren. Dabei zeigte sich, dass insbesondere die archaeelle Diversität in NawaRo-Anlagen sehr limitiert ist und die Organismen in den einzelnen Anlagen nah verwandt sind. Von großer Bedeutung waren dabei die Genera Methanoculleus und Methanosarcina (bei hoher Acetatkonzentration) bzw. Methanothrix (bei geringer Acetatkonzentration). Deren genaues Verhältnis in den verschiedenen Anlagen hing wesentlich von den zugesetzten Co-Substraten ab und vor allem in Anlage A konnte eine Abhängigkeit der Abundanz von Methanosarcina je nach Zusatz von Hühnertrockenkot bzw. Rindermist beobachtet werden. Im Gegensatz dazu spielten die untersuchten chemischen Parameter eine geringere Rolle und es konnten kaum Korrelationen zwischen der Abundanz einzelner archaeeller Genera und physikochemischen Parametern beobachtet werden. In der mit Klärschlamm betriebenen Anlage KA waren außerdem verschiedene Methanobacteriales und Organismen der WCHA1-57-Klade von Bedeutung. Neben methanogenen Archaea konnten in den Anlagen C, KA und KL außerdem Crenarchaeota nachgewiesen werden, die wahrscheinlich in das erst kürzlich neu postulierte Phylum der „Bathyarchaeota“ einzuordnen sind. Deren genaue physiologische Funktion im Biogasprozess ist jedoch noch ungeklärt. Zusätzlich zu den Archaea wurden exemplarisch auch Proben aus den einzelnen Anlagen hinsichtlich ihrer bakteriellen Population untersucht. Dabei waren in den NawaRo-Anlagen A, B, C und KL vor allem Firmicutes und Cloacimonetes zu finden. Letztere stellen dabei eine bisher nur wenig charakterisierte Gruppe dar, die wahrscheinlich im hydrolytischen Abbau von Cellulose und/oder Aminosäuren eine entscheidende Rolle spielen. Innerhalb der Firmicutes übten insbesondere die Clostridiales vielseitige physiologische Funktionen innerhalb der anaeroben Fermentation aus. Überraschend gering war jedoch der Anteil der syntrophen Organismen. Es ist möglich, dass einige der nicht näher klassifizierbaren OTUs dieser Gruppe angehören, da der syntrophe Abbau verschiedener Säuren von essentieller Bedeutung für den Biogasprozess ist. In der mit kommunalem Abwasserschlamm betriebenen Anlage KA war der Anteil der Firmicutes und Cloacimonetes deutlich geringer. Abundantestes Phylum waren hier die Proteobacteria, unter denen zusätzlich Syntrophe zu finden sind. Zusätzlich zur Untersuchung der mikrobiellen Gemeinschaft während des Normalbetriebes von Biogasanlagen wurden außerdem häufige Prozessstörungen simuliert. Dabei zeigte sich, dass es trotz rückläufiger Methanbildung häufig nicht zu Veränderungen in der Zusammensetzung der Mikroorganismen kommt und stattdessen deren Aktivität sinkt. Erst bei dramatischer Veränderung der physikochemischen Parameter, wie es im Rahmen des ersten Versuchs zur Ammonium-Intoxiaktion der Fall war, wurden deutliche Veränderungen in der archaeellen Biozönose detektiert. Die bakterielle Gemeinschaft blieb dabei kaum verändert. Bei geringerer Ammoniumkonzentration hingegen konnte eine gute Anpassungsfähigkeit der Archaea gezeigt werden. Dennoch war es nicht möglich, anhand dieser Versuche einen allgemein gültigen Grenzwert der Ammoniumkonzentration, ab der inhibitorische Effekte auftreten, festzulegen, da in Anlage A bei ähnlichen Konzentrationen die archaeelle Abundanz durchaus zurückging. Auch im Rahmen der Versuche zur Versauerung konnte eine gute Anpassungsfähigkeit der Archaea gegenüber erhöhten FOS-Konzentrationen gezeigt werden. Dabei war insbesondere eine erhöhte Abundanz der Syntrophomonadaceae mit Stressresistenz verbunden, während eine erhöhte Abundanz von Methanobacterium mit verminderter Säuretoleranz einher ging. Insgesamt liefern die Ergebnisse dieser Arbeit einen aufschlussreichen Einblick in die mikrobielle Population in NawaRo-Anlagen sowohl während des Normalbetriebes als auch im Verlauf von Prozessstörungen, die zu einem besseren Verständnis der ablaufenden Prozesse beitragen können. Dennoch ist die Rolle vieler beteiligter Organismen noch nicht gänzlich geklärt. Es sind daher weitere Studien nötig, um die mikrobiologischen Prozesse in Biogasanlagen vollständig zu verstehen.

