Probing the bacterial cell wall with chemical biology tools
Sminia, Tjerk J. - \ 2017
Wageningen University. Promotor(en): H. Zuilhof; W.M. de Vos, co-promotor(en): T. Wennekes. - Wageningen : Wageningen University - ISBN 9789463437080 - 196
bioengineering - sugars - labelling - synthesis - biochemical techniques - akkermansia muciniphila - gastrointestinal microbiota - carbohydrates - bioengineering - suikers - etiketteren - synthese - biochemische technieken - akkermansia muciniphila - microbiota van het spijsverteringskanaal - koolhydraten
After DNA and proteins, carbohydrates are the third language of life. Chapter 1 introduces the reader to this class of biomolecules, also called sugars or glycans, that can be found on the outer surface of almost all cells and plays a critical role as the social messengers of a cell. Although our knowledge about the role of glycans in eukaryotic cells has increased considerably in recent decades, our understanding of the glycan layer on bacterial cells is still very limited. Besides the carbohydrates that are present in both eukaryotes and prokaryotes an additional wide range of unique (e.g. microbial sialic acid), often very complex (e.g. pseudaminic acid), carbohydrates is present in prokaryotes. This chapter briefly introduces two research fields, carbohydrate chemistry and chemical biology, that when combined provide a powerful way to investigate the biological role of these unique bacterial carbohydrates at the molecular level. This chemistry-based approach, termed chemical microbiology, often starts with the development of a chemical synthesis for a target bacterial carbohydrate. Subsequently, the synthetic route towards this target allows for the introduction of unnatural functional groups, like chemical reporters, that result in the molecular tools needed to study their biological function. The studies described in this thesis, focus on developing such molecular tools to study the role of glycans and glycoconjugates in human gut bacteria and human-associated bacteria.
Chapter 2 provides an overview of metabolic oligosaccharide engineering (MOE) a popular chemical biology technique to label glycans in living cells. In MOE, carbohydrates derivatives are synthesised with unnatural chemical reporters and used to study their incorporation in glycans of eukaryote to prokaryote species. The progress in this field over the last 6 years is reviewed in detail with a special emphasis on the synthesis of the unnatural carbohydrates from commercially available sources. The principle behind MOE is that these unnatural carbohydrates with e.g. azide, alkyne, cyclopropene, or isonitrile chemical reporter groups, are still recognised by the endogenous enzymes in the cell that salvage this new carbohydrate. In this way they can enter the associated biochemical pathways and end up in newly biosynthesised cellular glycans. Subsequent labelling techniques, such as strain promoted azide alkyne cycloaddition or tetrazine ligation, enable the visualisation of these incorporated unnatural carbohydrates with for instance fluorescence microscopy.
Metabolic labelling is further explored in chapter 3. Key cell envelope glycoconjugates in the mucin-degrading gut microbiota member, Akkermansia muciniphila, were subjected to chemistry-based functional analysis, with Escherichia coli being used as a control species. Two novel non-toxic peptidoglycan (PG) probes were designed and synthesised to investigate the presence of PG in this species. Their design was based on the natural d-alanine dipeptide motif found in PG. Inspired by the fact that d-alanine dipeptide-derivatives were previously reported to be incorporated in newly synthesised PG, we synthesised a cyclopropene and isonitrile d-alanine dipeptide. Our probes proved to be non-toxic, as shown by growth and viable count analysis, and were therefore superior over existing PG probes. Another beneficial property was that the probes also did not influence the specific growth rate of A. muciniphila or E. coli. The PG probes were successfully incorporated into the peptidoglycan layer of A. muciniphila and visualised using a tetrazine click-ligation with a fluorophore. Our analysis proved for the first time that A. muciniphila has a PG layer. Besides PG labelling, we also investigated metabolic labelling of other glycoconjugates on the outer surface of A. muciniphila. This part of the study showed that azido-monosaccharide derivatives of N-acetylglucosamine, N-acetylgalactosamine, and fucose are successfully processed by A. muciniphila salvage pathways and incorporated into its surface glycoconjugates. Especially 6-azido-fucose was readily processed by the recently discovered l-fucose salvage pathway of A. muciniphila. The two compatible labelling techniques were next combined in a dual labelling experiment. Our isonitrile dipeptide peptidoglycan probe and 6-azido-fucose were successfully incorporated into A. muciniphila. Subsequent fluorescent labelling with bio-orthogonal techniques resulted in dual labelling of peptidoglycan and fucose-containing glycans in live A. muciniphila cells.
