This is an investigation into alcohol dehydrogenases (ADHs) from Geobacillus thermoglucosidasius. Eighteen ADHs have been studied, with seven taken for closer inspection. Characterisation was carried out to determine the industrial significance of these enzymes, starting with the substrate scope of the ADHs. The key results obtained are as follows: ADH A is the alcohol dehydrogenase domain of the bifunctional ADHE enzyme (Extance, 2012; Extance et al., 2013). It has been determined that the substrate scope, whilst restricted to linear aliphatic aldehydes, extends at least to dodecanal. Also, with a specificity constant of 167 mM-1 min-1 it appears that ADH A could prefer butanal to shorter-chain aldehydes such as ethanal and propanal with specificity constants of 38 mM-1 min-1 and 35 mM-1 min-1, respectively. Thus ADH A may have a preference for longer aldehydes than previously believed due to its native role in the production of ethanol from acetyl-coA. ADH B was previously investigated for its potential role in the production of butanol. Here it was confirmed as an NADH-dependent ADH, with a substrate scope limited to five carbon length substrates and smaller, with residual activity with C6 substrates. ADH B demonstrated activity with ethyl 4-chloroacetoacetate, an intermediate in the production of statins. Further, an estimated half-life whilst stored at 4°C of 770 days; retention of 86% activity with 10vol% ethyl acetate and 92% activity with 10vol% acetonitrile; and a specific activity of 27 U mg-1 with 3M 2-butanone are all indications that ADH B is a potentially useful enzyme for industry. The last enzyme to be previously investigated was ADH C, which in this work was confirmed to be an acetoin reductase with a very small substrate scope exclusively based around the acetoin motif, and therefore no further work was conducted. ADH D and ADH F both have broad substrate scopes including the industrially-relevant substrates, 5-norbornene-2-carboxaldehyde, 1-phenyl-1,2-propanedione, ethyl 4-chloroacetoacetate and ethyl-2-oxo-4-phenylbutyrate. ADH D is an NADPH-dependent enzyme whereas ADH F can utilise both NADH and NADPH. Both enzymes are annotated as aldo-keto reductases, which is further indicated by multiple sequence alignment with the most similar available protein sequences and crystal structures. Thus, these two enzymes are the first aldo-keto reductases to be examined from moderate thermophiles, and are tentatively assigned in the AKR family as AKR6D1 and AKR5G4 respectively. ADH D has a very low KM (≤0.1 μM) with NADPH, giving a specificity constant of 2,800,000 mM-1 min-1, substantially higher than any other noted. ADH D showed >80% activity from pH 5.0 - 8.0. The enzyme was resistant to solvents DMSO (at 5 vol%) and ethyl acetate, acetonitrile and cyclopentyl methyl ether (at 20vol%). ADH F had the broadest substrate scope of any ADH tested, with 1-phenyl-1,2-propanedione the most preferred substrate with a KM of 0.010 mM and a specificity constant of 54,000 mM-1 min-1. It greatly preferred sodium phosphate at pH 7.0, as almost any deviation resulted in a substantial loss of activity. Activity of ≥70% was recorded in 5vol% DMSO, ethyl acetate, acetonitrile, cyclopentyl methyl ether and 50vol% hexane . Both ADH D and F have optimal activities at 70 °C and both may have application in the biotechnology industry for the production of pharmaceutical intermediates and other high value chemicals. ADH E acts solely as an aldehyde reductase, with Vmax using NADH of 74, 331, 320 and 281 U mg-1 for methanal, ethanal, propanal and butanal, respectively. Activity with NADPH was limited (< 1% compared with NADH). Activity was also noted with higher aldehydes such as octanal and furfural. ADH G is an NADPH-dependent ADH utilizing aldehydes only. It has an optimal temperature of 60°C with a half-life of under two hours at that temperature. In conclusion, this thesis reports a feasibility study into the potential industrial use of specific enzymes for a variety of purposes ranging from the production of pharmaceutical intermediates to bioremediation. ADHs D and F are most likely to have use in the biotechnology industry, and ADHs B and E may be suitable for cofactor regeneration. ADH E may additionally be useful in the bioremediation industry. In addition, the anticipated biological significance of these enzymes is described.

