Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Bacteriophage: from exploration to exploitation
    Nobrega, Franklin L. - \ 2017
    Wageningen University. Promotor(en): John van der Oost, co-promotor(en): J. Azeredo; Stan Brouns. - Wageningen : Wageningen University - ISBN 9789463430524 - 338
    bacteriophages - hosts - interactions - genetic engineering - methodology - screening - bacteriofagen - gastheren (dieren, mensen, planten) - interacties - genetische modificatie - methodologie - screenen

    Over the past decades, bacteriophage research has revealed the abundance of phages in nature, their morphological and genomic diversity, their influence in the regulation of microbial balance in the ecosystem and their impact on the evolution of microbial diversity. Since the 1950s, phages have also played a central role in some of the most significant fundamental discoveries in biological sciences that have been crucial for the development of molecular biology. More recently, phage research has resulted in the development of genome editing tools, and it has generated the renewed interest of using phages and phage-related products as therapeutic agents. Although major progress has been made, basic understanding on phage biology is still lacking. The number of phage genes with unknown function still largely outnumber those with established roles. Therefore, further progress depends on a deeper understanding on phage biology.

    The present thesis aims at developing tools to support phage research, explores the use of phages for therapeutic purposes, and expands our insights into the biology of phages. A literature review on the molecular, structural and evolutionary determinants of phage-host interaction (Chapters 1 and 2) underlines the relatively poor understanding of the subject. A great variety of structures and mechanisms of infection are being revealed, but no correlations have yet been established between these and host interaction. Furthermore, so far no evolutionary model accurately describes the coevolution of phages and bacteria. A particular interest of evolutionary studies concerns the understanding of the prevalence of broad-host range phages in natural environments, since these are rarely isolated using standard laboratory isolation procedures. Indeed, we have tried to isolate broad host range phages targeting the Escherichia coli reference collection (Chapter 3), but found narrow-host range phages to be more prevalent. Only one phage of relatively broad host range was found (S2-36s), being able to infect 14 of the 72 strains. Proteins of interest for further exploration were found, such as depolymerases and colanic acid-degrading proteins, both with potential anti-biofilm activity.

    The isolation procedures against the ECOR collection proved to be challenging due to the amount of strains and samples to be evaluated. Consequently, a high-throughput methodology was developed to simplify these isolation procedures (Chapter 4). By automated monitoring of cell growth in 96-well plates it is possible to use differences in optical densities (plotted as heatmaps) between cells subjected to the samples and in control conditions to screen for the presence of phages. The method revealed an accuracy of 98% and reduced the workload by 90%. The method developed can also be used to screen for broad-host range phages or to screen collections of phages for variants or cocktails that are suitable for treating bacterial infections. A discussion is provided of the advantages and limitations of phages for therapeutic applications (Chapter 5). It is suggested that phages in their natural state cannot be used in therapeutic applications. The future of phage therapy may possibly be genome engineering for tailoring of phage properties. Subsequently, the genetic modification of phage T7 was shown to improve (2-log) the capacity of the phage to resist to the strongly acidic conditions and enzymatic challenges of the gastrointestinal tract (Chapter 6). This was achieved by modifying the phage to express a signal peptide on its capsid to which phospholipids attach forming a protective coating. The removal of the phospholipid coating using phospholipase caused reversion to the pH-sensitive phenotype of the wild-type phage. In case of orally-delivered phages, this may improve the efficacy of phage therapy.

    Engineering of phage genomes can also support evolutionary studies and basic phage research, e.g. analyzing if a certain gene is essential. A strategy developed for the random recombination of phage genomes (Chapter 7) demonstrated that it is possible to create novel productive phages by combining elements of different phage families. The findings reveal an unexpected level of flexibility and adaptability of phage genomes to accommodate and re-arrange genetic information, reflecting the pre-existing evolutionary compatibility of genes from different phages. The method is further expected to serve as a platform for improving our understanding of phage gene function and importance, where the random recombination of a single phage genome may be the preferred approach.

    A different approach for the therapeutic application of phages was explored. Using phage display it was possible to identify peptides targeting claudin-low breast cancer cells (Chapter 8) and osteoarthritis cells (Chapter 9) with high levels of specificity. The peptides identified may contribute to an early detection of claudin-low breast carcinomas, and to develop more individualized therapies for both breast cancer and osteoarthritis.

    In summary, the work developed in this thesis has resulted in new methodologies and biological data, thereby contributing to an increased understanding of phage biology and of the opportunities for the use of phages for diagnosis and therapy.

    Mixed culture engineering for steering starter functionality
    Spuś, Maciej - \ 2016
    Wageningen University. Promotor(en): E.J. Smid; Tjakko Abee. - Wageningen : Wageningen University - ISBN 9789462578333 - 170
    bacteriophages - predation - microorganisms - starters - genetics - diversity - bacteriofagen - predatie - micro-organismen - zuursels - genetica - diversiteit

    Undefined mixed complex starter cultures are broadly used in Gouda-type cheese production due to their robustness to phage predation, resilience for changes in environmental conditions and aroma compounds production ability during ripening. These microbial communities of lactic acid bacteria prior their isolation and deposition in starter culture collections were continuously used at the farm-level production facilities. Thus, one can consider undefined mixed complex starters as domesticated microbial communities. The process of domestication was facilitated by humans who have been continuously repeating successful fermentations using part of previous batch as inoculum (i.e. back-slopping). Therefore, a term ‘community breeding’ can describe this human-driven domestication of microbial communities. Community breeding of a model complex starter Ur led to establishment of a simple two-species composition of Lactococcus lactis and Leuconostoc mesenteroides represented by, in total, 8 genetic lineages. At the same time, this simple microbial community displays a high degree of intraspecies diversity, presumably caused by evolutionary processes of horizontal gene transfer, genome decay and mutations. Such diversity at strain level is particularly interesting in the context of continuous bacteriophage predation pressure present in this microbial community. It is thought that constant-diversity (CD) dynamics, based on the ‘kill-the-winner’ principles, is operational in Ur starter at the strain level. According to CD model, the fittest strain(s), which feed on the most abundant substrate, will be selected against due to density-dependent phage predation. The control of the fittest strain abundance by bacteriophages opens space for differentiation of strains via eco-evolutionary feedbacks. In particular, strains of complex starter culture not only adapted to quickly acidify milk (via efficient consumption of lactose and protein to peptides degradation), but concurrently, to consume other substrates present in milk. In addition, throughout the process of community breeding microbe-microbe interactions between community members have evolved. These interactions have led to division of metabolic labor among strains present in the culture, and eventually to better starter microbial community functioning.

    The aim of this thesis was to investigate the factors impacting the formation of compositionally and functionally stable undefined mixed complex starter cultures to further use this knowledge in steering its functionality, and potentially in developing new strategies for robust starter culture design. To facilitate this study, well-characterized Ur culture strain isolates were used to systematically reconstitute the starter culture into multi-strain blends with increasing level of strain and genetic lineage diversity. The investigation of factors such as phage predation, level of strain and genetic lineage diversity as well as environmental conditions, was performed during experimental evolution studies in milk. The functionality of the (evolved) starter cultures was tested in an adapted lab-scale MicroCheese model system. The specific approach used in each of the research chapters is described below in more detail.

