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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    Community-level interactions between plants and soil biota during range expansion
    Koorem, Kadri ; Snoek, Basten L. ; Bloem, Janneke ; Geisen, Stefan ; Kostenko, Olga ; Manrubia, Marta ; Ramirez, Kelly S. ; Weser, Carolin ; Wilschut, Rutger A. ; Putten, Wim H. van der - \ 2020
    Journal of Ecology 108 (2020)5. - ISSN 0022-0477 - p. 1860 - 1873.
    bacteria - climate change - fungi - nematodes - plant–plant interactions - plant–soil interactions

    Plant species that expand their range in response to current climate change will encounter soil communities that may hinder, allow or even facilitate plant performance. It has been shown repeatedly for plant species originating from other continents that these plants are less hampered by soil communities from the new than from the original range. However, information about the interactions between intra-continental range expanders and soil communities is sparse, especially at community level. Here we used a plant–soil feedback experiment approach to examine if the interactions between range expanders and soil communities change during range expansion. We grew communities of range-expanding and native plant species with soil communities originating from the original and new range of range expanders. In these conditioned soils, we determined the composition of fungi and bacteria by high-throughput amplicon sequencing of the ITS region and the 16S rRNA gene respectively. Nematode community composition was determined by microscopy-based morphological identification. Then we tested how these soil communities influence the growth of subsequent communities of range expanders and natives. We found that after the conditioning phase soil bacterial, fungal and nematode communities differed by origin and by conditioning plant communities. Despite differences in bacterial, fungal and nematode communities between original and new range, soil origin did not influence the biomass production of plant communities. Both native and range expanding plant communities produced most above-ground biomass in soils that were conditioned by plant communities distantly related to them. Synthesis. Communities of range-expanding plant species shape specific soil communities in both original and new range soil. Plant–soil interactions of range expanders in communities can be similar to the ones of their closely related native plant species.

    Review of insect pathogen risks for the black soldier fly (Hermetia illucens) and guidelines for reliable production
    Joosten, Lotte ; Lecocq, Antoine ; Jensen, Annette Bruun ; Haenen, Olga ; Schmitt, Eric ; Eilenberg, Jørgen - \ 2020
    Entomologia Experimentalis et Applicata 168 (2020)6-7. - ISSN 0013-8703 - p. 432 - 447.
    bacteria - biocontrol agents - black soldier fly - Diptera - entomopathogens - epidemiology - feed and food - fungi - guidelines - Hermetia illuscens - immune system - protozoa - Stratiomyidae - viruses

    Black soldier fly [BSF; Hermetia illucens L. (Diptera: Stratiomyidae)] larvae are very effective in transforming low-grade food waste into valuable high-end proteins and fat, in intensive production facilities. The production output of this species is growing quickly, but upscaling brings risks to the health status of the reared insects. Until now, not a single major case of disease outbreak caused by a pathogen in a BSF production unit has been reported. This contrasts with data on other species of mass-produced insects, which have experienced various disease outbreaks, indicating that BSFs are comparatively resistant to insect diseases. Further, there are no records of natural infections caused by entomopathogens in BSF. In this review, the known entomopathogens of Diptera, especially BSF, and their potential risks for causing disease in these insects are summarized.

    An Assessment of Climate Induced Increase in Soil Water Availability for Soil Bacterial Communities Exposed to Long-Term Differential Phosphorus Fertilization
    Randall, Kate C. ; Brennan, Fiona ; Clipson, Nicholas ; Creamer, Rachel E. ; Griffiths, Bryan S. ; Storey, Sean ; Doyle, Evelyn - \ 2020
    Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
    bacteria - climate change - phosphorus - rhizosphere - soil moisture

    The fate of future food productivity depends primarily upon the health of soil used for cultivation. For Atlantic Europe, increased precipitation is predicted during both winter and summer months. Interactions between climate change and the fertilization of land used for agriculture are therefore vital to understand. This is particularly relevant for inorganic phosphorus (P) fertilization, which already suffers from resource and sustainability issues. The soil microbiota are a key indicator of soil health and their functioning is critical to plant productivity, playing an important role in nutrient acquisition, particularly when plant available nutrients are limited. A multifactorial, mesocosm study was established to assess the effects of increased soil water availability and inorganic P fertilization, on spring wheat biomass, soil enzymatic activity (dehydrogenase and acid phosphomonoesterase) and soil bacterial community assemblages. Our results highlight the significance of the spring wheat rhizosphere in shaping soil bacterial community assemblages and specific taxa under a moderate soil water content (60%), which was diminished under a higher level of soil water availability (80%). In addition, an interaction between soil water availability and plant presence overrode a long-term bacterial sensitivity to inorganic P fertilization. Together this may have implications for developing sustainable P mobilization through the use of the soil microbiota in future. Spring wheat biomass grown under the higher soil water regime (80%) was reduced compared to the constant water regime (60%) and a reduction in yield could be exacerbated in the future when grown in cultivated soil that have been fertilized with inorganic P. The potential feedback mechanisms for this need now need exploration to understand how future management of crop productivity may be impacted.