Aigarchaeota, a deeply branching archaeal lineage with no cultivated representatives, is found in geothermal and hydrothermal systems worldwide and consists of at least 9 genus-level groups, each predicted to have diverse metabolic capabilities. This candidate archaeal phylum is part of the TACK superphylum, members of which possess eukaryotic-signature proteins, thus suggesting that they may represent evolutionary steps along the way to the genesis of the first eukaryotic cells. Cultivating members of Aigarchaeota would elucidate how eukaryotes arose in evolutionary history and provide biotechnological applications. Aigarchaeota Group 4 (AigG4), one genus in Aigarchaeota, was previously found to be abundant in corn stover in situ enrichments in Great Boiling Spring (GBS). AigG4 has been maintained in mixed laboratory cultures, where it composes ~ 0.5-1% of the community. However, these cultures could only be maintained when GBS water, which contains ~300 nM tungsten, was included in the medium. In addition, AigG4 metagenome bins from the in situ enrichments and laboratory cultures contained multiple genes encoding putative tungsten-containing aldehyde:ferredoxin oxidoreductases (TAORs). These observations led to the hypothesis that tungsten was the key component in GBS water that allowed for growth of AigG4. The requirement of tungsten for AigG4 long-term maintenance in mixed culture was tested using three different approaches: (1) Assessing the phylogeny of tungsten transporters and TAORs across the Aigarchaeota lineage, followed by confirmation of transcription of hypothesized AigG4 tungsten-associated genes in lab cultures, (2) Measuring tungsten levels in Great Boiling Spring (GBS) using ICP-OES (Inductively coupled plasma-optical emission spectroscopy), and (3) determining minimum amount of tungsten for long-term AigG4 maintenance in corn stover and in a defined mix of sugars (1% glucose, 1% xylose, 1% D-arabinose, 1% L-arabinose, 1% mannose). In addition, FISH (Fluorescent in situ hybridization) probes were designed to target the AigG4 lineage in the hopes that in conjunction with nanometer-scale secondary ion mass spectroscopy (Nano-SIMS), this would test whether tungsten has indirect or direct effects on AigG4 to track carbon substrate intake. Most Aigarchaeota lineages possess a tungsten transporter complex (TTC). AigG4 TTC groups most closely with the Tup family while other Aigarchaeota group with the Wtp family. Group 4 and Group 5 Aigarchaeota contained TAORs that grouped to other hypothesized TAORs but not to characterized counterparts suggesting diverse functional capabilities. Group 4 and Group 5 TAORs clustered together suggesting that these are conserved within these lineages. Gene expression of predicted AigG4 tungsten-associated enzymes was detected in culture. Tungsten was detected in GBS water as previously observed in 2005. In both corn stover and sugar mix, 1 nM tungsten was sufficient for long term AigG4 maintenance. In corn stover, AigG4 decreased to levels below detection after three 3-week transfer periods in 0 nM and 20 nM tungsten. In sugar mix, AigG4 abundance levels varied wildly in 0 nM tungsten after the fifth transfer period suggesting tungsten contamination. Two newly designed FISH probes exhibited lower fluorescence signal intensity than the previously designed FISH probe suggesting issues with either target site accessibility or conjugation of the fluorescent moiety to oligonucleotide probes.

Freie Universiẗat, Diss., 2006--Berlin. / Dateiformat: zip, Dateien im PDF-Format.