    Strains isolated from Ur starter culture were characterized in terms of their resistance against bacteriophages isolated from the same starter (Chapter 2). This test confirmed high diversity in phage resistance among strains belonging to different genetic lineages as well as among strains of the same lineage. Next, selected strains, which represented different levels of bacteriophage predation: resistant, moderately resistant, sensitive and no detectable sensitivity, were mixed in simple blends containing 4 strains representing 3 genetic lineages of Ur starter (3 such blends were designed). These blends were exposed to phage predation (one phage per blend) at the onset of prolonged sequential propagation experiment or propagated without phage addition (control). Throughout the serial propagation the genetic lineage composition was monitored. During the propagation of control blends we detected quick domination of a single lineage. This dominating lineage contained strains sensitive to phages. Genetic lineage level composition of the phage-challenged blends was much more dynamic suggesting the impact of phage predation. The relatively low strain diversity introduced in these blends was not high enough to sustain maximal diversity at the level of lineages.

    Chapter 3 describes a study using defined blends with higher complexity by extending the number of strains used. In total, 24 strains representing all 8 Ur starter lineages were exposed in sequential propagation experiment to a cocktail of 3 phages isolated from Ur starter. The propagation in milk of this multi-strain blend was executed for more than 500 generations and the abundance of genetic lineages was monitored throughout. Similarly as in the simple blends experiment, control blends were not exposed to bacteriophages. In control blends we observed a domination of one genetic lineage upon serial propagation, which resembles a periodic-selection-like (PS) behavior, where the fittest strains are dominating the microbial community and in result genetic-lineage diversity is being substantially reduced. In contrast, the composition of phage-challenged blends was again more dynamic than in control blends. In one of the phage-challenged blends behavior characteristic for a constant-diversity dynamics model was observed; throughout the serial transfer experiment, genetic lineage diversity was maintained by the presence of phage predation at relatively high level. In case of the second phage-challenged blend, due to a stochastic event, which likely caused a reduction in phage pressure, we observed a gradual recovery of the fittest strains, which again resembled a periodic-selection behavior. Therefore, phage predation, among other factors, can lead to shifts in microbial community population dynamics resulting in alternative stable states.

    The experimental evolution approach, resembling traditional process of back-slopping, was used in a Long-term experimental evolution of Undefined Mixed Starter Culture (LUMSC) study described in Chapter 4. The aim of this study was to investigate the compositional and functional stability ascribed to the undefined mixed Ur starter during enclosed prolonged propagation without any possible external influx of bacterial or phage material. Surprisingly, during this 1000-generation long experiment the enforced conditions of specific incubation temperature and propagation regime resulted in enrichment of previously not detected strain of Lactococcus laudensis. This strain was found to consume a by-product of metabolism of another strain present in the community, in particular, D-mannitol produced by Le. mesenteroides. Thus, a new putative interaction in the microbial community of the complex starter culture was found. This new interaction and the possible ability of L. laudensis to efficiently use peptides released by caseinolytic L. lactis ssp. cremoris resulted in a relatively high abundance of L. laudensis in all evolved LUMSC cultures. The high abundance of L. laudensis had a certain effect on the functionality of the cultures. The aroma profiles of model lab-scale milli-cheeses manufactured with LUMSC cultures, showed significant differences in formation of esters and alcohols when compared to cheeses produced with the original Ur starter. Moreover, L. laudensis strain was not only under the radar of previously used culture-dependent and culture-independent methods, but as well, under the radar of phage predation continuously present throughout the LUMSC experiment. This observation sheds new light on the possibility of how a strain can emerge to relatively high abundance in an enclosed serially propagated microbial community operating in accordance with CD dynamics model.

    Finally, the aspect of adaptation to environmental conditions was addressed by the study of an adjunct strain of Lactobacillus helveticus DSM 20075 described in Chapter 5. The aim was to develop a strain with increased autolytic capacity in conditions resembling the cheese matrix to possibly improve cheese ripening. The approach used here was based on a previously reported study, where the incubation of Lactococcus lactis MG1363 at high temperature resulted in spontaneous mutations causing stable heat-resistant and, in some cases, salt-hypersensitive phenotypes. In present study, after incubation of the Lb. helveticus DSM 20075 adjunct at different high temperatures (45-50 °C), heat-sensitive variants were recovered from plates. These variants were further characterized in terms of their growth rates at elevated temperatures (42-45 °C) and their autolytic capacity in low pH buffer with addition of NaCl. One of the variants (V50) showed substantially increased intracellular lactate dehydrogenase enzyme activity in the buffer suggesting its increased autolytic capacity. Next, both wild type and variant V50 were tested as adjuncts in lab-scale model milli-cheeses to determine their possible impact on the cheese aroma profiles. Indeed, adjunct strains, both WT and the variant, impacted the aroma profiles by producing benzaldehyde. In case of the variant strain the relative abundance of this compound was 3-fold higher. The applied strategy of incubating Lb. helveticus DSM20075 at high temperature resulted in specific, but different than in case of L. lactis MG1363, mutations suggesting another, yet to be elucidated, mechanisms for increasing the autolytic capacity of industrially-relevant strains. The approach of high-temperature incubation can be applied in dairy industry for the selection of (adjunct) cultures targeted at accelerated cheese ripening and aroma formation.

    In conclusion, the work presented in this thesis highlights the importance of co-evolution of strains in compositional and functional stability of the complex undefined mixed starter culture. In particular, the factors such as heterogeneity of bacteriophage resistance among highly related strains, microbe-microbe interactions and division of metabolic labor are crucial for optimal functioning of a complex starter microbial community. Further investigation of the factors impacting the composition of starter cultures is crucial to steer the functionality in a desired direction. With straightforward methods, such as changing the incubation temperature or the propagation regime it is possible to induce shifts in strain composition and thereby obtain cultures with new characteristics. Moreover, experimental evolution studies with microbial communities used in food fermentation can lead to the discovery of new strains with potentially new characteristics. Additionally, the study of microbial communities of starter cultures not only delivers industrially applicable knowledge but also reveals the action of basic principles in microbial ecology and evolution.