    The ecological role of bacterial seed endophytes associated with wild cabbage in the United Kingdom
    Tyc, Olaf ; Putra, Rocky ; Gols, Rieta ; Harvey, Jeffrey A. ; Garbeva, Paolina - \ 2020
    MicrobiologyOpen 9 (2020)1. - ISSN 2045-8827
    bacteria - endophytes - fungal pathogens - insect herbivory - plant growth - plant resistance - plant-insect interactions - seed germination

    Endophytic bacteria are known for their ability in promoting plant growth and defense against biotic and abiotic stress. However, very little is known about the microbial endophytes living in the spermosphere. Here, we isolated bacteria from the seeds of five different populations of wild cabbage (Brassica oleracea L) that grow within 15 km of each other along the Dorset coast in the UK. The seeds of each plant population contained a unique microbiome. Sequencing of the 16S rRNA genes revealed that these bacteria belong to three different phyla (Actinobacteria, Firmicutes, and Proteobacteria). Isolated endophytic bacteria were grown in monocultures or mixtures and the effects of bacterial volatile organic compounds (VOCs) on the growth and development on B. oleracea and on resistance against a insect herbivore was evaluated. Our results reveal that the VOCs emitted by the endophytic bacteria had a profound effect on plant development but only a minor effect on resistance against an herbivore of B. oleracea. Plants exposed to bacterial VOCs showed faster seed germination and seedling development. Furthermore, seed endophytic bacteria exhibited activity via volatiles against the plant pathogen F. culmorum. Hence, our results illustrate the ecological importance of the bacterial seed microbiome for host plant health and development.

    Trophic Regulations of the Soil Microbiome
    Thakur, Madhav P. ; Geisen, Stefan - \ 2019
    Trends in Microbiology 27 (2019)9. - ISSN 0966-842X - p. 771 - 780.
    bacteria - climate change - food webs - fungi - predators - top-down control

    The soil microbiome regulates vital ecosystem functions ranging from primary production to soil carbon sequestration. Yet, we have only begun to understand the factors regulating the soil microbiome. While the importance of abiotic factors is increasingly recognized, the roles of trophic regulations in driving the structure and function of the soil microbiome remain less explored. Here, we review the current understanding of how and when microbial and top predators of the soil shape the community structure and function of the soil microbiome via both direct and indirect effects. We finally highlight that the structure and function of the soil microbiome depend on the interactive effects among predation, plant inputs, and abiotic variables present in the soil.

    Differently Pre-treated Alfalfa Silages Affect the in vitro Ruminal Microbiota Composition
    Hartinger, Thomas ; Edwards, Joan E. ; Gómez Expósito, Ruth ; Smidt, Hauke ; Braak, Cajo J.F. ter; Gresner, Nina ; Südekum, Karl Heinz - \ 2019
    Frontiers in Microbiology 10 (2019). - ISSN 1664-302X
    16S rRNA gene sequencing - anaerobic fungi - archaea - bacteria - lucerne silage - qPCR - rumen microbiota - Rusitec

    Alfalfa (Medicago sativa L.) silage (AS) is an important feedstuff in ruminant nutrition. However, its high non-protein nitrogen content often leads to poor ruminal nitrogen retention. Various pre-ensiling treatments differing with respect to dry matter concentrations, wilting intensities and sucrose addition have been previously shown to improve the quality and true protein preservation of AS, and have substantial effects on in vitro ruminal fermentation of the resulting silages. However, it is unknown how these pre-ensiling treatments affect the ruminal microbiota composition, and whether alterations in the microbiota explain previously observed differences in ruminal fermentation. Therefore, during AS incubation in a rumen simulation system, liquid and solid phases were sampled 2 and 7 days after first incubating AS, representing an early (ET) and late (LT) time point, respectively. Subsequently, DNA was extracted and qPCR (bacteria, archaea, and anaerobic fungi) and prokaryotic 16S rRNA gene amplicon sequence analyses were performed. At the ET, high dry matter concentration and sucrose addition increased concentrations of archaea in the liquid phase (P = 0.001) and anaerobic fungi in the solid phase (P < 0.001). At the LT, only sucrose addition increased archaeal concentration in the liquid phase (P = 0.014) and anaerobic fungal concentration in the solid phase (P < 0.001). Bacterial concentrations were not affected by pre-ensiling treatments. The prokaryotic phylogenetic diversity index decreased in the liquid phase from ET to LT (P = 0.034), whereas the solid phase was not affected (P = 0.060). This is suggestive of a general adaption of the microbiota to the soluble metabolites released from the incubated AS, particularly regarding the sucrose-treated AS. Redundancy analysis of the sequence data at the genus level indicated that sucrose addition (P = 0.001), time point (P = 0.001), and their interaction (P = 0.001) affected microbial community composition in both phases. In summary, of the pre-ensiling treatments tested sucrose addition had the largest effect on the microbiota, and together with sampling time point affected microbiota composition in both phases of the rumen simulation system. Thus, microbiota composition analysis helped to understand the ruminal fermentation patterns, but could not fully explain them.

