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Microbial communities in a dynamic in vitro model for the human ileum resemble the human ileal microbiota
Stolaki, Maria ; Minekus, Mans ; Venema, Koen ; Lahti, Leo ; Smid, Eddy J. ; Kleerebezem, Michiel ; Zoetendal, Erwin G. - \ 2019
FEMS microbiology ecology 95 (2019)8. - ISSN 0168-6496
in vitro model - gut health - ileum - microbial diversity - microbiota - short chain fatty acids
The important role for the human small intestinal microbiota in health and disease has been widely acknowledged. However, the difficulties encountered in accessing the small intestine in a non-invasive way in healthy subjects have limited the possibilities to study its microbiota. In this study, a dynamic in vitro model that simulates the human ileum was developed, including its microbiota. Ileostomy effluent and fecal inocula were employed to cultivate microbial communities within the in vitro model. Microbial stability was repetitively achieved after 10 days of model operation with bacterial concentrations reaching on average 107 to 108 16S rRNA copy numbers/ml. High diversities similar to those observed in in vivo ileum samples were achieved at steady state using both fecal and ileostomy effluent inocula. Functional stability based on Short Chain Fatty Acid concentrations was reached after 10 days of operation using fecal inocula, but was not reached with ileostomy effluent as inoculum. Principal Components and cluster analysis of the phylogenetic profiles revealed that in vitro samples at steady state clustered closest to two samples obtained from the terminal ileum of healthy individuals, independent of the inoculum used, demonstrating that the in vitro microbiota at steady state resembles that of the human ileum.
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.
Impact of microbial variability on food safety and quality
Aryani, D.C. - \ 2016
Wageningen University. Promotor(en): Marcel Zwietering, co-promotor(en): Heidy den Besten. - Wageningen : Wageningen University - ISBN 9789462577381 - 190
listeria monocytogenes - lactobacillus plantarum - growth analysis - kinetics - growth models - inactivation - heat stress - strain differences - food safety - milk - ham - microbial diversity - food quality - listeria monocytogenes - lactobacillus plantarum - groeianalyse - kinetica - groeimodellen - inactivatie - warmtestress - stamverschillen - voedselveiligheid - melk - ham - microbiële diversiteit - voedselkwaliteit
The fish egg microbiome : diversity and activity against the oomycete pathogen Saprolegnia
Liu, Y. - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): Irene de Bruijn. - Wageningen : Wageningen University - ISBN 9789462577671 - 169
salmon - fish eggs - marine microorganisms - microbial diversity - bioinformatics - genomics - saprolegnia - oomycota - fish diseases - suppression - fungal antagonists - zalm - visseneieren - mariene micro-organismen - microbiële diversiteit - bio-informatica - genomica - saprolegnia - oömycota - visziekten - onderdrukking - schimmelantagonisten
Prof. dr. F. Govers (promotor); Prof. dr. J.M. Raaijmakers (promotor); Dr. I. de Bruijn (co-promotor); Wageningen University, 13 June 2016, 170 pp.
The fish egg microbiome: diversity and activity against the oomycete pathogen Saprolegnia
Emerging oomycete pathogens increasingly threaten biodiversity and food security. This thesis describes the study of the microbiome of Atlantic salmon (Salmo salar L.) eggs and analyses of the effects of infections by the oomycete pathogen Saprolegnia on the microbial architecture. A low incidence of Saprolegniosis was correlated with a relatively high abundance and richness of specific commensal Actinobacteria. Among the bacterial community, the isolates Frondihabitans sp. 762G35 (Microbacteriaceae) and Pseudomonas sp. H6 significantly inhibited hyphal attachment of Saprolegnia diclina to live salmon eggs. Chemical profiling showed that these two isolates produce furancarboxylic acid-derived metabolites and a lipopeptide viscosin-like biosurfactant, respectively, which inhibited hyphal growth of S. diclina in vitro. Among the fungal community, the fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride. Both a quantitative and qualitative difference in the Trichoderma population between Saprolegnia-infected and healthy salmon eggs was observed, which suggested that mycoparasitic Trichoderma species could play a role in Saprolegnia suppression in aquaculture. This research provides a scientific framework for studying the diversity and dynamics of microbial communities to mitigate emerging diseases. The Frondihabitans, Pseudomonas and Trichoderma isolates, and/or their bioactive metabolites, are proposed as effective candidates to control Saprolegniosis.