With the positive results of MOE in A. muciniphila in hand, chapter 4 describes the further investigation of MOE. After successful validation of our Ac4FucAz probe for MOE in Bacteroides fragilis we continued their application in other human gut microbiota members, including the butyrate-producing Anaerostipes rhamnosivorans, Intestimonas butyriciproducens, and Eubacterium hallii. Labelling of these human gut microbes proved to be rather challenging with a-specific cellular labelling with the fluorophore being the major problem. Initial results, however, did show that a 6-azido-l-rhamnose probe resulted in fluorescent labelling of A. rhamnosivorans, which provides initial evidence for the existence of an as of yet undocumented salvage pathway. In this species the 6-azido-fucose probe was not salvaged. Via confocal microscopy and flow cytometry analysis we observed that the 6-azido-rhamnose probe was selective for A. rhamnosivorans in the presence of A. muciniphila. Such a co-culture experiment is a first step in mimicking the complex human gut microbiome. For E. hallii Ac4GalNAz gave clear metabolic labelling and the majority of the cell population could be labelled with the fluorescent dye after a strain-promoted azide alkyne cycloaddition. Other glycan probes (Ac4GlcNAz, Ac4FucAz, and Neu5Az) also resulted in labelling, but not as prominent as Ac4GalNAz. Surprisingly, MOE has never been reported for the common lab strain Escherichia coli MG1655. Curious to investigate this in more detail we started MOE in E. coli. However, no labelling was obtained when Ac4GlcNAz probe was added to E. coli, most likely due to the fast growth, metabolism and turnover. Only, when fresh Ac4GlcNAz probe was added every 30 minutes, metabolic labelling in E. coli was observed. To further investigate the influence of GlcNAc metabolism in E. coli on MOE, single-gene knock-outs of E. coli GlcNAc metabolism from the Keio collection were investigated. Labelling was observed for NagA (N-acetyl glucosamine 6 P deacetylase) and NagK (N-acetyl-d-glucosamine kinase) E. coli mutants. Both enzymes are involved in the last step of the biosynthesis towards UDP-N-acetylglucosamine. When the overall E. coli metabolism was inhibited, after addition of the respiration inhibitor sodium azide, no metabolic labelling was observed. These results indicate that MOE in E. coli is possible, but challenging and can only be performed under specific circumstances.
An investigation into the total synthesis of pseudaminic acid, a sialic acid produced by specific human-associated prokaryotes, is described in chapter 5. Sialic acids are typically found at the terminal positions of surface glycoconjugates in both eukaryotes and prokaryotes. Other related microbial sialic acids are legionaminic and acinetaminic acid. The total synthesis of these microbial sialic acids is notoriously difficult, as exemplified by the fact that only a few chemical synthesis routes towards them are currently known. Our total synthesis of pseudaminic acid started from the readily available amino acid l-threonine that was transformed into a key versatile Garner aldehyde derivative intermediate. With this aldehyde in hand, the Henry nitro-aldol condensation reaction was investigated. After studying numerous conditions, such as asymmetric catalysis or elongated reaction times, and extensive optimisation efforts we were never able to obtain the Henry reaction product to continue with this route. As an alternative, a tethered aminohydroxylation was investigated for its ability to introduce the key functional group and stereochemistry onto an intermediate obtained from the Garner aldehyde derivative. This reaction indeed gave the desired amino-alcohol motif in the correct stereochemistry, but another diastereomer proved very difficult to separate from the desired product. After some additional transformations and protection steps we obtained a derivative in which the primary alcohol could be oxidised to provide a hexose intermediate that resembles the hexose intermediate present in pseudaminic acid biosynthesis. This key hexose intermediate will likely enable a subsequent Barbier reaction, a chain elongation step, in future studies. With most of the key transformations accomplished, the completion of a pseudaminic total synthesis based on l-threonine should soon be possible. Besides finishing the total synthesis, future work should also focus on adapting this synthesis route to allow installation of chemical reporter groups on pseudaminic acid for its application in MOE.