    Successional Dynamics in the Gut Microbiome Determine the Success of Clostridium difficile Infection in Adult Pig Models
    Jurburg, Stephanie D. ; Cornelissen, Jan J.B.W.J. ; Boer, Paulo de; Smits, Mari A. ; Rebel, Johanna M.J. - \ 2019
    Frontiers in Cellular and Infection Microbiology 9 (2019). - ISSN 2235-2988 - 11 p.
    animal models - bacteria - Clostridium difficile - microbiome - pig

    Clostridium difficile infections (CDI) are a major cause of antibiotic-associated diarrhea. It is hypothesized that CDI develops due to the antibiotic-induced disruption of the intestinal microbial community structure, which allows C. difficile to flourish. Here, we pre-treated weaned pigs with the antibiotics Clindamycin or Ciprofloxacin for 1 day, and subsequently inoculated them with a human and pig enteropathogenic C. difficile strain 078 spores. Body temperature, clinical signs of disease, and the fecal microbiome were monitored daily for 15 days. Clindamycin had a stronger effect on the pigs than Ciprofloxacin, resulting in drastic shifts in the fecal microbiome, decreases in microbial diversity and significant increases in body temperature, even in the absence of C. difficile. Fecal shedding of C. difficile was detectable for 3 and 9 days in Ciprofloxacin and Clindamycin treated pigs inoculated with C. difficile, respectively, and in both cases decreased cell proliferation rates were detected in colon tissue. The timing of C. difficile shedding coincided with the decrease in a large cluster of Firmicutes following Clindamycin treatment, a pattern which was also independent of C. difficile inoculation. The observed community patterns suggest that successional dynamics following antibiotic treatment facilitate C. difficile establishment. The similarities between the microbiome responses observed in our study and those previously reported in CDI-infected humans further support the utility of adult pigs as models for the study of CDI.

    Fermentations great promise
    Smid, E.J. - \ 2019
    biobased economy - microorganisms - bacteria - chemicals - food - fatty acids - kerosene - fermentation
    Addiction systems antagonize bacterial adaptive immunity
    Sluijs, Lisa van; Houte, Stineke van; Oost, John van der; Brouns, Stan J.J. ; Buckling, Angus ; Westra, Edze R. - \ 2019
    FEMS Microbiology Letters 366 (2019)5. - ISSN 0378-1097
    adaptive immunity - bacteria - CRISPR - plasmid - TA - toxin

    CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-anti-toxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.

    The Genome of the Fungal Pathogen Verticillium dahliae Reveals Extensive Bacterial to Fungal Gene Transfer
    Shi-Kunne, Xiaoqian ; Kooten, Mathijs van; Depotter, Jasper R.L. ; Thomma, Bart P.H.J. ; Seidl, Michael F. - \ 2019
    Genome Biology and Evolution 11 (2019)3. - ISSN 1759-6653 - p. 855 - 868.
    Verticillium - ascomycete - bacteria - fungus - horizontal gene transfer

    Horizontal gene transfer (HGT) involves the transmission of genetic material between distinct evolutionary lineages and can be an important source of biological innovation. Reports of interkingdom HGT to eukaryotic microbial pathogens have accumulated over recent years. Verticillium dahliae is a notorious plant pathogen that causes vascular wilt disease on hundreds of plant species, resulting in high economic losses every year. Previously, the effector gene Ave1 and a glucosyltransferase-encoding gene were identified as virulence factor-encoding genes that were proposed to be horizontally acquired from a plant and a bacterial donor, respectively. However, to what extent HGT contributed to the overall genome composition of V. dahliae remained elusive. Here, we systematically searched for evidence of interkingdom HGT events in the genome of V. dahliae and provide evidence for extensive horizontal gene acquisition from bacterial origin.