    Onderzoek naar middelen tegen Dickeya dianthicola in Sedum
    Bulle, A.A.E. ; Trompert, J.P.T. ; Hollinger, T.C. - \ 2013
    Lisse : Praktijkonderzoek Plant en Omgeving BBF - 21
    houtachtige planten - sedum - bacterieziekten - dickeya dianthicola - fysische gewasbeschermingsmethoden - elicitoren - antagonisten - bacteriofagen - methodologie - veldproeven - woody plants - sedum - bacterial diseases - dickeya dianthicola - physical control - elicitors - antagonists - bacteriophages - methodology - field tests
    Bacteriën veroorzaken in de teelt van diverse siergewassen steeds vaker grote problemen. Er worden niet alleen nieuwe bacterieziekten gekarakteriseerd, maar ook worden meer gewassen door al bekende bacteriesoorten aangetast. Een aantasting door bacteriën verloopt vaak desastreus voor het gewas en de verliezen door vernietiging van de oogst kunnen gigantisch zijn. Er zijn momenteel geen gewasbeschermingsmiddelen beschikbaar voor de bestrijding (doding) van bacteriën in een gewas. Daarom is het voorkómen van een besmetting door preventie en goede hygiënemaatregelen veelal de enige optie en dus van groot belang. Maar dit voorkomt helaas niet altijd dat een bacterie-infectie optreedt. Er zijn verschillende stoffen (elicitors) bekend die het afweermechanisme van de plant stimuleren waardoor de plant weerbaarder wordt tegen ziekten en plagen. In de literatuur zijn veel elicitors beschreven. Op basis hiervan is een aantal elicitors getest in het zomerbloemgewas Sedum, waarin de bacterie Dickeya dianthicola desastreus kan toeslaan. In 2012 is een veldproef in Sedum uitgevoerd bij PPO in Lisse, waarin een besmetting met de bacterie Dickeya dianthicola (veroorzaker van rot) is aangebracht. Op een praktijkperceel waar in vorige jaren aantasting was gezien, is een tweede proef aangelegd. Er is in deze proeven geen duidelijk en consistent effect van de onderzochte middelen gezien. Afhankelijk van het waarnemingsmoment lijken enkele middelen effect te kunnen hebben. Mogelijk dat het effect van de elicitors groter zou zijn geweest als de toepassingen eerder in het groeiseizoen mogelijk waren geweest.
    Faagtherapie is alternatief voor antibiotica
    Willemsen, P.T.J. - \ 2011
    Kennis Online 2011 (2011)14 juni. - p. 21 - 21.
    veehouderij - antibioticaresistentie - pluimveehouderij - kuikens - bacterieziekten - bacteriofagen - antibacteriële eigenschappen - livestock farming - antibiotic resistance - poultry farming - chicks - bacterial diseases - bacteriophages - antibacterial properties
    De zoektocht naar alternatieven voor het gebruik van antibiotica in de veehouderij is nog steeds urgent. Bacteriofagen kunnen uitkomst bieden. De faagtherapie bleek o.a. succesvol in een proef met 120 kuikens, waarvan de helft besmet was. Na toediening was de ziekteverwekkende bacterie voldoende gereduceerd.
    Virussen succesvol als bacteriedoders, Thema: Innovaties duurzame gewasbescherming BO-12.03-003.02-012
    Vlugt, R.A.A. van der; Wolf, J.M. van der; Doorn, J. van; Dees, R.H.L. ; Schoorl-Nijhuis, E.H. ; Verbeek, M. - \ 2011
    biologische bestrijding - erwinia - bacteriofagen - gewasbescherming - bloembollen - aardappelen - biological control - erwinia - bacteriophages - plant protection - ornamental bulbs - potatoes
    Poster met onderzoeksinformatie over het gebruik van bacteriofagen voor de bestrijding van de Erwinia-bacteriesoort Dickeya.
    CRISPR-mediated antiviral defence in prokaryotes
    Jore, M.M. - \ 2010
    Wageningen University. Promotor(en): John van der Oost, co-promotor(en): Stan Brouns. - S.l. : s.n. - ISBN 9789085857815 - 154
    prokaryoten - bacteriofagen - antiviruseigenschappen - prokaryotes - bacteriophages - antiviral properties - cum laude
    cum laude graduation (with distinction)
    Schonere kip dankzij fagen
    Bergen, M.A.P. van - \ 2009
    Kennis Online 6 (2009)aug. - p. 10 - 10.
    bacteriologie - bacteriofagen - campylobacter - salmonella - pluimveehouderij - pluimvee - bacteriology - bacteriophages - campylobacter - salmonella - poultry farming - poultry
    Antibiotica zijn door de toename van resistenties geen wondermiddel meer. Omdat er alternatieven moeten komen voor de bestrijding van schadelijke bacteriën, doet het Centraal Veterinair Instituut (CVI) onderzoek naar een, in de westerse wereld, in de vergetelheid geraakte methode met fagen. De eerste testen bij campylobacter en salmonella zijn hoopgevend
    Structure, function and subcellular localization of the potato Resistance protein Rx1
    Slootweg, E.J. - \ 2009
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Aska Goverse. - [S.l. : S.n. - ISBN 9789085854678 - 176
    solanum tuberosum - planteiwitten - pathogenesis-gerelateerde eiwitten - bindende eiwitten - resistentiemechanismen - ziekteresistentie - plantenvirussen - plantenparasitaire nematoden - bacteriofagen - genexpressie - faag display - solanum tuberosum - plant proteins - pathogenesis-related proteins - binding proteins - resistance mechanisms - disease resistance - plant viruses - plant parasitic nematodes - bacteriophages - gene expression - phage display
    Resistance proteins are part of the plant’s immune system and mediate a defence response upon recognizing their cognate pathogens. They are thought to be present in the cell as part of a larger protein complex. The modular architecture of R proteins suggests that they form a scaffold for various interacting proteins, involved in pathogen recognition, downstream signalling or protein stabilization. However, few common interactors have been found for the CC-NB-ARC domains despite extensive screenings for downstream interactors. The objective of thesis was to get new insights in the structure, function and localization of R proteins by using the potato resistance genes Rx1 and Gpa2 as a model system. Initially, a novel T7 phage display method was developed to facilitate high throughput selection of interacting molecules (Chapter 2). However, the use of a T7 cDNA phage library to identify interactors of the CC-NB-ARC domains of Rx1 resulted in the discovery of a large set of highly basic peptides (Chapter 3). In Chapter 4, the functional role of the CC-NB-ARC domain in mediating disease resistance was explored by creating chimeric proteins between Rx1 and Gpa2. This resulted in the observation that the CC-NB-ARC is able to confer both virus and nematode resistance in potato. Furthermore, it was shown that the CC-NB-ARC of Rx1 and the LRR of Gpa2 are incompatible and vice versa. This phenomenon was studied in more detail in Chapter 5, in which a docking model for the interacting surface of these domains was constructed based on the individual structural domains. Finally, the subcellular localisation was investigated to get a better understanding about the R proteins function in the cell (Chapter 6).

    The lytic T7 phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for strong interactions with a variety of substrates. To visualise these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein (Chapter 2). By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3’-end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. As far as we know this is the first report of using a translational frameshift for a biotechnological purpose. The phages formed in this way have capsids composed of three different variants of their capsid protein; EYFP-fused versions derived by frameshift translation, non-fused versions derived by regular translation from the helper plasmid, and versions that display peptides encoded in the library ligated in the phage genome. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. Closely monitoring the effect of the selection procedure enables fine tuning, and obviates the need for more laborious ELISA or plaque lift assays. Furthermore, with the fast pace of developments in single molecule detection technologies and sorting systems, these fluorescent phages open the way to high throughput platforms for the direct selection of binding molecules.

    In Chapter 3, cDNA phage display was applied as an alternative method to identify additional downstream Rx1 interactors, which could further resolve the Rx1 signalling pathway. In a pilot experiment the value of T7 phage display to identify specific interactors was demonstrated by using an antibody raised against the PVY coat protein. Screening of a PVY-infected N. benthamiana cDNA phage display library resulted in the selection of peptides harbouring the known PRIKAI epitope. Next, phage display was explored as technique to discover proteins interacting with the potato R protein Rx1. The system turned out to be prone to pick up interactors binding to matrices like Ni-NTA or to fusion proteins like thioredoxine. A possible way to circumvent this weakness was to design the selection procedure in such a way that it alternates between different matrices and to limit the number of selection round. This adapted approach resulted in the identification of a series of highly basic protein fragments and random peptides, for which a specific interaction could be shown. Two cDNA sequences encoded the ribosomal proteins L19 and L36a, which showed a stunted growth phenotype upon gene silencing in N. benthamiana using VIGS and a slightly reduced Rx-mediated HR.

    The nematode resistance protein Gpa2 and the virus resistance protein Rx1 provide an excellent test system to investigate the exchangeability of recognition and signalling domains and explore the evolutionary flexibility of R proteins, for they confer resistance to completely unrelated pathogens (Globodera pallida and potato virus X, respectively). In Chapter 4, we provide evidence for the hypothesis, that, via intergenic sequence exchanges and various types of mutations, NB-LRR proteins have the potential to alter resistance specificities towards taxonomically unrelated pathogens in relatively short evolutionary time periods. Both the regulatory sequences and CC-NB domains of the paralogs Gpa2 and Rx1 are non-pathogen specific and exchangeable. Remarkably, the genetic fusions of the CC-NB of Rx1 with the LRR of Gpa2 (Rx1CN/Gpa2L) and the reciprocal domain swap (Gpa2CN/Rx1L) were not functional when driven by the endogenous promoters or 35S promoter. Gain of wild type resistance was obtained by re-introducing the first five LRRs of Rx1 in Rx1CN/Gpa2, restoring the compatibility between the N-terminal part of the LRR and the ARC2 domain. Decreasing the expression levels for Gpa2CN/Rx1L resulted in extreme resistance against PVX, indistinguishable from wild type plants. Our results indicate that not only coding sequences, but that also optimizing the expression levels may play a role in generating novel resistances.