Environmental proxies of antigen exposure explain variation in immune investment better than indices of pace of life
Horrocks, N.P.C. ; Hegemann, A. ; Ostrowski, S. ; Ndithia, H. ; Shobrak, M. ; Williams, J.B. ; Matson, K.D. ; Tieleman, B.I. - \ 2015
Oecologia 177 (2015)1. - ISSN 0029-8549 - p. 281 - 290.
female pied flycatchers - tropical birds - trade-offs - ecological immunology - microbial diversity - natural antibodies - aridity gradient - south-africa - history - patterns
Investment in immune defences is predicted to covary with a variety of ecologically and evolutionarily relevant axes, with pace of life and environmental antigen exposure being two examples. These axes may themselves covary directly or inversely, and such relationships can lead to conflicting predictions regarding immune investment. If pace of life shapes immune investment then, following life history theory, slow-living, arid zone and tropical species should invest more in immunity than fast-living temperate species. Alternatively, if antigen exposure drives immune investment, then species in antigen-rich tropical and temperate environments are predicted to exhibit higher immune indices than species from antigen-poor arid locations. To test these contrasting predictions we investigated how variation in pace of life and antigen exposure influence immune investment in related lark species (Alaudidae) with differing life histories and predicted risks of exposure to environmental microbes and parasites. We used clutch size and total number of eggs laid per year as indicators of pace of life, and aridity, and the climatic variables that influence aridity, as correlates of antigen abundance. We quantified immune investment by measuring four indices of innate immunity. Pace of life explained little of the variation in immune investment, and only one immune measure correlated significantly with pace of life, but not in the predicted direction. Conversely, aridity, our proxy for environmental antigen exposure, was predictive of immune investment, and larks in more mesic environments had higher immune indices than those living in arid, low-risk locations. Our study suggests that abiotic environmental variables with strong ties to environmental antigen exposure can be important correlates of immunological variation.
Exploring microbial diversity of marine sponges by culture-dependent and molecular approaches
Naim, M.A. - \ 2015
Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462572867 - 220
sponsen - microbiële diversiteit - symbiose - gastheerspecificiteit - zeeschimmels - biodiversiteit - sponges - microbial diversity - symbiosis - host specificity - marine fungi - biodiversity
Discovery of sponge-grade metazoans dated 650 million years ago proved that sponges have been around since the Precambrian era. Their resilience to ever-changing environmental conditions and their global distribution is one of the features attributed to the symbionts in sponges, which include Archaea, Bacteria and Eukarya. It is yet unknown how sponges attract and select their bacterial associates but mechanisms to maintain or newly acquire their symbionts have been demonstrated, such as vertical and horizontal transmission.
Discovery of species-specific bacterial communities in the marine sponges H. panicea, H. oculata and H. xena which are dominated by an alpha, beta- and gammaproteobacterium, respectively, confirmed host-specificity of bacterial associates in marine sponges from the North Sea, although their function remains unknown. Detection of Chlamydiae in high relative abundance raised the question as to what is their function in the sponge holobiont as they were only distantly related to other known Chlamydiae.
Little is known about the fungal community in marine sponges. This prompted the study of sponge-associated fungi based on molecular analysis. This was previously a difficult enterprise due the large amount of ‘contaminating’ sponge DNA, which is susceptible to amplification with fungi-specific PCR primers as well. The advent of next generation sequencing technology now for the first time allowed to overcome this hurdle by the sheer numbers of sequences that can be generated. This lead to discovery of novel yeast lineages from the phyla Ascomycota and Basidiomycota in North Sea and Mediterranean marine sponges, indicating a much higher diversity of fungi yet to be explored. For instance, yeasts from the order Malasseziales, which are common pathogens of marine animals, were found as the dominant yeasts in many of the sponges tested that were without apparent disease.
A complementary cultivation-dependent approach provided access to fungal isolates. Fungi belonging to the genus Penicillium were found to be the dominant fungi recovered by isolation from the Mediterranean sponges Aplysina aerophoba, Petrosia ficiformis and Corticium candelabrum. In addition, fungi belonging to the order Alternaria and yeasts affiliated to the genus Rhodotorula were isolated multiple times. No overlap was found with the fungal species observed through the molecular study, which indicates that the great plate anomaly also exists for fungi. Many of the fungal Pencilillium and Alternaria strains isolated were shown to have the genetic capacity for producing polyketide synthases (PKS) or PKS-non ribosomal peptide synthase (PKS-NRPS) hybrids. These enzyme complexes are generally responsible for the production of secondary metabolites with a high biological activity.
Isolation of bacteria from H. panicea in a cultivation experiment with a large diversification of media and growth conditions and subsequent comparison of the retrieved microorganisms to bacteria found in the sponge tissue by a molecular approach revealed the presence of bacterial genera that dominate the cultivation library, but comprise of represent minor components of the sponge microbiome. This includes genera such as Bacillus, Paracoccus and Shewanella. Another genus that was commonly isolated from many marine sponges, but only is found at low relative abundance in the sponge microbiome is Pseudovibrio. Phenotypic characterization based on antibiotic resistance and genotypic differentiation based on bacterial BOX elements and presence of halogenase-encoding genes could discriminate closely related strains that could not be distinguished based on their 16 rRNA gene sequence.
In conclusion, this thesis helps to bridge the gap between cultivation-dependent and cultivation-independent studies of sponge-associated bacteria and fungi by clearly defining the frontiers of the gap. The knowledge derived from this thesis could serve as a scientific foundation and inspiration for future microbial diversity studies and provides perspective for analysing and exploiting sponge symbionts.