    Fecal Bacterial Communities in Insectivorous Bats from the Netherlands and Their Role as a Possible Vector for Foodborne Diseases
    Wolkers-Rooijackers, Judith C.M. ; Rebmann, Katharina ; Bosch, Thijs ; Hazeleger, Wilma C. - \ 2018
    Acta Chiropterologica 20 (2018)2. - ISSN 1508-1109 - p. 475 - 483.
    bacteria - bats - DGGE - microbial diversity

    Bats are commonly regarded as vectors for viruses, but little is known about bacterial communities in bats and the possible role of bats in the transmission cycle of foodborne diseases. To gain more insight, microbial communities in fecal samples from 37 insectivorous bats of different species from the Netherlands were investigated by polymerase chain reaction and denaturant gradient gel electrophoresis (PCR-DGGE). Subsequently, 10 samples from the following bat species: common pipistrelle (Pipistrellus pipistrellus; n = 3), Daubenton's bat (Myotis daubentonii; n = 3), serotine bat (Eptesicus serotinus; n = 1), whiskered bat (Myotis mystacinus; n = 1), Geoffroy's bat (Myotis emarginatus; n = 1) and Natterer's bat (Myotis nattereri; n = 1) were selected and used in bacterial 16S rDNA cloning and sequencing. The fecal microbiota in bats was found to be diverse with predominant bacterial genera Carnobacterium, Serratia, Pseudomonas, Enterococcus and Yersinia. The presence of opportunistic pathogens Citrobacter freundii, Escherichia coli, Enterococcus faecalis, Serratia fonticola and Rahnella aquatilis was also recorded. Based on cloning results, we found no proof that bats in the Netherlands are a major vector for the transmission of bacterial zoonotic diseases, although previous findings in literature reported isolation of foodborne pathogens from bats.

    Nieuwe aanpak medicijnresten in afvalwater
    Langenhoff, A.A.M. - \ 2018
    water - waste water treatment - sludges - water treatment - bacteria
    Kan onze wereld bestaan zonder bacteriën? | WURcast
    Schraa, G. - \ 2017
    Detmer Sipkema - Are sponges full of bacteria?
    Sipkema, D. - \ 2017
    luffa acutangula - bacteria - oceans
    Fermentatie is hot: nieuwe toepassingen van een oeroude techniek
    Smid, E.J. ; Hugenholtz, J. - \ 2017
    biofuels - biobased economy - bioenergy - chemical industry - nutrition - fermentation - cellulose - bacteria - biomass
    Botryococcus braunii for the production of hydrocarbons and exopolysaccharides and the role of associated bacteria
    Gouveai, João Diogo Guimarães - \ 2017
    Wageningen University. Promotor(en): R.H. Wijffels, co-promotor(en): M.J. Barbosa; D. Sipkema. - Wageningen : Wageningen University - ISBN 9789463436960 - 157
    biomass production - algae - algae culture - hydrocarbons - bacteria - biomassa productie - algen - algenteelt - koolwaterstoffen - bacteriën

    Microalgae are photosynthetic organisms that are found worldwide in many different aquatic environments and therefore display an immense biological diversity. They are a promising source of many useful polymers that have industrial applications such as food, fuel, material and pharmaceutical. One microalga that has gathered quite a research community is Botryococcus braunii. The reason for its scientific club is the fact it can synthetize long chain hydrocarbons molecules from C20 to C40. These hydrocarbons have been found in oil-shales and tests show that it can be used as aviation fuel. Besides producing hydrocarbons, some strains of B. braunii can produce exopolysaccharides (EPS) composed mainly of galactose and a small fraction of fucose. The EPS has interesting rheological properties for the food industry and potential active compounds that could be used in the pharmaceutical industry .

    Like many other microorganisms, microalgae in the natural environment are usually in the presence of bacteria. The presence of bacteria with microalgae can either have a beneficial or an antagonistic effect. For B. braunii little is known about the bacteria community present especially for the EPS producing strain. For that reason, the aim of this thesis was to investigate B. braunii’s associated bacteria with the aim of improving B. braunii’s biomass growth and hydrocarbon and EPS content. In chapter one, we introduced the topic of microalgae as a potential source of sustainable polymers and we introduced the species B. braunii, describing its characteristics and scientific interest. It is also introduced the topic of microalgae and bacteria associations by looking at other studies from literature.