Darmstadt, Techn. Univ., Diss., 2005. / Dateien in unterschiedlichen Formaten

La fermentation sombre et les piles à combustible microbiennes (MFC) sont deux technologies émergentes respectivement pour la conversion biologique de l'énergie chimique des composés organiques en hydrogène (H2) et en électricité. En raison des avantages cinétiques et thermodynamiques, la température élevée peut être la clé pour augmenter à la fois la production d'H2 de fermentation sombre et la production d'électricité dans les MFC. Par conséquent, cette thèse se concentre sur la manière dont la température influence la production biologique de H2 et d'électricité à partir d'eaux usées contenant du carbone organique. Deux inocula traités thermiquement (à boues activées fraîches et digérées) ont été comparés pour la production de H2 à partir de xylose à 37, 55 et 70 °C. A la fois à 37 et 55 °C, on obtient un meilleur rendement en H2 par les boues activées fraîches comparé aux boues digérées tandis qu'un très faible rendement en H2 est obtenu par les deux inocula à 70 °C. Ensuite, quatre prétraitements d'inoculum différents (chocs acides, alcalins, thermiques et de congélation) ont été évalués pour créer une efficace communauté productrice de H2 mésophile (37 °C) ou thermophile (55 °C). Les chocs acides et alcalins ont sélectionné des micro-organismes producteurs de H2, appartenant aux Clostridiaceae, au détriment des bactéries produisant du lactate, ce qui a donné respectivement le rendement en H2 le plus élevé à 37 et 55 °C. Bien que le choc thermique ait abouti à un faible rendement en H2 dans un seul lot, il a été montré que la production de H2 par les boues activées fraîches traitées thermiquement augmentait dans l'expérience avec quatre cycles consécutifs. Des boues activées fraîches et traitées thermiquement ont été sélectionnées comme inoculum pour la production continue de H2 à partir d'une eau usée synthétique contenant du xylose dans un réacteur à lit fluidisé (FBR) mésophile (37 °C) et thermophile (55-70 °C, augmenté par étapes). Un rendement en H2 plus élevé a été obtenu dans le FBR thermophile que dans le FBR mésophile. En outre, la production de H2 à 70 °C, qui a échoué dans l'étude précédente, a été couronnée de succès dans le FBR, avec un rendement stable de 1.2 mol H2 mol-1 xylose. La température de fonctionnement de 70 °C s'est également révélée optimale pour la production de H2 à partir d'eaux usées thermomécaniques (TMP) dans un incubateur à gradient de température, car la culture en batch à 70 ° C. Une approche de l'ARN a été utilisée pour étudier la structure et le rôle des communautés microbiennes attachées à l'anode, attachées à la membrane et planctoniques dans un MFC mésophile (37 °C) et thermophile (55 °C) alimenté au xylose. Une communauté anodine dominée par Geobacteraceae a soutenu la production d'électricité à 37 °C, alors que l'établissement de micro-organismes méthanogènes et H2 oxydants a entraîné une faible production d'électricité à 55 °C. Cependant, le développement d'une communauté exoélectrogène thermophile peut être favorisé en appliquant une stratégie de démarrage qui comprend l'imposition d'un potentiel négatif à l'anode et l'inhibition chimique des méthanogènes. Une communauté exoélectrogénique mésophile a également été montré pour produire de l'électricité à partir d'eaux usées de TMP dans un MFC à flux ascendant exploité à 37 °C. En conclusion, une production de H2 plus élevé et plus stable peut être obtenu dans une fermentation sombre thermophile plutôt que mésophile. La fermentation sombre à 70 °C est particulièrement appropriée pour le traitement des eaux usées de TMP car elle est libérée à haute température (50-80 °C) et pourrait être traitée sur site. Les eaux usées de TMP peuvent également être utilisées comme substrat pour la production d'électricité dans les MFC mésophiles. La production d'électricité dans les MFC thermophiles est faisable, mais l'enrichissement des micro-organismes exoélectrogènes thermophiles peut nécessiter une longue période de démarrage / Dark fermentation and microbial fuel cells (MFCs) are two emerging technologies for biological conversion of the chemical energy of organic compounds into hydrogen (H2) and electricity, respectively. Due to kinetic and thermodynamic advantages, high temperature can be the key for increasing both dark fermentative H2 production and electricity production in MFCs. Therefore, this thesis focuses on delineating how temperature influences biological production of H2 and electricity from organic carbon-containing wastewaters. Two heat-treated inocula (fresh and digested activated sludge) were