    The CC, NB-ARC, and LRR domains of the Rx1 and Gpa2 proteins interact with each other and recognition of the elicitor mediated by the LRR is translated in an activation of the NB-ARC. The available functional and evolutionary data make Gpa2 a suitable candidate for structural modelling of the individual domains and their interaction (Chapter 5). A structural model of the NB-ARC / LRR interaction could function as a framework for the interpretation of known empirical data and the design of new experiments to test R protein operational mechanisms (Zhang 2009). Therefore, computer aided modelling of the 3D structure domain models for the NB-ARC and the LRR domains were obtained and used as basis for a domain docking study. The functional interaction between the domains was studied via a detailed analysis of their incompatibility in chimeric Gpa2 and Rx1 proteins. A large set of sequence exchanges between the two proteins was created for that purpose. Both in the LRR and in the ARC2 domain small regions could be identified in which the amino acids differing between Gpa2 and Rx1 led to domain incompatibility. Five of the ARC2 positions required for LRR compatibility and three known autoactivating positions from the RX1 LRR were used as constraints in domain docking computation to limit the potential search space. The resulting docking model indicated an important role in the NB-ARC-LRR interaction for electrostatic and hydrophobic interactions. A loop region rich in acidic residues in the ARC2 domain was found close in space to a patch of basic residues grouped together in the LRR. Hydrophobic residues on both the NB and the ARC2 contacted hydrophobic residues on the surface of the LRR. A correlation analysis of the NB-ARC and LRR subdomains detected coevolution between the interacting surfaces, which supports a direct interaction between these two domains. Site-directed mutagenesis and pull-down experiments were used to test the role of surface features that might play an important role in the interdomain docking interface.

    In Chapter 6, we have made use of the characteristic of the Rx1 protein that it remains functional when its domains are co-expressed as separate polypeptides. This allowed us to create fluorescent constructs, not only of the full length protein, but also of the separate subdomains. Most of these tagged constructs still form functional proteins. C- and N-terminal fusions of Green Fluorescent Protein (GFP) variants to Rx, made it possible to study its subcellular localization in Nicotiana benthamiana cells. Contrary to our expectations we observed the presence of Rx1 in both the cytoplasm and the nucleus. Rx1 does not contain known nuclear localization signals and the size of the protein (140 kDa including GFP) exceeds the limit for passive diffusion through the nuclear pore. Fluorescent fusions of a series of deletion constructs, CC-NB-ARC, NB-ARC, NB-ARC-LRR, CC and LRR showed three distinct patterns of subcellular localization. The NB-ARC-LRR and LRR constructs have a cytoplasmic localization and are mostly absent in the nucleus. The NB-ARC and CC-NB-ARC constructs showed equal fluorescence intensities in both the nucleus and the cytoplasm. The CC alone fused to GFP, however, seems to preferentially accumulate in the nucleus resulting in a three to four times higher fluorescence intensity in the nucleus compared to the cytoplasm. The diffusion behaviour inside the nucleus for both the complete CC and a CC fragment containing the two predicted helices downstream of the central turn, showed that their nuclear accumulation coincides with a significantly reduced nuclear diffusion as compared to unfused GFP and the other CC fragments. This difference might point to a potential interaction between the CC and an unknown nuclear component. Furthermore, SGT1 and Rar1 are thought to function as chaperones involved in stabilizing R proteins. Both the silencing experiments with these two proteins and the P-loop mutation show that the nuclear localisation of Rx1 is probably conformation dependent. Two approaches were followed to see if CP recognition or Rx1 signalling pathway were linked to a certain cellular compartment. At one hand the Rx1 protein itself or its subdomains were directed to either the nucleus or the cytoplasm by fusion to exogenous targeting signals (Nuclear Export Signals or Nuclear Localization Signals). On the other hand the elicitor, the PVX coat protein, was directed to the nucleus or cytoplasm. The PVX coat protein is a much smaller protein and can under normal circumstances diffuse freely between the cytoplasm and the nucleus. The surprising result was that no effect was found for retargeting Rx1, but when the elicitor was targeted to the nucleus, it could not activate Rx1 anymore, indicating that recognition might to take place in the cytoplasm.

    In the final chapter, the results obtained in this thesis are put into perspective by studying parallels in scientific literature on NB-LRR proteins with similar functions in other organisms.