Despite general concern that the massive deposits of methane stored under permafrost underground and undersea could be released into the atmosphere due to rising temperatures attributed to global climate change, little is known about the methanogenic microorganisms in permafrost sediments, their role in methane emissions, and their phylogeny. The aim of this thesis was to increase knowledge of uncultivated methanogenic microorganisms in submarine and terrestrial permafrost deposits, their community composition, the role they play with regard to methane emissions, and their phylogeny. It is assumed that methanogenic communities in warmer submarine permafrost may serve as a model to anticipate the response of methanogenic communities in colder terrestrial permafrost to rising temperatures. The compositions of methanogenic communities were examined in terrestrial and submarine permafrost sediment samples. The submarine permafrost studied in this research was 10°C warmer than the terrestrial permafrost. By polymerase chain reaction (PCR), DNA was extracted from each of the samples and analyzed by molecular microbiological methods such as PCR-DGGE, RT-PCR, and cloning. Furthermore, these samples were used for in vitro experiment and FISH. The submarine permafrost analysis of the isotope composition of CH4 suggested a relationship between methane content and in situ active methanogenesis. Furthermore, active methanogenesis was proven using 13C-isotope measurements of methane in submarine permafrost sediment with a high TOC value and a high methane concentration. In the molecular-microbiological studies uncultivated lines of Methanosarcina, Methanomicrobiales, Methanobacteriacea and the Groups 1.3 and Marine Benthic from Crenarchaeota were found in all submarine and terrestrial permafrost samples. Methanosarcina was the dominant group of the Archaea in all submarine and terrestrial permafrost samples. The archaeal community composition, in particular, the methanogenic community composition showed diversity with changes in temperatures. Furthermore, cell count of methanogens in submarine permafrost was 10 times higher than in terrestrial permafrost. In vitro experiments showed that methanogens adapt quickly and well to higher temperatures. If temperatures rise due to climate change, an increase in methanogenic activity can be expected as long as organic material is sufficiently available and qualitatively adequate. / Trotz allgemeiner Bedenken, dass auf Grund des Temperaturanstieges im Zusammenhang mit der globalen Klimaerwärmung große Mengen des in terrestrischen und submarinen Permafrostsedimenten gespeicherten Methans freigesetzt werden könnte, ist bisher wenig über die in diesen Böden lebenden methanogenen Mikroorganismen, ihre Phylogenese und sowie ihre Bedeutung hinsichtlich der Methanemissionen bekannt. Das Ziel dieser Doktorarbeit war die Erweiterung der bisherigen Kenntnisse über unkultivierte methanogene Mikroorganismen in submarinen und terrestrischen Sedimentablagerungen, die Zusammensetzung ihrer Lebensgemeinschaft, ihrer Phylogenese und ihrer Bedeutung bei der Emission von Methan. Es wird vermutet, dass methanogene Gemeinschaften submarines Permafrostes zur Erstellung von Modellen genutzt werden können, um Aussagen bezüglich potenzieller Reaktionen methanogener Gemeinschaften des kälteren terrestrischen Permafrostes auf steigende Temperaturen, zu ermöglichen. Die Zusammensetzung der methanogenen Gemeinschaft wurde in terrestrischen und submarinen Permafrostproben untersucht. Der im Rahmen dieser Forschungsarbeit untersuchte submarine Permafrost wies eine im Vergleich zum terrestrischen Permafrost um circa 10°C höhere Temperatur auf. Mittels Polymerasenkettenreaktion (PCR) wurde von jeder der Proben DNA extrahiert und mittels weiterer molekular-mikrobiologischen Methoden wie DGGE, RT-PCR und Klonierung analysiert. Des Weiteren wurden die Proben für in vitro Experimente und Zellzählungen (DAPI und FISH) verwendet. Die Analyse der Isotopenzusammensetzung von CH4 in submarinen Permafrostsedimenten ließ einen Zusammenhang zwischen Methangehalt und aktiver in situ Methanogenese vermuten. Überdies konnte aktive Methanogenese, mittels 13C-Isotopenmessungen von Methan in submarinem Permafrostsediment mit hohem TOC-Wert