    In chapter two, 16 publically available strains of B. braunii were ordered in culture banks and screened for biomass productivity, hydrocarbon and total carbohydrate content. The aim of the study was to identify one or more good strains that displayed high biomass productivity as well as hydrocarbon or total carbohydrate content. In seven strains out of 16 cultivated in 250 mL volume Erlenmeyer flaks, we detected 5 to 42 % content of hydrocarbons of the dry biomass with four strains producing botryococcenes (C30-C34) and three strains producing alkanes (C20-C25). Two strains showed high amounts of EPS content above 50 % per dry biomass. Seven strains comprising of the strains with higher biomass productivity plus the highest hydrocarbons and EPS content, were tested for scalability using bench scale 800 mL volume bubble column reactors. Two strains, AC761 which produces botryococcenes and CCALA778 which produces EPS, were selected as the most promising B. braunii strains for industrial production of hydrocarbon and EPS.

    In chapter three, we studied the bacterial community associated with B. braunii. We cultured 12 strains from the initial 16 from chapter 2 and extracted the DNA from samples taken over a time period of 12 days. It was clear from this study that B. braunii hosts a variety of bacterial species and still maintain its growth. The bacteria families Rhizobiaceae, Bradyrhizobiaceae and Comamonadaceae were found in all 12 strains. These families which belong to the phylum Proteobacteria could have an important role regarding B. braunii growth. Each strain displayed a different bacterial community composition but all the strains from the CAEN culture collection clustered near each other suggesting that the algae culture collection could have an influence on the bacterial community composition. Bacteria genus identification based on 16S rRNA gene amplicon similarity showed several genera present including Rhizobium spp. and Variovorax spp.. Two genera were found that are possibly linked to hydrocarbon degradation: Sphingomonas spp. and Rhodobacter spp..

    In chapter four, we investigated further B. braunii CCALA778 which was shown in chapter 2 to accumulate high amounts of EPS. We investigated the effects of antibiotics on algal growth, EPS accumulation and bacterial community composition of CCALA778. Taxonomical identification by 16S rRNA gene analysis indicated that most of the bacteria present with CCALA778 were Gram-negative. Of all antibiotics and antibiotic mixes, only the treatment with Penicillin did not affect the growth of B. braunii. The remaining antibiotics halted the growth of CCALA778 while they were active. The exceptions were with the antibiotics Chloramphenicol, Gentamycin and Linezolid which permanently ceased the growth of CCALA778. The accumulation of EPS seemed to be related to biomass growth, but we did also observe a reduction of EPS with the cultures treated with Penicillin suggesting that bacteria could have an effect on the EPS content. Antibiotics had specific effects on the bacterial community with all treatments showing significant changes over time. The most efficient treatment in removing bacteria were the mixes Metronidazole-Rifampicin-Penicillin and Penicillin-Rifampicin which were the only treatments to show significant changes in the bacterial community when compared to the untreated cultures after 10 days of cultivation. Antibiotics and antibiotic mixes can create changes in the bacterial community but it is unlikely that they alone can lead to axenic B. braunii cultures.

    In chapter five, we used Ultra Violet-C light (UVC) to reduce bacteria diversity and abundance present in B. braunii CCALA778. UVC is highly effective in inactivating bacteria and for that reason is being investigated further in medicinal applications. After applying the UVC to B. braunii CCALA778, we were able to reduce the relative abundance of 16S rRNA genes assigned to bacteria to less than 1 % compared to the 70% in the non-treated cultures. With the UVC treated CCALA778 we observed several physiological changes. The UV treated cultures with reduced bacterial load showed nearly double the EPS accumulation when compared to the untreated. To confirm that we did not see an artefact in our results due to the UVC treatment, UVC treated cultures were also inoculated with bacteria from the untreated and we observed a reduction of EPS similar to what we saw with the untreated cultures. There were no changes to the EPS composition after the removal of the bacteria. Other physiological changes were observed, namely that colony size of B. braunii CCALA778 significantly increased when compared to the untreated culture and the UV treated with bacteria. We hypothesise that the increase in colony size was probably due to the fact there was more EPS accumulated which helped with cell aggregation. We also observed an increase on the biomass growth in the UV-treated CCALA778 which we hypothesized being related to the fact that there was none or hardly any competition for essential micronutrients such as phosphate. From this study we concluded that the associated bacteria present with B. braunii CCALA778 were antagonistic. We believe the reason why the bacteria were antagonistic is because of the readily available EPS which is a rich source of organic compounds that bacteria could use for their own proliferation allowing them to compete with B. braunii for essential nutrients.