compared, for H2 production from xylose at 37, 55 and 70 °C. At both 37 and 55 °C, a higher H2 yield was achieved by the fresh than digested activated sludge, whereas a very low H2 yield was obtained by both inocula at 70 °C. Then, four different inoculum pretreatments (acidic, alkaline, heat and freezing shocks) were evaluated for creating an efficient mesophilic (37 °C) or thermophilic (55 °C) H2 producing community. Acidic and alkaline shocks selected known H2 producing microorganisms belonging to Clostridiaceae at the expenses of lactate producing bacteria, resulting in the highest H2 yield at 37 and 55 °C, respectively. Although a heat shock resulted in a low H2 yield in a single batch, H2 production by the heat-treated fresh activated sludge was shown to increase in the experiment with four consecutive batch cycles.Heat-treated fresh activated sludge was selected as inoculum for continuous H2 production from a xylose-containing synthetic wastewater in a mesophilic (37 °C) and a thermophilic (55-70 °C, increased stepwise) fluidized bed reactor (FBR). A higher H2 yield was obtained in the thermophilic than in the mesophilic FBR. Furthermore, H2 production at 70 °C, which failed in the earlier batch study, was successful in the FBR, with a stable yield of 1.2 mol H2 mol-1 xyloseadded. Operation temperature of 70 °C was also found optimal for H2 production from thermomechanical pulping (TMP) wastewater in a temperature gradient incubator assay.A RNA approach was used to study the structure and role of the anode-attached, membrane-attached and planktonic microbial communities in a mesophilic (37 °C) and a thermophilic (55 °C) two-chamber, xylose-fed MFC. An anode attached community dominated by Geobacteraceae sustained electricity production at 37 °C, whereas the establishment of methanogenic and H2 oxidizing microorganisms resulted in a low electricity production at 55 °C. However, the development of a thermophilic exoelectrogenic community can be promoted by applying a start-up strategy which includes imposing a negative potential to the anode and chemical inhibition of methanogens. A mesophilic exoelectrogenic community was also shown to produce electricity from TMP wastewater in an upflow MFC operated at 37 °C. In conclusion, a higher and more stable H2 yield can be achieved in thermophilic rather than mesophilic dark fermentation. Dark fermentation at 70 °C is particularly suitable for treatment of TMP wastewater as it is released at high temperature (50-80 °C) and could be treated on site. TMP wastewater can be also used as substrate for electricity production in mesophilic MFCs. Electricity production in thermophilic MFCs is feasible, but enrichment of thermophilic exoelectrogenic microorganisms may require a long start-up period

Nearly all viruses encapsulate their genomes in protective protein shells known as capsids. Capsids self-assemble from repeating protein subunits, which surround the viral genome. Many viruses use a powerful biomotor to pump DNA into preformed capsid shells. Therefore, not only does the capsid protect the genome from environmental stress, it additionally stabilizes against high internal pressure caused by the tightly-packaged genome inside. To understand how capsids remain stable despite extreme conditions, I use thermophilic bacteriophage P74-26 as a model to probe the structural mechanisms that govern capsid assembly and stability. P74-26 capsids have a similar architecture to capsids of mesophilic tailed bacteriophages, allowing direct comparison to elucidate the structural basis of enhanced thermostability. Here I determine the structure of the P74-26 capsid decoration protein, which contains a core beta-barrel domain termed the ‘beta-tulip’ domain. The beta-tulip domain is conserved in structural proteins from both Herpesviruses and phage, as well as a broad-spectrum Cas9 inhibitor, providing evidence of shared evolutionary ancestry. Additionally, my high-resolution structure of the P74-26 virion capsid reveals unique interdigitated architectural features that contribute to enhanced stability in the thermophile. P74-26 has a significantly larger capsid than related mesophiles yet retains the same icosahedral geometry, demonstrating a novel mechanism for increasing capsid capacity. Furthermore, my thesis work explores capsid assembly and maturation mechanisms in vitro, establishing P74-26 as a platform for future development of novel nanoparticles and therapeutic delivery systems. Taken together, this work illuminates the incredible stability of a thermophilic virus and illustrates its utility as a powerful tool for studying viral maturation.