Chapter 6 is the general discussion about all the work mentioned in the other chapters. It also contains additional information and suggestions for further research in the field of chemical microbiology.
Multiplex-detectie van Phytophthora: "padlock-based Universal Multiplex detection Array" (pUMA)
Gaszczyk, K. ; Mendes, O. ; Verstappen, E.C.P. ; Bonants, P.J.M. ; Schoen, C.D. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 145 - 146.
phytophthora - detectie - identificatie - biochemische technieken - nucleotidenvolgordes - polymerase-kettingreactie - phytophthora - detection - identification - biochemical techniques - nucleotide sequences - polymerase chain reaction
Plant Research International heeft een diagnostische methode ontwikkeld die toe te passen is 'in planta', en ook de meest recent beschreven (quarantaine-) soorten omvat. De methode omvat de ontwikkeling van een generieke Phytophthora-methode gevolgd door een Phytophthora-identificatie.
Multiplex-detectie van plantenpathogenen
Bergervoet, J.H.W. ; Peters, J. ; Vlugt, R.A.A. van der; Wolf, J.M. van der; Weerdt, M. de; Beekhoven, J. van - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 145 - 145.
plantenvirussen - detectie - elisa - besparingen - biochemische technieken - plant viruses - detection - elisa - savings - biochemical techniques
Een manier om de kosteneffectiviteit van de detectie van plantenpathogenen te verbeteren is door het tegelijkertijd detecteren van meerdere pathogenen in één monster (zgn. multiplexen). Het gebruik van de Luminex xMAP-technologie biedt een uitgelezen platform om bestaande reagentia in te zetten in een multiplex-setting.
Biochemical and structural analysis of thermostable esterases
Levisson, M. - \ 2009
Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Servé Kengen. - [S.l. : S.n. - ISBN 9789085854593 - 167
esterasen - fysicochemische eigenschappen - chemische structuur - biochemische technieken - kristallisatie - esterases - physicochemical properties - chemical structure - biochemical techniques - crystallization
Biocatalysts play an important role in modern biotechnology because of their specificity, selectivity, efficiency and sustainability. One of the industrially most exploited and important groups of biocatalysts are the esterases. These enzymes catalyze, in the presence of water, the hydrolysis of an ester-bond resulting in the formation of an alcohol and a carboxylic acid. The use of enzymes in industrial processes also has its restrictions. Many processes are operated at elevated temperatures or in the presence of organic solvents. These conditions are detrimental to most enzymes and therefore there is a growing demand for enzymes with an improved stability. In this regard, there is a special interest from industry in enzymes of thermophilic origin since these enzymes generally display a high intrinsic thermal and chemical stability. This thesis describes the results of biochemical and structural analyses of thermostable esterases.
Bioinformatics was used to identify new ester-hydrolyzing enzymes in the genomes of the hyperthermophilic bacterium Thermotoga maritima and the hyperthermophilic archaeon Archaeoglobus fulgidus. These potential esterases were cloned and heterologously expressed in Escherichia coli. Different types of ester hydrolyzing enzymes were found, including carboxylesterases, an acetyl esterase and a lipase. The biochemical properties of these enzymes were studied in detail. In addition, crystallization trials were performed, resulting in the three-dimensional structures of several of these enzymes. New structural features were revealed, such as the combination of an esterase domain with an immunoglobulin-like domain.
The information obtained in this study provides fundamental knowledge, which may act as a basis for modern methods of enzyme engineering, with the aim to improve the applicability of these enzymes.