    Bacteriophages: therapeuticals and alternative applications : Onderzoeksrapport commissie genetische modificatie
    Vlugt, R.A.A. van der; Verbeek, M. - \ 2008
    Bilthoven : Cogem (CGM 2008-03) - 75
    bacteriofagen - taxonomie - geneesmiddelenbehandeling - medische behandeling - bacteriophages - taxonomy - drug therapy - medical treatment
    Faag-display als bron voor diagnostische antistoffen
    Speksnijder, A.G.C.L. ; Beekwilder, M.J. ; Doorn, J. van - \ 2008
    detectie - schimmelsporen - bacteriofagen - antilichamen - recombinant dna - moleculaire detectie - detection - fungal spores - bacteriophages - antibodies - recombinant dna - molecular detection
    In deze poster informatie over de ontwikkeling van procedures voor de selectie van specifieke fagen voor de herkenning van schimmelsporen en de ontwikkeling van een recombinant antilichaam voor de detectie van Tulp virus X
    Stress response and virulence in Salmonella Typhimurium: a genomics approach
    Hermans, A.P.H.M. - \ 2007
    Wageningen University. Promotor(en): Tjakko Abee; Marcel Zwietering, co-promotor(en): H.J.M. Aarts. - [S.l.] : S.n. - ISBN 9789085045724 - 180
    salmonella typhimurium - stressreactie - virulentie - bacteriofagen - transcriptie - genexpressieanalyse - salmonella typhimurium - stress response - virulence - bacteriophages - transcription - genomics
    Since 1995 the number of human infections with Salmonella serovar Typhimurium DT104 increased in The Netherlands and abroad. The multi antibiotic resistance of this strain has been often proposed as plausible reason for this increase. Within his PhD research, Armand Hermans found novel DT104 specific DNA, that is most likely involved in virulence and might be an additional reason for the DT104 increase. In addition, the possibility for DT104 to survive certain process- and product conditions has been studied in this genomics based research. A better survival of stress such as acid or heat treatment could be another additional explanation for the DT104 increase. To enlarge the current insights on this topic, the activities of stress survival and virulence genes were studied by using the microarray technology. During exposure of DT104 to different stresses, similar stress survival mechanisms were activated, while different virulence mechanisms were stress specific activated. Bacterial cell communication also may play a role in regulation of these genes.
    Simulation and analysis of FRET in the study of membrane proteins
    Nazarov, P.V. - \ 2006
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga; V.V. Apanasovich. - [S.l.] : S.n. - ISBN 9789085045533 - 135
    oppervlakte-eiwitten - fluorescentie-emissiespectroscopie - energie-uitwisseling - neurale netwerken - bacteriofagen - surface proteins - fluorescence emission spectroscopy - energy exchange - neural networks - bacteriophages
    Membrane proteins play an important role in almost all cell activities. However, the characterization of the structure of membrane proteins in lipid bilayers is still at the frontier of structural biology. While 30-40% of all proteins are situated at or in membranes, yet less than 1% of the known protein structures are of membrane proteins. The complexity and delicacy of membrane-protein systems impedes the application of standard methods of protein study, such as X-ray crystallography and NMR. Furthermore, these techniques are mainly aimed at short-range structural information, and seem to be not very useful for the study of long-range interactions, for instance in the case of protein assemblies. These factors urge to find other biophysical approaches to study proteins incorporated into lipid bilayers. A successful alternative is Förster (or fluorescence) resonance energy transfer (FRET) spectroscopy in combination with site-directed labeling with fluorescent probes. This technique provides distance information within a range of 10-100 Å, which is sufficient to study the structure of membrane proteins and their complexes. The crucial point in the extraction of structural information from FRET data is an advanced and robust data analysis. This work is devoted to the development of such methods for analysis of fluorescence data, based on simulation modeling, global analysis and artificial neural networks. Especially the advances and problems of the simulation-based fitting (SBF) approach to fluorescence data analysis are considered. The methodologies of global analysis and SBF are applied to obtain information about the position, aggregation and structure of M13 major coat protein in DOPC:DOPGvesicles. The resulting physical parameters, that describe the embedment and orientation of the protein in the membrane, such as protein-protein aggregation, protein depth, tilt angle, and tilt direction, are in good accordance with previously reported values. Based on the FRET data, it was found that M13 major coat protein (having 50 amino acid residues) in its bilayer conformation could be described as a singlea-helix between amino acid positions 10-46. Additional work was performed on the methodological aspects of improving the SBF data analysis technique. Here it is proposed to use an artificial neural network to speed up the parameter identification and to make the process of fitting less sensitive to noise. The main idea of this method is the substitution of a time-consuming simulation model by an artificial neural network, specifically a multi-layer perceptron. The method results in a speeding up of the simulation by about a factor of 10 4 for the developed FRET model.
    Detection and sorting of micro-beads and bacteriophages in a microfluidic biochip
    Kunst, B.H. - \ 2006
    Wageningen University. Promotor(en): Ton Visser; Sacco de Vries, co-promotor(en): Arjen Schots. - [S.l.] : S.n. - ISBN 9789085045632 - 100
    bacteriofagen - fluorescerende tracers - fluorescentie-emissiespectroscopie - bacteriophages - fluorescent tracers - fluorescence emission spectroscopy
    The objective of the research described in this thesis was to design amicrofluidicsplatform for the screening ofbiolibraries. Thismicrofluidicplatform can be considered as a technology demonstrator for the screening and sorting of particles smaller than cells like fluorescent-labelled (submicrometer-sized) beads, bacteria and viruses. The particles can even have a relatively low fluorescence yield that can be compensated by longer interrogation times as compared to the ones used in conventional flowcytometrythat is now capable of screening many thousands of cells per minute.
    The membrane-bound form of gene 9 minor coat protein of bacteriophage M13
    Houbiers, M.C. - \ 2002
    Wageningen University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : S.n. - ISBN 9789058086921 - 118
    bacteriofagen - manteleiwitten - bacteriophages - coat proteins

    Bacteriophage M13 is a virus that infects the bacteria Escherichia coli ( E. coli ), a single cell organism that resides in our intestines. It consists of the cytoplasm (contents) and a double membrane that keeps the contents together (the barrier to the outside world). The membrane is formed by lipid molecules which consist of a head group that very much likes water (hydrophilic) and two fatty tails that dislike water (hydrophobic). In order to avoid contact with water the fatty tails group together in such a way that a planar double layer (bilayer) of the lipid molecules results. In this lipid bilayer also proteins are present to fulfill different functions, e.g. regulate transport across this barrier. The lipids and proteins together form the membrane. However, this membrane is not a complete barrier for certain intruders in the E. coli cell. A virus, such as M13 bacteriophage, can enter the E. coli cell. It uses the E. coli cell as a host by using the cell's components to multiply itself. And finally the bacteriophage is able to cross the membrane and put itself together on its way out. During this latter process, called the assembly, the new bacteriophage is formed out of its components. It is a special feature of bacteriophage M13 that the assembly takes place in the membrane. Even more special is that bacteriophage M13 does not kill its host. Normally, many other viruses kill their hosts by lysing the cells. Bacteriophage M13 consists of a single DNA molecule, which codes the genomic information, and a "coat" of proteins, which surrounds and protects the DNA. Bacteriophage M13 looks like a rod of almost 1 micrometer length and about 6.5 nanometer diameter. The coat proteins along the long side are all identical and present in many copies (about 2700) and therefore are called major coat proteins. On either end of the bacteriophage are a few different coat proteins, called minor coat proteins. One of these is the topic of this work: the gene 9 protein (g9p). G9p consists of only 32 amino acids. It is located on the end of the bacteriophage that emerges first from the E. coli cell during the assembly. Therefore, g9p is expected to play a role early in the assembly process. Genetic experiments also showed that g9p is a possible candidate to interact with the tip of the DNA molecule. This makes g9p an interesting object of study, since it might lift up some of the veil over the assembly process. The assembly process is still like a black box: the components that go in and the outcome (intact bacteriophage) are known, but what happens in between is still a mystery, although parts of it have been revealed. By taking a closer look of g9p we hope to contribute a small piece in unraveling this mystery. Up to now the structure and position of g9p before assembly were based on speculation. In this thesis we aimed to characterize g9p in more detail by analysis of the structure it adopts in a membrane. In addition, we aimed to determine how and which side enters the membrane.


    To study g9p we used chemically synthesized protein and since biological membranes are very complex structures we studied g9p in simpler artificial membranes.

    G9p has been predicted to be hydrophobic (Chapter 2) and is therefore difficult to handle. It is difficult to remove the protein once it sticks to a surface, and it has a tendency to form aggregates by sticking to each other. It can be dissolved in the organic solvent TFE. In an aqueous solution, detergents are necessary to solubilize the protein. In Chapter 2 conditions for solubilizing the protein were found. In a weak detergent g9p appeared to aggregate. This was accompanied by a conformational change froma-helix tob-sheet, as measured by CD and FTIR. The protein was incorporated in phospholipid membranes via TFE or detergent. When using detergent, care had to be taken to avoid aggregation intob-sheets. Phospholipid membranes appeared to preserve the state of the protein, either aggregated or non-aggregated. The work described in Chapter 2 resulted in the production of reproducible protein-lipid systems, which were necessary to study g9p in the membrane-bound state.

    Information about the structure of g9p is described in Chapter 2 and 3. CD and FTIR revealed that g9p is predominantlya-helical in the phospholipid membrane. By analysis of the amide I band of the FTIR spectrum secondary structure elements were quantified. About 67% of the protein structure wasa-helix, and the remaining part was assigned to be turn structure. In addition, the orientation of thea-helix axis with respect to the membrane normal was determined by measuring ATR-FTIR spectra of the protein incorporated in oriented bilayers. G9p appeared to be oriented preferentially parallel to the membrane normal, which means that the orientation is mainly transmembrane (Chapter 3). Based on these results and general principles that govern the positioning of protein domains in bilayers, we proposed possibilities for a tentative structural model of g9p in a membrane (Chapter 3 and 5). One model assumes a single N-terminal transmembrane helix of 21 amino acid residues long and a C-terminally located turn structure. An alternative model assumes a 15 amino acid trans-membrane helix and a second shorter amphipathic helix, which is more in line with the membrane plane. Turn structure links the two helices. The remaining turn structure is located C-terminally according to this model. This part was shown to contain the antigenic site (Chapter 4). The latter helix-turn-helix model remarkably resembles models of the major coat proteins of bacteriophage M13 and related bacteriophages.