Depuis la découverte des cheminées hydrothermales, de multiples travaux ont été menés afin d’en étudier la diversité microbienne. Les inventaires moléculaires réalisés ont ainsi mis en évidence une grande diversité d’espèces qui contraste avec la faible proportion (1 %) d’espèces isolées par approche culturale. Une nouvelle approche d’immobilisation cellulaire par inclusion dans une matrice de polymères (gellane et xanthane) a ainsi été développée pour permettre l’étude de ces communautés thermophiles anaérobies marines. Le système, basé sur la formation d’une émulsion entre une solution de polymères inoculée et de l’huile, permet le piégeage de cellules dans des billes de gel de 1 à 2 mm de diamètre. Les conditions optimales d’immobilisation ont été obtenues pour une émulsion réalisée à 80 °C sous agitation (150 tr/min) à partir d’une solution de gellane (2,5 %) et de xanthane (0,25 %) avec 12 g/L de NaCl et 4 g/L de citrate de sodium, bullée à l’azote et réduit au Na2S avant inoculation. Les billes ont montré une bonne résistance mécanique après 5 semaines d’incubation à des pH compris entre 5,4 et 8, des températures allant jusqu’à 90 °C et des concentrations en NaCl et soufre allant jusqu’à respectivement 80 et 5 g/L. Des cultures en batch de Thermosipho sp. AT1272 et Thermococcus kodakarensis KOD1 immobilisées ont permis d’obtenir des concentrations allant jusqu’à 107 cellules/g de billes et 108 cellules/mL de fraction liquide. Une culture en continu réalisée en bioréacteur gas-lift pendant 41 jours à partir d’une communauté synthétique immobilisée composée de 8 souches (hyper)thermophiles a démontré la capacité de l’immobilisation cellulaire à protéger les cellules face à un stress oxique et à les maintenir (3 des 8 souches) dans le bioréacteur jusqu’à ce que les conditions de culture soient propices à leur croissance. La réactivité de la communauté immobilisée face aux changements environnementaux (température) a également été démontrée. Enfin, la culture en continu réalisée pendant 64 jours d’un échantillon immobilisé de diffuseur du site Rainbow a permis la croissance de plusieurs espèces bactériennes et archéennes (Oceanithermus sp., Thermococcus sp.) dont une partie n’a été détectée que dans les billes (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) par clonage-séquençage. L’ensemble de ces résultats ont permis de valider l’utilisation d’un protocole d’immobilisation par inclusion dans une matrice de polymères pour l’étude des communautés hydrothermales, de leur diversité et de leur dynamique. / Since the discovery of hydrothermal vents, multiple studies have been conducted in order to study microbial diversity. Molecular inventories realized have thus demonstrated a great diversity of species that contrasts with the low proportion (1%) of species isolated by culture approach. A new cell immobilization approach by inclusion in a polymer matrix (gellan and xanthan) has been developed for the study of these thermophilic anaerobic marine communities. The system, based on the formation of an emulsion between an inoculated polymer solution and oil, allows the entrapment of cells in gel beads with a diameter between 1 and 2 mm. The optimal immobilization conditions were obtained for emulsion performed at 80 °C with stirring (150 rpm) with a polymer solution composed of gellan (2.5%) and xanthan (0.25%) with 12 g/L of NaCl and 4 g/L of sodium citrate, bubbled with nitrogen and reduced with Na2S before inoculation. The beads showed a good mechanical stability after a 5-week incubation at pH between 5.4 and 8, temperatures up to 90 °C and NaCl and sulfur concentrations up to respectively 80 and 5 g/L. Batch cultures of immobilized Thermosipho sp. AT1272 and Thermococcus kodakarensis KOD1 yielded concentrations up to 107 cells/g of beads and 108 cells/mL of liquid fraction. A continuous culture performed in a gas-lift bioreactor for 41 days of an immobilized synthetic community composed of 8 (hyper)thermophilic strains demonstrated the capacity of cell immobilization to protect cells from oxique stress and to maintain them (3 of 8 strains) in the bioreactor until having suitable culture conditions for their growth. The reactivity of the immobilized community to environmental change (temperature) was also demonstrated. Finally, the continuous culture performed for 64 days of an immobilized diffuser sample from Rainbow site allowed the growth of several bacterial and archaeal species (Oceanithermus sp., Thermococcus sp.), part of which was detected only in the beads (Sulfurimonas sp., Nitratifractor sp., Vibrio sp.) by cloning-sequencing. All these results have validated the use of an immobilization protocol by inclusion in a polymer matrix for the study of hydrothermal communities, of their diversity and their dynamics.