Nieuwe aangrijpingspunten voor bestrijding van Phytophthora infestans
Meijer, H.J.G. ; Govers, F. - \ 2009
aardappelen - solanum tuberosum - phytophthora infestans - biochemische technieken - ziektepreventie - gewasbescherming - milieubeheer - moleculaire detectie - potatoes - solanum tuberosum - phytophthora infestans - biochemical techniques - disease prevention - plant protection - environmental management - molecular detection
Posterpresentatie over de beheersing van de aardappelziekte die in belangrijke mate isgebaseerd op preventieve bespuitingen met fungiciden die moeten voorkomen dat Phytophthora infestans de plant binnendringt. Om de druk op het milieu te verminderen, vindt onderzoek naar nieuwe aangrijpingspunten voor Phytophthora-bestrijding plaats
Voorbeelden van biobased toepassingen
Bolck, C.H. ; Bos, H.L. - \ 2009
biochemische technieken - type voorbeelden - productontwikkeling - biobased economy - bioplastics - biochemical techniques - type specimens - product development - biobased economy - bioplastics
Deze info sheet geeft een aantal voorbeelden van toepassingen uit de biobased economy. De laatste jaren is er een fors aantal ontwikkelingen geweest, waaruit nieuwe toepassingen en/of producten zijn voortgekomen
Specificity, pathogenicity and population dynamics of the endoparasitic nematode Heterodera arenaria in coastal foredune
Stoel, C.D. van der - \ 2001
Wageningen University. Promotor(en): L. Brussaard; J.W. Woldendorp; W.H. van der Putten. - S.l. : S.n. - ISBN 9789058084361 - 135
plantenparasitaire nematoden - heterodera - ammophila arenaria - plantenziekteverwekkers - populatiedynamica - plantensuccessie - pathogeniteit - vastleggen van duinen - identificatie - biochemische technieken - plant parasitic nematodes - heterodera - ammophila arenaria - plant pathogens - population dynamics - plant succession - pathogenicity - sand dune stabilization - identification - biochemical techniques
Key words: Heterodera , plant-parasitic nematodes, soil pathogens, Ammophila arenaria , occurrence, abundance, specificity, population dynamics, life history, pathogenicity, PCR-SSCP, molecular method, escape, sand burial, dispersal, migration, fitness, development time, survival, reproductive success, bottom-up, top-down.
In natural ecosystems hardly any attention has been given to the population dynamics of plant-parasitic nematodes. In coastal foredunes, plant-parasitic nematodes are supposed to be involved in the degeneration and succession of the dominant sand-fixing grass Ammophila arenaria (Marram grass). The specificity, pathogenicity and population dynamics of the sedentary endoparasitic nematode Heterodera arenaria have been studied to determine if this species might be a key component of the soil pathogen complex of A. arenaria.
H. arenaria was found to be specific to Elymus farctus and A. arenaria in the mobile area of the coastal foredunes. Colonisation of the newly deposited sand layer by H. arenaria corresponded well with the development of pathogenicity in a series of bioassays. However, direct addition of the nematode to A. arenaria did not result in growth reduction of the plant. So, H. arenaria behaves like a biotrophic parasite, which has a high specificity but is not aggressive. Therefore, H. arenaria did not seem to be directly involved in the degeneration of A. arenaria .
Each year, the majority of the population of new H. arenaria cysts develops in the newly deposited sand layers. These layers are colonised by A. arenaria roots throughout the growing season. Migration to the new root layer may offer an individual nematode the benefit of early development and a larger potential offspring. The continuous release of juveniles in the field and their development in experiments indicate that release of juveniles from cysts is an ultimately determined process. Juveniles were found to emerge in November and many eggs or juveniles did not survive the winter period. The strategy of release, however, seems effective; the distance of migration could be too large to detect specific cues from the plant and the start of root formation in the field is highly variable. The emergence of juveniles late in the growing season could result in a second generation within the same year. The constant number of cysts per gram of roots suggests that the population density of H. arenaria is most likely a bottom-up directed process.