    In Chapter 4 g9p was found to be able to insert spontaneously into phospholipid monolayers and bilayers. In addition, the N-terminal side of the protein was determined to be the part to insert, whereas the C-terminal part was determined to remain outside the membrane (N in -C out orientation). This was shown by cleavage experiments. After cleavage, the presence or absence of the antigenic site, which was found to be located at the C-terminal end of the protein, functioned as a determinant of the topology. Negatively charged phospholipids may enhance the efficiency of the adoption of a N in -C out orientation, which is in agreement with our experimental results and with literature (Chapter 5).

    The relevance of these experimental results with model systems is that they may provide indications for the in vivo situation. Up to now, no evidence was available regarding the conformation of g9p. The predominantlya-helical conformation we observed seems likely to be present in vivo also (Chapter 5). The finding that g9p can insert spontaneously in model membranes may be relevant for the in vivo situation. Our orientational and topological results show what part of the protein is buried in the membrane and what part is accessible. Translated to the in vivo situation this implies that the C-terminal side is sticking out in the cytoplasm, thus making it a good candidate to interact with the viral DNA in the assembly process.

    Membrane-anchoring interactions of bacteriophage major coat proteins
    Meijer, A.B. - \ 2001
    Wageningen University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : S.n. - ISBN 9789058084071 - 102
    manteleiwitten - laserfluorescentiespectroscopie - paramagnetische elektronenresonantiespectroscopie - bacteriofagen - coat proteins - laser fluorescence spectroscopy - electron paramagnetic resonance spectroscopy - bacteriophages

    The major coat proteins of the filamentous bacteriophages Pf3 and M13 are stored in the inner membrane of the cell during the reproductive cycle. In this process, protein-lipid anchoring interactions are important for the formation of the correct structure of these proteins in the membrane, enabling fast and efficient phage assembly. The focus of this thesis is on the role of domains and specific amino acids on the position of these proteins in the membrane. Both proteins are studied using site-specific probing using fluorescence and ESR spectroscopy. This biophysical approach provides information about the relative depth and dynamics of specific sites of the protein in the membrane. In previous structural views of the membrane-bound state of the M13 coat protein, the protein is thought to be in an L-shape, in which the N-terminal arm of the protein is positioned along the membrane surface. The spectroscopic studies described in this thesis show that the amphipathic N-terminus of the protein is not firmly associated with the membrane surface, but is also present in a more extended configuration. It is demonstrated that the hydrophobic amino acids in the N-terminal arm play an important role in the topology of the helix at the membrane surface. Furthermore, it is found that at the C-terminal side of the helical transmembrane domain, the charged lysines and hydrophobic phenylalanines are involved in strong anchoring interactions with the membrane interface region. These amino acids affect the location of the entire helical transmembrane domain. The results of a site-specific probing study on the Pf3 major coat protein reveals striking similarities with those obtained from the M13 major coat protein. Both proteins exhibit a strong structural coherence, in spite of the low primary sequence homology. Therefore, the tertiary structure of the Pf3 major coat protein should closely resemble the membrane-bound structure of the M13 coat protein. Apparently, domains of amino acids, which are comparable in physico-chemical characteristics, but lack sequence homology, are able to assemble in a structural coherent manner. In conclusion, the relatively simple coat proteins are positioned in the membrane via a complicated set of protein-lipid interactions provided by individual amino acids as well as domains of amino acids.

    Solid-state 31P NMR spectroscopy of bacteriophage M13 and tobacco mosaic virus
    Magusin, P. - \ 1995
    Agricultural University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : Magusin - ISBN 9789054853558 - 65
    kernmagnetische resonantie - kernmagnetische resonantiespectroscopie - virologie - tabaksmozaïekvirus - bacteriofagen - nuclear magnetic resonance - nuclear magnetic resonance spectroscopy - virology - Tobacco mosaic virus - bacteriophages

    In this thesis, the results of various 31P NMR experiments observed for intact virus particles of bacteriophage M13 and Tobacco Mosaic Virus (TMV), are presented. To explain the results in a consistent way, models are developed and tested. 31P nuclei in M13 and TMV are only present in the phosphodiesters of the encapsulated nucleic acid molecule. Therefore, 31P NMR spectroscopy reveals structural and dynamic properties of the nucleic acid backbone selectively without isotope labeling, even though the virus particles largely consist of coat proteins. In the Introduction (Chapter 1), it is discussed that the 31P chemical shift is sensitive to local nucleic acid backbone geometry and that the 31P NMR relaxation is dependent on the isolated and collective backbone motions. As shown in Chapter 3, high-power 1H-decoupled one-dimensional 31P NMR spectra observed for nonspinning samples of M13 and TMV contain a single, broad line dominated by the 31P chemical shift anisotropy (CSA), which masks any structural inequivalence among the encapsulated phosphodiesters. However, these spectra do contain interesting mobility information. On the one hand, they show that the nucleic acid molecule in each of the viruses is strongly immobilized in comparison to free nucleic acids in solution, as a result of interactions with the protein coat. On the other hand, the 31P resonance lineshapes; show clear signs of motional narrowing, which is indicative for (restricted) motion with frequencies in the order of the static linewidth or larger (≥10 4Hz). In contrast, the nonspinning 31P transversal relaxation measured for M13 indicates motion in the slow or intermediate frequency region as compared to the static linewidth (≤10 4Hz), because T 2e becomes shorter as the viscosity of the gel decreases

    To analyze the results in a more quantitative manner, three different rotational diffusion models for the phosphorus motion are developed in Chapter 2. These models are first tested at a theoretical level to get a feeling for their accuracy and to check their correspondence with standard theories under appropriate limiting conditions. Simulations show that a clear distinction between the effect of motional amplitude and frequency cannot be made within experimental error on the basis of one-dimensional spectra or transversal relaxation alone. However, these parameters can be extracted from the combined data. For fast motions, the transversal 31P NMR relaxation predicted by our models is consistent with standard Redfield relaxation theory. The relaxation effects caused by ultraslow rotational diffusion closely resemble the effects of translational diffusion of water protons in an inhomogeneous magnetic-field gradient. It is discussed in Chapter 3, that simple models, like isotropic and rigid-rod diffusion cannot reproduce the experimental data. Instead, a consistent description is offered by a combined diffusion model, in which the 31P NMR lineshape is dominated by fast internal DNA or RNA motions, and transversal relaxation reflects slow overall rotation of the rod-shaped virions about their length axis.

    To obtain more specific structural information, magic angle spinning (MAS) NMR spectroscopy is employed, which breaks up the broad 31P NMR lineshape into a sharp centerband at the isotropic chemical shift position flanked by rotational sidebands (Chapter 4). MAS 31P NMR spectra of TMV show two resolved sideband patterns with an overall intensity ratio of approximately 2, which are assigned to the three types of phosphodiesters in TMV on the basis of RO-P-OR' bondangles and supposed arginine bonding effects. In contrast, MAS 31P NMR spectra of M13, only contain a single, relatively broad centerband flanked by sidebands, indicating that a continuous distribution of phosphodiester conformations, rather than a few distinguishable, exists within the phage. The observed decrease of inhomogeneous linewidth at increasing temperature and hydration could perhaps be caused by some sort of "conformational averaging" as a consequence of nucleic acid backbone motion. This is illustrated by use of a simple model, which shows the lineshape effects caused by fast restricted fluctuation of the dihedral angles between the POC and the OCH planes on both sides of the 31P nucleus in the nucleic acid backbone. The presence of internal phosphodiester motions with frequencies ≥10 5Hz, as concluded from the motional narrowing of nonspinning 31P NMR lineshapes in Chapter 3, is confirmed by the deviation of sideband intensities in MAS 31P NMR spectra of dilute M13 gels from the theoretical values for solid powders. No dramatic broadening of the sidebands is observed, indicating that motions with frequencies in the order of the spinning rates applied (10 3Hz) are absent. Backbone motions also seem to be the main cause of transversal relaxation measured at spinning rates of 4 kHz or higher. At spinning rates below 2 kHz, transversal relaxation is significantly faster. This dependence of T 2e on the spinning rate is assigned to slow, overall rotation of the rod-shaped M13 phage about its length axis.