Les virus peuvent être retrouvés dans tous les écosystèmes où de la vie est présente. Ils constituent l’entité biologique la plus abondante de la biosphère. Si de nombreuses données sont disponibles sur l’abondance et la dynamique virale dans les écosystèmes aquatiques tempérés, peu d’études ont été menées sur ces aspects dans les milieux extrêmes, dont les sources hydrothermales. Dans l’étude présentée dans ce manuscrit, les communautés procaryotiques et virales des sources hydrothermales des Terres australes et antarctiques françaises (TAAF) ont été explorées. Dans un premier temps, les cellules procaryotiques et les particules de type viral (VLP) ont été dénombrées dans plusieurs sources chaudes terrestres et marines côtières. L’abondance microbienne et virale est de l’ordre de 105 - 106 particules/ml dans les deux types de sources avec des rapports VLP/procaryotes (VPR) qui sont généralement faibles, concordant ainsi avec rares les données disponibles actuellement dans la littérature. Dans un second temps, la diversité morphologique des VLP a été analysée par observation au microscope électronique à transmission. La présence de VLP de morphologies différentes a pu être constatée dans quelques échantillons bruts, mais également dans des cultures d’enrichissement, où elles étaient associées à des Thermococcales et des Thermotogales. Finalement, quelques souches isolées de ces échantillons ont été criblées pour la présence de virus aboutissant à la description d’un nouveau bactériovirus tempéré associé à une bactérie thermophile Geobacillus. L’effet d’un choc osmotique en présence de NaCl et l’effet d’un stress anoxique sur la production virale ont également été étudiés. La caractérisation du virus GTV1 a ensuite été entamée. Il appartient à la famille des Myoviridae et a un génome composé d’ADN double brin de 38841 pb, composé de 71 ORF prédits. Enfin, l’étude de la diversité microbienne a permis de décrire une nouvelle espèce bactérienne hautement thermophile, Calditerricola clavaformis sp.nov. / Viruses thrive in all types of ecosystems where life is found. They represent the most abundant biological entity of our biosphere. Though several studies have been conducted on viral abundance and dynamics in mesophilic aquatic ecosystems, these aspects remain largely unexplored in extremophilic environments, such as hot springs. In this study, prokaryotic and associated viral communities of the French Southern and Antarctic Lands hot springs were explored. First, prokaryotic cells and Virus-like particles (VLP) were enumerated in several terrestrial and inshore hot springs. The results reveal an abundance of 105 - 106 particles/ml in both types of hot springs studied. The virus-to-prokaryote ratios (VPR) were generally low, confirming thus actual knowledge in these types of ecosystems. The morphological diversity of VLP was then studied in raw samples as well as in enrichment cultures containing Thermococcales and Thermotogales. Several isolates obtained from these samples were then screened for viral particles which led to the discovery and description of a temperate phage (GTV1) of a thermophilic bacterium belonging to the genus Geobacillus. The effect of NaCl and anoxic stress on the viral production was studied. The genomic characterization of the GTV1 was started and revealed a 38441 bp genome with 71 predicted ORF. Finally, microbial diversity studies led also to the discovery of a new extremely thermophilic bacterium, Calditerricola clavaformis sp.nov.

‘Aigarchaeota’, a deeply branching lineage in the domain Archaea with no cultivated representatives, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. The ‘Aigarchaeota’ consists of at least nine proposed genus-level groups that have been confirmed via 16S rRNA sequencing, with ‘Aigarchaeota’ Group 1 (AigG1) being the focus of this study. Based on cultivation-independent genomic data available from several AigG1 members in Great Boiling Spring (GBS), NV, and Yellowstone National Park, 22 different types of growth media were designed and tested for their ability to support growth of AigG1. One of these cultures, G1-10, was found to contain AigG1 at ~5% abundance, as well as other novel thermophilic microbial groups including a new species of the genus Pyrobaculum, members of the candidate phyla ‘Calescamentes’ and ‘Fervidibacteria’, and the novel archaeal lineage NAG1 (‘Geoarchaeota’). To attempt to obtain pure cultures of AigG1 and other novel thermophiles, a single-cell sorting system using an optical trap and a microfluidic device was constructed. The system was validated by sorting E. coli cells, which demonstrated that single, viable cells could be reliably obtained. Using this single cell sorting device on the G1-10 culture, a pure culture of a member of the genus Pyrobaculum was obtained, which was shown to represent a distinct species in this phylum by whole genome sequencing and in silico DNA-DNA hybridization. Additionally, a pure culture of the first representative of the candidate phylum ‘Fervidibacteria’ from an enrichment culture derived from G1-10. Additionally, to aid in morphology-based sorting of AigG1 and stable isotope labeling studies, fluorescence in situ hybridization (FISH) based on catalyzed reporter deposition (CARD-FISH) were developed and an AigG1-specific probe was tested. CARD-FISH was successfully used to detect AigG1 in both the G1-10 culture and in natural sediment samples from GBS. Stable isotope labeling incubations were performed with a variety of 13C-labeled substrates (bicarbonate, amino acids, sugars, and short chain fatty acids) on GBS sediments and G1-10 culture samples, and CARD-FISH was used to specifically detect AigG1 in the fixed samples. Nanometer-scale secondary-ion mass spectrometry (nano-SIMS) will then be used to determine whether AigG1 was capable of taking up the different carbon substrates tested. Overall, the results and accomplishments from this project and follow up nano-SIMS analysis will allow a better understanding of the metabolic potential of AigG1 and will aid future efforts to attempt to obtain pure cultures of this novel lineage.