    In chapter 6, we discuss the implications from our previous 4 experimental chapters. The aim of the study was to improve the biomass productivity and hydrocarbon and EPS content of the microalgae B. braunii. In brief, B. braunii displayed a wide range of physiological traits regarding biomass productivity and hydrocarbon and total carbohydrate content. We showed that B. braunii can co-habit with a wide range of bacteria diversity and abundance and that the associated bacteria were antagonistic to CCALA778 by affecting its biomass growth. We also showed that by removing the associated bacteria we can increase the EPS accumulation. Currently most of the research on microalgae and bacteria interactions, focus on the positive side, but we must understand also how bacteria can be antagonistic to microalga growth. Bacteria can be antagonistic to microalgae by competing for nutrients and also being detrimental to industrial process by degrading the product of interest in the case of organic carbons such as EPS. Therefore it is unlikely we can use the benefits that bacteria can provide such as enhancing growth to improve the cultivation of B. braunii and other similar microalgae species that secrete EPS. Since bacteria can be antagonistic to microalgae that secrete large amounts of organic compounds such as EPS, it is imperative to minimize contamination in large scale photobioreactors (PBR). It is important because in large scale PBR, contamination can occur leading to downtime of the reactors. If microalgae industry is to advance, it must develop PBR units that prevent contamination of bacteria from the surrounding environment.

    Adapting to change : on the mechanism of type I-E CRISPR-Cas defence
    Künne, Tim A. - \ 2017
    Wageningen University. Promotor(en): J. van der Oost, co-promotor(en): S.J.J. Brouns. - Wageningen : Wageningen University - ISBN 9789463436649 - 239
    immunity - defence mechanisms - rna - bacteria - escherichia coli - analytical methods - priming - immuniteit - verdedigingsmechanismen - rna - bacteriën - escherichia coli - analytische methoden - zaadbevochtiging

    Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery.

    Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times.

    Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis.

    Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders.

    In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

    In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming.

    Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.

    Mutation supply and the repeatability of selection for antibiotic resistance
    Dijk, Thijs van; Hwang, Sungmin ; Krug, Joachim ; Visser, Arjan de; Zwart, Mark P. - \ 2017
    Physical Biology 14 (2017)5. - ISSN 1478-3967
    antibiotic resistance - bacteria - Evolution - predictability and repeatability of evolution - TEM-1 Beta-lactamase

    Whether evolution can be predicted is a key question in evolutionary biology. Here we set out to better understand the repeatability of evolution, which is a necessary condition for predictability. We explored experimentally the effect of mutation supply and the strength of selective pressure on the repeatability of selection from standing genetic variation. Different sizes of mutant libraries of antibiotic resistance gene TEM-1 β-lactamase in Escherichia coli, generated by error-prone PCR, were subjected to different antibiotic concentrations. We determined whether populations went extinct or survived, and sequenced the TEM gene of the surviving populations. The distribution of mutations per allele in our mutant libraries followed a Poisson distribution. Extinction patterns could be explained by a simple stochastic model that assumed the sampling of beneficial mutations was key for survival. In most surviving populations, alleles containing at least one known large-effect beneficial mutation were present. These genotype data also support a model which only invokes sampling effects to describe the occurrence of alleles containing large-effect driver mutations. Hence, evolution is largely predictable given cursory knowledge of mutational fitness effects, the mutation rate and population size. There were no clear trends in the repeatability of selected mutants when we considered all mutations present. However, when only known large-effect mutations were considered, the outcome of selection is less repeatable for large libraries, in contrast to expectations. We show experimentally that alleles carrying multiple mutations selected from large libraries confer higher resistance levels relative to alleles with only a known large-effect mutation, suggesting that the scarcity of high-resistance alleles carrying multiple mutations may contribute to the decrease in repeatability at large library sizes.

    Assessing the impact of socio-economic development and climate change on faecal indicator bacteria in the Betna River, Bangladesh
    Islam, Majedul - \ 2017
    Wageningen University. Promotor(en): R. Leemans, co-promotor(en): N. Hofstra. - Wageningen : Wageningen University - ISBN 9789463436304 - 137
    climatic change - environmental impact - water quality - rivers - contamination - bacteria - coliform bacteria - faecal coliforms - bangladesh - south asia - klimaatverandering - milieueffect - waterkwaliteit - rivieren - besmetting - bacteriën - coliformbacteriën - fecale coliformen - bangladesh - zuid-azië

    Consumption of water that is contaminated with pathogens still causes high numbers of death and disease. Understanding the factors that influence the dynamic distribution of waterborne pathogens is important, as this will help understanding improvements and possible solutions. Such understanding is particularly important in a developing country like Bangladesh, where large proportions of the population often have little or no access to clean water. Despite the high relevance for public health, few studies currently exists on the fate and transport of pathogens and the so-called Faecal Indicator Bacteria (FIB, e.g. E. coli, enterococci) in (sub)tropical systems. FIB are susceptible to shifts in water flow and quality. The predicted increases in rainfall and floods due to climate change will exacerbate the faecal contamination scenarios. This could be further compounded by the rapid change in socio-economic conditions (population growth, urbanization, sanitation and agricultural management) in the developing countries. Therefore, to reduce future health risks, understanding the influence of changes in socio-economic conditions and climate on microbial dynamics is important.