    Both nonspinning and MAS 31P NMR spectra are analyzed in Chapter 2 and 3, respectively, to study possible mobility differences among the phosphodiesters in M13 and TMV. The nonspinning lineshape of 30% TMV is best simulated, if it is assumed that one of the three binding sites is more mobile than the other two. It is shown that this is compatible with the reduced CSA reflected by the major sideband pattern in MAS spectra of TMV as compared to the minor one. A large mobility of one of the three binding sites would agree with structural models based on x-ray diffraction data, in which two of the binding sites are interacting with arginine residues, whereas no arginine is close to the third one. Two-component analysis of the nonspinning 31P NMR data of 30% M13 suggests that the encapsulated DNA molecule perhaps contains 83% immobile and 17% mobile phosphodiesters. This would shed new light on the nonintegral ratio 2.4:1 between the number of nucleotides and protein coat subunits in the phage: if 83% of the viral DNA is less mobile, the binding of the DNA molecule to the protein coat would actually occur at the integral ratio of two nucleotides per protein subunit. However, MAS NMR spectra provide no additional evidence for such a two-component model.

    Finally, in Chapter 5, the slow overall motion of M13 and TMV is investigated using 2D-exchange 31P NMR spectroscopy. 2D-exchange 31P NMR spectra recorded for TMV with mixing times t m ≤1 sec do not show any offdiagonal broadening indicating that the value of 3 Hz for the overall motion of TMV determined in Chapter 3 from nonspinning transversal relaxation, is an overestimation. For 30% M13, a log-Gaussian distribution around 25 Hz of coefficients mainly spread between 1 and 10 3Hz must be introduced to reproduce the 2D-exchange spectra recorded at various mixing times in a consistent way. Motional inhomogeneity in gels of M13 is probably caused by the tendency of the bacteriophages in solutions to form variously sized aggregates. Taking the same coefficient distribution and a minor relaxation contribution caused by fast backbone motion into account, nonspinning transversal relaxation can even be better simulated for inhomogeneous overall motion, than it was done for homogeneous motion in Chapter 3. The shrinking of the σ 22 -discontinuity on the diagonal with respect to the lineshape as a whole for t m ≥0.1 sec, cannot be explained by slow overall motion, but seems to be caused by restricted spindiffusion between 31P nuclei with chemical shifts that differ less than 1 ppm.

    The interaction of M13 coat protein with lipid bilayers : a spectroscopic study
    Sanders, J.C. - \ 1992
    Agricultural University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : Sanders - 102
    bacteriofagen - membranen - celmembranen - spectroscopie - resonantie - eiwitten - bacteriophages - membranes - cell membranes - spectroscopy - resonance - proteins

    In this thesis a small part of the reproductive cycle of the M13 bacteriophage is studied in more detail, namely the interaction of the major coat protein (MW 5240) with lipid bilayers. During the infection process is the major coat protein of M13 bacteriophage stored in the cytoplasm membrane of the E. coli cell, while DNA replication takes place in the cytoplasm. The M13 coat protein in model lipid bilayers is present in two forms, which were previously called the alfa-oligomeric (b-state) and the β-polymeric (c-state) form. The secondary structure of the strongly aggregated M13 coat protein in the β-polymeric form is dominated by a high percentage of β-sheet conformation whereas the non-aggregated M13 coat protein in the α-oligomeric form was previously estimated to consist of 50% α-helix.

    A more precise knowledge of the secondary structure of the M13 coat protein in both forms helps to understand how protein-lipid interactions take place. Therefore, Circular Dichroism, Raman and Fourier Transform Infrared spectroscopy has been applied on lipid M13 coat protein systems reconstitutes. Care was taken to study either the aggregated or non aggregated form of the M13 coat protein. The β-polymeric form of M13 coat protein has a secondary structure of 13% α-helix, 57% β-sheet, 13% turn and 16% remainder and the α-oligomeric M13 coat protein is in a 91% α-helix, 5% β-sheet, 3% turn and 1% remainder conformation. The overall conformation of the M13 coat protein in the α-oligomeric form corresponds well with the conformation of virion bound coat protein.

    In a more theoretical study it was tried to relate the conformation and aggregation state of the M13 coat protein. MD simulations were performed on M13 coat protein in the β-polymeric and the α-oligomeric form. The simulations were started from initial conformations of M13 coat protein as monomers or dimers of alfa-helices or U-shaped β-sheets. The M13 coat protein in the U-shaped βstructure changes from a planar to a twisted form with larger twist for the M13 coat protein as monomer than as a dimer. The M13 coat protein in the beta-sheet conformation β-polymeric is much more flexible than the M13 coat protein in an a-helix conformation (α-oligomeric) as monitored by the rms fluctuations of the Cαatoms. A comparison of the energies after 100 ps MD simulation shows that of the monomers, M13 coat protein in an a-helix has the lowest energy. The energy difference between α- and β-structures decreases from 266 kJ/mol to 148 kJ/mol when going from monomers to dimers. It is expected that this difference will decrease with higher aggregation numbers, suggesting that the M13 coat protein in the β-sheet, conformation must be aggregated, in agreement with observations performed with HPLC.

    Using 2H-NMR and 31P-NMR the interaction of the M13 coat protein in both forms with specific headgroup and chain deuterium labelled phospholipids is studied. It can be concluded from the spectra that the protein in the predominant βsheet conformation causes a fraction of lipids to be trapped. Together with the information obtained from the structure and aggregation state of this form (chapters 2 and 3) it is suggested that the trapped lipids are arranged in a non bilayer structure, probably induced by a misfitting of the hydrophobic core of the protein and the membrane bilayer. The protein in the predominant a-helix conformation perfectly fits in the lipid bilayer and has only minor influences on the surrounding lipid matrix.

    However, because in the inner membrane only α-helical proteins are found, the correspondence with the conformation of the virion bound protein and the fact that the aggregation of the β-polymeric protein is irreversible it is suggested that the α-oligomeric protein is the more likely form of the M13 coat protein to be found in the E. coli. Therefore, a more detailed study was performed on the interaction of this form of the M13 coat protein with lipid bilayers. The 2H-NMR quadrupolar splittings of the αhead-group methylene deuterons of deuterated phosphatidylcholine and phosphalidylethanolamine decrease, whereas the quadrupolar splittings of the βhead-group methylene deuterons of the two lipids increase with increasing protein content. All deuterated segments in the phosphatidylglycerol headgroup show the same relative decrease of the NMR quadrupolar splittings. These results are interpreted in terms of a change in torsion angles of the methylene groups, induced by positive charges, probably lysine residues of the protein at the membrane surface. For all lipid bilayer compositions studied the head-group perturbations are similar. It is concluded that there is no strong specific interaction between one of the lipid types examined and the M13 coat protein.