und hoher Methankonzentration, bewiesen werden. Im Rahmen der molekular-mikrobiologischen Untersuchungen wurden in allen submarinen und terrestrischen Permafrostproben unkultivierte Linien von Methanosarcina, Methanomicrobiales, Methanobacteriacea und den Gruppen 1.3 und Marine Benthic von Crenarchaeota gefunden. Methanosarcina war in allen submarinen und terrestrischen Permafrostproben die dominierende Gruppe der Archaeen. Die Zusammensetzung der archaealen Gemeinschaft, insbesondere die Zusammensetzung der methanogenen Gemeinschaft, variierte zwischen den submarinen und terrestrischen Proben. Des Weiteren fand sich bei der Zellzählung der Methanogenen im submarinen Permafrost eine 10-fach höhere Zellzahl als im terrestrischen Permafrost. Die in vitro Experimente zeigten, dass Methanogene sich schnell und gut an höhere Temperaturen anpassen können. Im Falle weiter steigender Temperaturen auf Grund der Klimaveränderungen, kann – bei ausreichender Verfügbarkeit und Qualität organischen Materials – mit einer Zunahme der methanogenen Aktivität gerechnet werden.

Les microorganismes anaérobies représentent la population majoritaire de notre tube digestif et ont un impact remarquable sur notre santé. Leur culture demeure à ce jour longue, fastidieuse et coûteuse et nombreux sont ceux qui restent incultivables. Or la culture est un outil indispensable pour l'étude du microbiote digestif. Ainsi, le laboratoire dans lequel ma thèse s’est déroulée a créé un nouveau concept de culture « Microbial Culturomics » qui a permis d’isoler 193 nouvelles espèces bactériennes anaérobies. Un travail sur l’utilisation des antioxydants pour permettre la culture aérobie des bactéries anaérobies a également été amorcé : une optimisation des techniques de culture prometteuse autour de laquelle mes travaux ont vu le jour. Notre premier projet a consisté à développer un dispositif de culture innovant permettant la culture des archaea méthanogènes en aérobiose et en absence de source externe de dihydrogène. Notre deuxième projet a consisté à élaborer un flacon d’hémoculture unique dans lequel la croissance de toutes les bactéries, aérobies et anaérobies, pouvaient être détectées. Notre troisième projet quant à lui repose sur la comparaison du mode de culture anaérobie et de celui en aérobie avec les antioxydants à travers l’exemple de trois souches bactériennes strictement anaérobies. L’utilisation des antioxydants pour faciliter la culture des microorganismes anaérobies a donc apporter des résultats très prometteurs qui pourrait être utilisés, après validation par des études multicentriques dans les laboratoires de microbiologie clinique et environnementaux. / Anaerobic microorganisms are characterized by their ability to grow and survive in the absence of oxygen. Indeed free oxygen molecules are not used for their metabolism and can be toxic to varying degrees, sometimes leading to cell death. Although it is known that these microorganisms are the predominant in our digestive microbiota and that they have a great impact on our health, their culture remain long, fastidious, costly, and in most cases impossible. Becteria culture is an indispensable tool for isolating strains, performing studies from living models, and identifying new ones. Thus, the laboratory in which my thesis tooks place created a new concept of culture "Microbial Culturomics" which made it possible to isolate 193 new anaerobic bacterial species. A work based on the use of antioxidants to enable the aerobic culture of anaerobic bacteria was also initiated: a promising optimization of the culture techniques from which my work was born. Our first project consisted in developing an innovative culture device allowing the cultivation of methanogenic archaea in aerobic and without an external source of dihydrogen. In our second project, we performed a single culture bottle in which the growth of all bacteria, aerobic and anaerobic, could be detected. Our third project was based on the comparison of anaerobic and aerobic culture with antioxidants through the example of three strictly anaerobic bacterial strains.Therefore the use of antioxidants enable to facilitate anaerobic bacteria cultivation. These results are very encouraging for clinical and environmental microbiology laboratories.