    Very few studies have quantified the relationship between the waterborne pathogens/FIB concentrations and climate and socio-economic changes. In this study a process-based model was developed and a scenario analysis was performed based on the new combined climate and socio-economic changes scenarios, to assess the present and future river hydrodynamics, FIB sources, die-off processes and concentrations. We used FIB, because measuring FIB are cheaper than pathogens. FIB are usually not pathogenic but their presence indicates the likely presence of waterborne pathogens. These pathogens are expected to respond to climate change in a comparable way to FIB. The present study is based on the Betna River basin in southwestern Bangladesh, where faecal contamination is not monitored and very little knowledge exists on the distribution of contaminants.

    First of all, FIB concentrations of the river water were measured to identify the river’s faecal contamination levels that can be used to validate the water-quality model. In the study area, wastewater is not treated and this untreated wastewater is discharged directly into the river. This is evident from the measured FIB data. In 88% of the E. coli and all enterococci samples, the USEPA bathing water quality standards were violated (Chapter 2). Such violation indicates potential health risks associated with the use of the river water for domestic, bathing and irrigation purposes. The correlation between environmental variables (water temperature, precipitation and salinity) and FIB concentrations was also determined. A positive correlation was found with water temperature and precipitation, and a negative correlation with salinity. The positive correlation with temperature is due to the co-occurrence of high summer temperature with abundant monsoon rainfall. The positive correlation with precipitation can be explained by the increased runoff from agricultural lands and urban areas. This runoff contains many bacteria. In the study area, during the rainy season (July to September) precipitation increases and as a result water salinity decreases. The observed negative correlation with salinity is more likely due to the typical weather patterns during the rainy season when low salinity coincides with increased precipitation and high temperature, than to salinity dependent die-off of bacteria. A regression model was applied that explained almost half of E. coli and enterococci variability in river water. This, however, only considers water temperature and precipitation (Chapter 2).

    Then, the present and future hydrodynamics of the river were simulated using a two dimensional hydrodynamic model (MIKE 21 FM). Although the main goal of this thesis is to assess the river’s present and future FIB concentrations, the reasons for this hydrodynamic modelling are twofold. Firstly, outputs of the hydrodynamic model are used as input into the water-quality model (Chapter 4). Secondly, hydrodynamics (i.e. water level and discharge) are simulated because increased water level and discharge together with sea level rise stimulate floods in the river basin. These floods are related to outbreaks of waterborne diseases. The modelled results corresponded very well with the measured water levels and discharges. The model was applied to simulate baseline and future water levels and discharge for Representative Concentration Pathway RCP4.5 and RCP8.5 scenarios using bias-corrected downscaled data from two climate models (IPSL-CM5A and MPI-ESM). The model results showed an expected increase in water level up to 16% by the 2040s and 23% by the 2090s (Chapter 3). The monsoon daily maximum discharge was expected to increase up to 13% by the 2040s and 21% by the 2090s. These model results also showed that the duration of the water level above the danger level and extreme discharge periods can increase by half a month by the 2040s and over a month by the 2090s. The coincidence of the water danger level with extreme discharge may cause disastrous floods in the study area.

    Next, the hydrodynamic model was coupled with a water-quality module (ECOLab). The fate and transport of FIB was simulated, the influence of different processes tested and the contribution from different sources to the total contamination quantified (Chapter 4). The model outputs corresponded very well with the measured FIB data. The present river microbial water quality based on measured and simulated results indicated, once again, noncompliance with bathing water standards. Primary and secondary levels of wastewater treatment were not sufficient to reach the standards most of the time, and discharges from sewer drains and incoming concentrations from the upstream boundary were found to be a major cause of water contamination. Tide, wind and diffuse sources (urban and agricultural runoff) contributed little. The high FIB inputs from the upstream open boundary come from untreated point source discharges from upstream urban areas and accumulation of diffuse contaminants from the large upstream areas. Therefore, this study underlines the need for establishment of wastewater treatment plants both in the studied basin and upstream urban areas. This study provides insight into bacterial fate and transport mechanisms, contribution of different sources to the faecal contamination and applicability of wastewater treatment in a river of a subtropical developing country where this type of study is lacking. Uncertainties are related to the lack of high temporal resolution measured FIB data and the lack of available data for contaminant loads from septic tank leakages, open defecation and sediment resuspension. However, the model well captured the measured FIB variability, suggesting that it can be applied for microbial water quality assessments in other watersheds of the world with similar characteristics.