Biological nitrogen fixation of soybean in acid soils of Sumatra, Indonesia
Waluyo, S.H. - \ 2000
Agricultural University. Promotor(en): W.M. de Vos; L. 't Mannetje; L.T. An. - S.l. : S.n. - ISBN 9789058082954 - 151
glycine max - sojabonen - bodembiologie - stikstoffixatie - stikstofbindende bacteriën - rhizobium - bradyrhizobium - inoculatie - entstof - biochemische technieken - dna-fingerprinting - stamverschillen - stammen (biologisch) - zaadbehandeling - omhullen - zure gronden - bodemaciditeit - bekalking - sumatra - indonesië - glycine max - soyabeans - soil biology - nitrogen fixation - nitrogen fixing bacteria - rhizobium - bradyrhizobium - inoculation - inoculum - biochemical techniques - dna fingerprinting - strain differences - strains - seed treatment - pelleting - acid soils - soil acidity - liming - sumatra - indonesia
The aim of this study is to improve soybean cultivation in transmigration areas, especially in Sitiung, West Sumatra. However, these soils are very acid, and have a high P-fixing capacity. To reduce the amounts of fertilisers, normally 5 - 7 ton lime ha -1 and 100 kg P as TSP, seed, pelleted with lime (60 kg ha -1 ) and TSP (10 kg ha -1 ), was introduced. In this way only 2 ton lime ha -1 are required.
Soybean can fix nitrogen (BNF) in symbiosis with ( Brady ) Rhizobium bacteria. However, these acid soils in general, have low numbers of ( Brady ) Rhizobium . By inoculating the soils with ( Brady ) Rhizobium , BNF of soybean, and yield, were considerably improved.
A study was made of the indigenous ( Brady ) Rhizobium population in view of the following:
Using molecular techniques, indigenous strains derived from soil samples from old soybean areas (Java) and from new soybean areas (Sumatra) were classified in more detail. Most likely B. japonicum is the dominant strain in Java while in Sumatra B. elkanii is more present. A Sinorhizobium fredii -like strain was isolated from one soil sample from Java.
Massaspectrametrisch onderzoek van nortestosteron en nortestosteron di-heptafluorboterzuurderivaat
Schilt, R. ; Huf, F.A. - \ 1987
Wageningen : RIKILT (Rapport RIKILT 87.61) - 8
massaspectrometrie - biochemische technieken - derivaten - hormonen - mass spectrometry - biochemical techniques - derivatives - hormones
In dit rapport wordt het massaspectrametrisch gedrag van nortestosteron en het nortestosteron als di-heptafluorboterzuur derivaat besproken. Naast spectra zijn ook de fragmentatie mechanismen weergegeven. Van het derivaat zijn de fragmentionen met hoge resolutiemassaspectrometrie onderzocht.
Verslag 4th Cologne Workshop in Dope Analysis, 6-11 april 1986 te Keulen
Schilt, R. - \ 1986
Wageningen : RIKILT (Rapport / RIKILT 86.66) - 12
doping - biochemische technieken - analytische methoden - paarden - doping - biochemical techniques - analytical methods - horses
Verslag van het bezoek aan de vierde Workshop over dopinganalyse, gehouden van 6 tot en met 11 april 1986 te Keulen. In dit verslag wordt een overzicht gegeven van de gehouden lezingen en de praktische aspecten die tijdens de workshop aan de orde zijn geweest.
Haalbaarheidsstudie naar biologisch/fysisch-chemische nitraatverwijdering uit grondwater.
Hoek, J.P. van der; Klapwijk, A. - \ 1986
H2O : tijdschrift voor watervoorziening en afvalwaterbehandeling 19 (1986). - ISSN 0166-8439 - p. 53 - 58.