    In the ESR spectra of spin labels in lipid bilayers no second protein induced component can be observed upon incorporation of M13 coat protein in the α-oligomeric form. It is argued on the bases of ESR and time resolved fluorescence results that M13 coat protein in the α-oligomeric does not immobilize lipids but restricts the order and motion of the lipids, due to its cylindrical form, proportions and non specific interaction with lipids in the bilayer.

    Finally, from this work it can be concluded that the influences of the M13 coat protein in its probably natural form, the α-oligomeric form, on the lipid membranes are small. Positive charges are introduced at the bilayer surface and the hydrophobic area is less fluid in the presence of the M13 coat protein. This let us to understand that higher amounts of cardiolipin are produced when M13 is infecting E. coli. M13 coat protein changes the charge density at the membrane surface which is counteraffected by cardiolipin. In addition the machinery of E. coli is also capable in retaining the fluidity of its membranes simply by changing the lipid tail composition. This let us understand that even high concentrations of M13 coat protein are not lethal for its host, E. coli.

    Virus-membrane interactions : spectroscopic studies
    Datema, K.P. - \ 1987
    Agricultural University. Promotor(en): T.J. Schaafsma; M.A. Hemminga. - S.l. : Datema - 149
    bacteriofagen - biologie - cellen - bestraling - membranen - plantenvirussen - straling - relaties - virussen - bacteriophages - biology - cells - irradiation - membranes - plant viruses - radiation - relationships - viruses

    In this thesis some new aspects of the infection process of nonenveloped viruses are reported. The interaction of a rod-shaped (TMV) and three spherical (CCMV, BMV, SBMV) plant viruses, of the filamentous bacteriophage M13, and of their coat proteins with membranes have been investigated. A comparison is made between the infection mechanisms of these non-enveloped viruses.


    All plant viruses studied interact with membranes. This is demonstrated by turbidity measurements of small unilamellar vesicles with different surface charges (chapter 2). The interaction is either electrostatic or hydrophobic.

    Neutral vesicles always interact with viral capsids by indirect hydrophobic interaction. On the other hand, charged vesicles always interact with opposite charges at the capsids by electrostatic interaction. The location of the coat protein after interaction has been determined to test Durham's model for plant virus infection, in which the coat protein becomes an integral membrane protein, similarly as for M13 infection. The results indicate that as a result of the interactions, multilamellar vesicles are formed, containing all the protein in case of electrostatic interaction. The coat protein is associated at the bilayer surface. However, after hydrophobic interaction no protein is found in the multilamellar vesicles. For the latter type of interaction a mechanism is proposed, in which the coat protein behaves as a catalyst, only enhancing the rate of fusion and multilamellar vesicle formation. Since in either case no lipid-protein complex is formed, that is stabilized by direct hydrophobic lipid-protein interactions, Durham's model for plant virus infection is very likely to be incorrect.

    The recently proposed co-translational disassembly model for plant virus infection, in which the viral particle dissociates during translation of its RNA by the host ribosomes, does not include a specific role for membranes during disassembly. However, before the co-translational disassembly takes place, the particles need to be destabilized. At this moment no specific mechanism for this process has been proposed. Also the fate of the coat protein after particle disassembly remains unspecified in any model, with exception of Durham's model. The observed electrostatic interaction at the bilayer surface (e.g. with the N-terminal arm of the coat proteins, released upon assembly may play a significant role in the infection mechanism.

    From the experiments described in this thesis no results contradictory to co-translational disassembly have been found. Therefore, to our present opinion, the best model for plant virus infection is the co-translational disassembly model. In this view it is assumed implicitely that the virus particles arrive intact in the cytoplasm, for example by passage through the cell wall and the plasma membrane by local, transient wounding.


    M13 coat protein has been incorporated as an intrinsic protein in micelles and model membranes (chapter 3-4). The secondary structure of coat protein in SDS micelles is, predominantly α-helix (60%), while in membranes (DMPC/DMPA 80/20 w/w) the structure is entirely β-structure.

    In micelles at high detergent protein ratio the protein is dimeric with the central core in β-structure. The termini of the coat protein (30 residues or 60%) are in α-helix structure. The dynamics of the micellar system, investigated by time-resolved fluorescence anisotropy measurements, is characterized by rotation of the complex on the nanosecond timescale (10 ns at 20°C) and additional mobility of the Trp-26 sidechain on the subnanosecond timescale (0.5 ns). The complex has a temperature dependent overall rotation, that satisfies the Stokes-Einstein relation for spherical rotation. From this dependence it has been determined that the complex consists of two coat protein molecules and approximately 57 SDS molecules.

    In membranes, regardless of the lipid to protein ratio, the coat protein Is aggregated. This is concluded from three independent measurements. Fluorescence anisotropy decay measurements indicate that the single tryptophan-26 in the hydrophobic core is highly ordered on the nanosecond timescale in liquid-crystalline bilayers, whereas the surrounding lipids are not. 2H NMR measurements indicate that all the exchangeable sites at the backbone are ordered on the microsecond timescale. Both results are consistent with protein aggregation. Finally, the fraction of motionally restricted lipid, determined from spin label ESR is too low for a monomeric state of the coat protein in the membrane, also in agreement with protein aggregation.

    The state of the M13 coat protein in model membranes used in the experiments of this thesis is therefore best described as a β- polymeric state. Within the polymer the orientation of the coat protein is unknown. For comparison, invivo , the coat protein in the E . coli cytoplasmic membrane is known to be oriented. Its secondary structure and state of aggregation, however, are up to now unknown.


    In model membranes also the effect of M13 coat protein incorporation on the lipids has been investigated (chapter 4). Upon introduction of coat protein in the membrane as an intrinsic protein a fraction of the lipid molecules becomes motionally restricted.

    The spin labelled phospholipids show a difference in their selectivity for the coat protein: cardiolipin = phosphatidic acid>>stearic acid phosphatidylserine = phosphatidylglycerol>>phosphatidylcholine phosphatidylethanolamine. The selectivities found are related to the composition of the target E . coli cytoplasmic membrane. Typically, neutral phosphatidylethanolamine accounts for 74% of the lipid in the membrane, constituting the bulk of the lipid, while phosphatidylglycerol is present for 19% and cardiolipin for 3%. The high selectivity of cardiolipin for the coat protein forms direct, biophysical evidence for a previously suggested molecular association of cardiolipin with the coat protein. This was concluded from an increase in the cardiolipin synthesis after infection of E . coli by M13. No increase in phosphatidylglycerol synthesis, the major negatively charged lipid, is observed after infection.

    Using selectively deuterated palmitic acid as probe lipid, spectral broadening has been observed in presence of M13 coat protein. This result, as well as the ESR results, agrees with a two-site exchange model for the probe lipid between sites in the bulk of the membrane and motionally-restricted sites at the protein. The exchange rate is fast on the nanosecond timescale of the ESR technique, but slow on the microsecond timescale of the 2H NMR technique. The exchange rate of 10 +7Hz, deduced from simulation of the spin label ESR spectra, is in excellent agreement with these upper and lower limits.

    Bacteriophagen in zuursels
    Pette, J.W. - \ 1945
    's-Gravenhage : Algemeene Landsdrukkerij (Verslagen van landbouwkundige onderzoekingen no. 51 (8) C)
    bacteriofagen - kaaszuursels - coaguleermiddelen - zuivelindustrie - melkproducten - parasitologie - stremsel - bacteriophages - cheese starters - coagulants - dairy industry - milk products - parasitology - rennet
    Over het wezen van den bacteriophaag
    Söhngen, N.L. - \ 1929
    Wageningen : Veenman - 16
    bacteriofagen - fysiologie - morfologie - bacteriophages - physiology - morphology
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