Zwei Glykosylhydrolasen des acidothermophilen Bakteriums Alicyclobacillus acidocaldarius konnten charakterisiert werden. Die jeweiligen Gene wurden kloniert und sequenziert. Das intrazelluläre Enzym CelA und das extrazelluläre Enzym CelB könnten zusammen eine tragende Rolle im Abbau von beta-1,4-verknüpften Polysacchariden spielen. Ein Gen, welches für eine beta-1,4-Endoglucanase (CelA) kodiert, wurde kloniert und überexpremiert. Das Enzym wurde gereinigt. Das Protein besitzt eine Immunoglobulin-ähnliche Domäne, jedoch keine Cellulosebindedomäne. Zusammen mit den Sequenzähnlichkeiten der katalytischen Domäne zeigen diese Merkmale, daß das Protein zur Familie 9, Untergruppe E1 der Glykosylhydrolasen gehört. CelA zeigte höchste Aktivität gegenüber beta-1,4-Glucanen, besaß jedoch auch Aktivität gegen Haferspelzenxylan. Das Enzym hatte ein pH optimum von 5.5 und ein Temperaturoptimum von 70 °C. Im Protein waren zwei Zink- und zwei Calcium-Ionen gebunden, die wahrscheinlich wichtig für die Temperaturstabilität sind. Gegenüber p-Nitrophenylglykosiden ergab sich ein überraschendes Hydrolysemuster: Höchste Aktivität wurde auf dem Cellobiosidderivat gefunden, eine niedrigere Aktivität fand sich auf dem Cellotetraosederivat, wohingegen keine Aktivität auf den Glucose- und Cellotriosederivaten gemessen wurden. Das Hydrolysemuster führte zu dem Schluß, daß in CelA die Bindung von beta-1,4-Glucanen entweder auf der nicht reduzierenden Seite der -2 Substratunterbindestelle sterisch verhindert wird, oder alternativ zwei starke Substratunterbindestellen -1 und -2 vorhanden sind. Es wurde kein Signalpeptid zur Markierung des Proteins für die Translokation über die Membran gefunden. Zusammen mit der hohen Aktivität gegenüber Oligosacchariden, dem fast neutralen pH Optimum und der Inaktivierung bei niedrigem pH, deutet dies auf eine Rolle des Proteins als cytoplasmatisches Enzym zum Abbau importierter Oligosaccharide hin. Ein zweites Enzym mit Xylanase- und Cellulaseaktivität wurde zunächst aus A. acidocaldarius Kulturen gereinigt. CelB besaß eine molekulare Masse von 100 kDa und konnte nur mit Hilfe von Detergenz von den Zellen abgelöst werden. Klonierung und Sequenzanalyse des entsprechenden Gens ergaben einen offenen Leserahmen, welches für ein Präprotein kodierte, das ein typisches Sec-abhängiges Signalpeptid besaß. Gereinigtes, rekombinantes CelB und eine verkürzte Variante, der die letzten 203 Aminosäuren fehlten, zeigten Enzymaktivitäten, die dem Wildtypprotein ähnlich waren. Ein niedriges pH-Optimum von 4 wurde bestimmt. Auch die Stabilität war bei niedrigem pH hoch, wobei das Enzym nach Inkubation über nacht bei pH 1.5 bis 7 eine Restaktivität von 80 % aufwies. Das Temperaturoptimum betrug 80 °C. Bei dieser Temperatur war das Enzym auch stabil und zeigte nach 1 h 60 % Restaktivität. CelB hatte die Spezifität eines Endoenzyms, jedoch wurden nach längeren Inkubationszeiten Cellobiose und Xylobiose aus Cellulose bzw. Xylan freigesetzt. Das Enzym gehörte zur Familie 51 der Glykosylhydrolasen, aber es war erst der zweite Eintrag dieser Familie mit typischer Endoglucanaseaktivität. CelB hatte höchste Sequenzähnlichkeit mit der zweiten Endoglucanase, EGF aus Fibrobacter succinogenes, wobei diese beiden Proteine eine markante Gruppe im phylogenetischen Baum dieser Familie bildeten. Die Analyse der Aminosäurezusammensetzung der katalytischen Domänen ergab, daß CelB in Übereinstimmung mit der Anpassung an einen niedrigen pH-Wert, weniger geladene Aminosäuren als die neutrophilen Enzyme der gleichen Familie besitzt. Wildtyp-CelB und unverkürztes, rekombinantes CelB waren nur in Anwesenheit von Detergenz löslich. Dagegen war das verkürzte CelB Protein vollständig in Wasser löslich. Daher