    The developed model could be an ideal tool to forecast future impacts of climate and socioeconomic changes on FIB fate, transport and dynamics. Finally, future FIB concentrations were simulated using the coupled hydrodynamic and microbial model (MIKE 21 FM-ECOLab) and scenario analysis (Chapter 5). Scenarios have been developed building on the most recent Shared Socio-economic Pathways (SSPs) and Representative Concentration Pathways (RCPs) scenarios from the Intergovernmental Panel on Climate Change (IPCC). We developed a baseline scenario (October 2014–September 2015) reflecting the current conditions and two future scenarios, S1 (sustainability scenario) and S2 (uncontrolled scenario) mimicking different future developments of socio-economic (population, urbanization, sanitation, wastewater treatment development, land use) and climate-change factors (temperature, precipitation and sea-level rise). In S1 RCP4.5 was combined with socio-economic scenarios SSP1, and for S2 RCP8.5 was combined with SSP3 (S2). Assumptions on sanitation, waste water treatment and agricultural management in line with the storylines were made to quantify future changes in FIB concentrations and consequent health risk. Different future scenarios were found to have substantial impact on FIB concentrations in the river. By the 2090s, FIB concentrations are expected to decrease by 98% or increase by 75% for the sustainability scenario and uncontrolled scenario respectively. An uncontrolled future resulted in a deterioration of microbial water quality due to socio-economic developments, such as higher population growth, land-use change and increased sewage discharges and changes in rainfall patterns. Microbial water quality strongly improved under a sustainable climate and improved sewage treatment. FIB concentrations were much more sensitive to changes in socio-economic factors than to changes in climatic factors. This underlines the importance of socio-economic factors in assessing and improving microbial water quality.

    The results show the importance of improvements in sanitation and wastewater treatment in the Bangladeshi Betna River basin to ensure that future FIB concentrations in the river comply with the US-EPA bathing water quality standards. Major investments to construct wastewater treatment plants are necessary to compensate for the population growth and increased the volume of wastewater treatment. Although the current level of contamination is already too high, without wastewater treatment the water quality will further deteriorate.

    The thesis assesses the present and future FIB dynamics in the Betna River through sampling, statistical and process-based modelling, and scenario analysis. The results contribute to increase the knowledge base on the dynamic distributions of the FIB in surface water in a developing country and in a subtropical system, where this type of study is lacking. It also reduces the knowledge gaps regarding future flooding scenarios at the local scale. While some earlier studies focused on only assessing climate-change impacts on microbial water quality, this study for the first time assessed the influence of combined climate and socio-economic scenarios (using scenarios based on the new SSP-RCP scenario matrix) on river FIB concentrations. This combined modelling and scenario approach enables the assessment of faecal contamination sources and dynamics at present and in the future. The developed model and scenario analysis approach provides a basis for the water managers to reduce the widespread faecal contamination and the risks of waterborne disease outbreaks, which are still a leading cause of deaths in developing countries.

    Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9
    Mougiakos, Ioannis ; Bosma, Elleke F. ; Weenink, Koen ; Vossen, Eric ; Goijvaerts, Kirsten ; Oost, John van der; Kranenburg, Richard van - \ 2017
    ACS synthetic biology 6 (2017)5. - ISSN 2161-5063 - p. 849 - 861.
    Bacillus smithii - bacteria - CRISPR/Cas9 - genome editing - homologous recombination - thermophiles

    Well-developed genetic tools for thermophilic microorganisms are scarce, despite their industrial and scientific relevance. Whereas highly efficient CRISPR/Cas9-based genome editing is on the rise in prokaryotes, it has never been employed in a thermophile. Here, we apply Streptococcus pyogenes Cas9 (spCas9)-based genome editing to a moderate thermophile, i.e., Bacillus smithii, including a gene deletion, gene knockout via insertion of premature stop codons, and gene insertion. We show that spCas9 is inactive in vivo above 42 °C, and we employ the wide temperature growth range of B. smithii as an induction system for spCas9 expression. Homologous recombination with plasmid-borne editing templates is performed at 45-55 °C, when spCas9 is inactive. Subsequent transfer to 37 °C allows for counterselection through production of active spCas9, which introduces lethal double-stranded DNA breaks to the nonedited cells. The developed method takes 4 days with 90, 100, and 20% efficiencies for gene deletion, knockout, and insertion, respectively. The major advantage of our system is the limited requirement for genetic parts: only one plasmid, one selectable marker, and a promoter are needed, and the promoter does not need to be inducible or well-characterized. Hence, it can be easily applied for genome editing purposes in both mesophilic and thermophilic nonmodel organisms with a limited genetic toolbox and ability to grow at, or tolerate, temperatures of 37 and at or above 42 °C.

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