biochemische technieken - drinkwater - nitraten - verwijdering - waterzuivering - biochemical techniques - drinking water - nitrates - removal - water treatment
A qualitative and quantitative investigation of olfactory and nasal respiratory mucosal surfaces of cow and sheep based on various ultrastructural and biochemical methods
Menco, B.P.M. - \ 1977
Landbouwhogeschool Wageningen. Promotor(en): L.M. Schoonhoven, co-promotor(en): L.H. Bannister. - Wageningen : Veenman - 157
rundvee - epitheel - schapen - reuk - zoölogie - reukorganen - weefselultrastructuur - biochemische technieken - cattle - epithelium - sheep - smell - zoology - olfactory organs - tissue ultrastructure - biochemical techniques
Many hypotheses have been developed to account for the process of olfaction (for reviews see MOULTON and BEIDLER, 1967; DAVIES, 1971 and POYNDER, 1974), but at the present time none of them has been verified. The olfactory organ demonstrates very interesting receptor properties. It interacts with a great variety of compounds, called odorants (STAHL, 1973).Appropriate biochemical and biophysical methods including the separation of sensory surfaces should be able to provide important evidence about receptive mechanisms and attempts to use such techniques have begun in recent years (e.g. KONORRING,CH and NORRING, 1969; ASH and SKOGEN, 1970; KOYAMA et al., 1971; KOROLEV and FROLOV, 1973; MENCO et al., 1974; MARGOLIS, 1975). Techniques for separating receptor structures have also been suggested by OTTOSON (1970), but sofar none of these methods has yielded a pure fraction of receptor endings (see DODD, 1974). The present account will deal mainly with the anatomy of the bovine olfactory epithelium emphasizing prospects for isolating receptor moieties. The adjacent nasal respiratory epithelium has also been investigated since, like the olfactory bipolar nervous cells such respiratory cells bear cilia. In the latter case, however, they are motile, rather than sensory, thus enabling a determination of features which are specific for the olfactory cilia and a comparison between two adjacent epithelium types allows such specific characterizations (LUCAS and DOUGLAS, 1934; SLEIGH, 1974).The cow has been used as an experimental animal, since the size of its olfactory organ and the availability of samples seemed most convenient for this work. Some of the studies presented here were carried out on sheep. Indications for behaviour towards odorants in cows have been reviewed by ERNST and PUSHKARSKII (1975).In Chapter 1, the distal processes of both epithelium types are described, using different microscopical methods, including light microscopy, scanning electron microscopy, thin section transmission electron microscopy and thick section high- voltage transmission electron microscopy. This latter method was used in the hope that it would permit olfactory cilia to be followed over their whole lengths. In this chapter some attention will also be devoted to macroscopic observations.Chapter 2 deals with the results of freeze-etch and electron spin resonance studies on both epithelium types. Both techniques allow predictions of some of the molecular properties of the receptive area through investigation of intact tissue in vitro.Chapter 3 deals with a quantitative analysis of the morphological data. Statistical methods are used where it is possible. Several features of the olfactory nerve endings are compared for different nasal areas and for adult as opposed to juvenile animals. Special emphasis is placed on the ciliary processes. Olfactory and respiratory cilia are also compared with each other. Furthermore, estimates for possible receptor concentrations based chiefly on freezeetch results are presented.Such quantitative information is important for biochemical work, since it indicates whether one can consider nerve ending preparations as homogeneous or if one has to take into account that biochemical preparations may contain morphologically different nerve ending types. Furthermore, some ideas about receptor quantities which might be isolated can be obtained.Finally, Chapter 4 deals with attempts to isolate peripheral receptor membranes. The conventional criteria used by others for assessing the purity of the fractions have been shown to be inadequate (DODD, 1974); this prompted us to initiate anatomical studies on the bovine olfactory mucosa, so as to provide ourselves with a more adequate basis for future biochemical studies.
|Beproeving van enige zuurmengels op bactericide en fungicide werking in mengvoer
Wieringa, G.W. ; Viering, J. - \ 1974
Wageningen : [s.n.] (Mededelingen / Instituut voor bewaring en verwerking van landbouwprodukten I.B.V.L. no. 425) - 10
analyse - biochemische technieken - biologische technieken - mengvoer - analysis - biochemical techniques - biological techniques - compound feeds
|Kwaliteitsonderzoek met behulp van een taste - panel : verslag van de eerste orienterende proef in de periode van 5 maart 1962 - 26 juni 1962
Ludwig, J.W. - \ 1963
Wageningen : [s.n.] (Intern rapport / Instituut voor bewaring en verwerking van landbouwprodukten (I.B.V.L.) no. 181) - 20
analyse - biochemische technieken - biologische technieken - cassave - voedselbewaring - voedingsmiddelen - huishoudkunde - organoleptische kenmerken - aardappelen - wortelgewassen als groente - sensorische evaluatie - solanum tuberosum - analysis - biochemical techniques - biological techniques - cassava - food preservation - foods - home economics - organoleptic traits - potatoes - root vegetables - sensory evaluation - solanum tuberosum