wird eine Rolle der C-terminalen Region bei der Zellassoziation nahegelegt. Dieser hydrophobe Bereich zeigte lokale Übereinstimmungen der Aminosäuresequenz mit einer hydrophoben Region einer Amylase aus dem gleichen Organismus. / Two glycoside hydrolases from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius were characterized and the corresponding genes cloned and sequenced. Together the intracellular enzyme CelA and the extracellular enzyme CelB may play a major role in the degradation of beta-1,4-linked polysaccharides. A gene encoding a beta-1,4-endoglucanase (CelA) was cloned and the enzyme overexpressed and purified. The protein contained an immunoglobulin-like domain but lacked a cellulose-binding domain. In conjunction with sequence similarities of the catalytic domain these features demonsrated the protein to be a member of glycoside hydrolase family 9, subgroup E1. CelA was most active against substrates containing beta-1,4-linked glucans, but also exhibited activity against oat spelt xylan. It displayed a pH optimum of 5.5 and a temperature optimum of 70 °C. The protein was found to contain one zinc and two calcium ions, likely to be important for temperature stability. It showed a striking pattern of hydrolysis on p-nitrophenyl glycosides, with highest activity on the cellobioside derivative, some on the cellotetraoside derivative and none on the glucoside and trioside derivatives. The hydrolysis patterns led to the conclusion that CelA contained a steric block for beta-1,4-linked glucans on the non reducing side of subsite -2 or, alternatively, two strong binding sites -1 and -2. No signal peptide for transport of CelA across the membrane was detected. This, together with high activity on oligosaccharides, a near neutral pH optimum, and inactivation at low pH, suggests a role for the protein as a cytoplasmic enzyme for the degradation of imported oligosaccharides. A second enzyme with xylanase and cellulase activity was purified from A. acidocaldarius cultures. CelB displayed a molecular mass of 100 kDa and could only be removed from cells with the help of detergent. Cloning and sequence analysis of the corresponding gene revealed an ORF encoding a preprotein with a typical sec-dependent signal peptide. Purified recombinant CelB and a truncated varient lacking the C-terminal 203 amino acid residues displayed enzymatic properties similar to the wild-type protein. A low pH optimum of 4 was found. Stability was also high at low pH, the enzyme retaining 80 % of activity after incubation over night from pH 1.5 to 7. The temperature optimum was 80 °C, a temperature at which the enzyme was also stable, showing 60 % residual activity after 1 h. CelB displayed an endo mode of action, but release of cellobiose and xylobiose from cellulose and xylan, respectively, was observed after prelonged periods of incubation. CelB belonged to glycoside hydrolase family 51, but it was only the second entry in this family with activity typical of an endoglucanase. Highest sequence similarity was found towards the other endoglucanase, EGF from Fibrobacter succinogenes, the two forming a distinct group in the phylogenetic tree of this family. Analysis of the amino acid composition of the catalytic domains demonstrated that CelB contains fewer charged amino acids than its neutrophilic counterparts, which is in line with adaptation to low pH. Wild-type and full-length recombinant CelB were soluble only in detergent. In contrast, truncated CelB was completely water soluble, suggesting a role of the C-terminal region in cell association. This C-terminal hydrophobic region displayed local sequence similarities to a hydrophobic region of an amylase from the same organism.