Growth of anaerobic methane oxidizing archaea and sulfate reducing bacteria in a high pressure membrane-capsule bioreactor
Timmers, P.H.A. ; Gieteling, J. ; Widjaja-Greefkes, H.C.A. ; Plugge, C.M. ; Stams, A.J.M. ; Lens, P.N.L. ; Meulepas, R.J.W. - \ 2015
Applied and Environmental Microbiology 81 (2015)4. - ISSN 0099-2240 - p. 1286 - 1296.
cold-seep sediments - 16s ribosomal-rna - gradient gel-electrophoresis - guaymas basin - hydrothermal sediments - microbial diversity - marine-sediments - population-dynamics - community structure - gene database
Anaerobic methane oxidizing communities of archaea (ANME) and sulfate reducing bacteria (SRB) grow slowly, which limits physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated sulfate reducing bacteria (SRB) are affected by it. Here, we report growth of ANME/SRB in a membrane-capsule bioreactor inoculated with Eckernförde Bay sediment that combines high pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2 µm membrane. Results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. Labelled-methane oxidation rates were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was only enriched at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2, Eel-1 group and Desulforomonadales and not to the typically found SEEP-SRB1. Increase of ANME-2a/b occurred in parallel with increase of SEEP-SRB2 which was previously only found associated with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at high methane partial pressure.
Community dynamics of complex starter cultures for Gouda-type cheeses and its functional consequences
Erkus, O. - \ 2014
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): Eddy Smid. - Wageningen UR : Wageningen - ISBN 9789462570108 - 215
goudse kaas - lactococcus lactis - microbiële diversiteit - gouda cheese - lactococcus lactis - microbial diversity
Lactic acid bacteria (LAB) are used as starter and adjunct cultures for the production of artisanal and industrial fermented milk products such as yoghurt and cheese. Artisanal fermentations is propagated with the transfer of an inoculum from old batch of fermented food to the new batch (back-slopping) to initiate the fermentation with the activity of the indigenous microbiota present in the inoculum. In industrial production, these inocula with indigenous microbiota are replaced with the starter cultures that contain lower numbers of LAB species for better controlled fermentation process and consistent final product quality. Cheese manufacturing is still performed in both artisanal ways and with the use of starter cultures. Gouda cheese starter cultures constitute several strains from the subspecies of Lactococcus lactisand Leuconostocs mesenteroidesin different combinations. The mixed and undefined type of starter culture may harbour variable number of strains that contribute unique functionalities to the cheese manufacturing process. Therefore, understanding, controlling and predicting the cheese manufacturing processes require the determination of strain level diversity in the starter culture, their collective and specific metabolic complement, and their activity throughout the cheese manufacturing process, including the interactions between the strains. The first two studies that are covered in this thesis describes the development of a high resolution AFLP fingerprinting tool allowing the discrimination of closely related strains in the starter culture and the subsequent analysis of the microbial community of Gouda cheese starter with this implemented technique and with metagenomics. Furthermore, the thesis includes the development of another tool to selectively amplify DNA only from live fraction of the microbial community in cheese using propidium monoazide (PMA), which is required to study community dynamics with culture independent approaches. The last study in the thesis describes the effects of the variation in propagation regime on the community composition of a mixed starter culture and connects the composition change to the functionalities that impact on flavour development during cheese manufacturing. Overall, the approaches presented in this thesis are intended to eventually enable accurate prediction and control of the cheese manufacturing process using (un)defined starter cultures, but may also allow rational design and development of new starter cultures.
The rhizosphere zoo: An overview of plant-associated communities of microorganisms, including phages, bacteria, archaea, and fungi, and some of their structuring factors
Buée, M. ; Boer, W. de; Martin, F. ; Overbeek, L.S. van; Jurkevitch, E. - \ 2009
Plant and Soil 321 (2009)1-2. - ISSN 0032-079X - p. 189 - 212.
burkholderia-cepacia complex - gradient gel-electrophoresis - disease-suppressive soils - fine-scale distribution - field-grown wheat - real-time pcr - ectomycorrhizal fungi - microbial diversity - mycorrhizal fungi - ribosomal-rna
Rhizosphere microorganisms have two faces, like Janus the Roman god of gates and doors who symbolizes changes and transitions, from one condition to another. One face looks at the plant root, the other sees the soil. The ears and the nose sense the other gods around and the mouths are wide open, swallowing as much as they can, and as described in Chapter 11, they also are busy talking. These faces may as well represent Hygieia (the Greek god of Health and Hygiene, the prevention of sickness and the continuation of good health) and Morta (the Roman god of death) for rhizosphere microbes can be beneficial, and promote plant growth and well being (Chapter 12) or detrimental, causing plant sickness and death (Chapter 13). It can be argued that many rhizosphere microbes are “neutral”, faceless saprophytes that decompose organic materials, perform mineralization and turnover processes. While most may not directly interact with the plant, their effects on soil biotic and abiotic parameters certainly have an impact on plant growth. Maybe they are Janus’ feet, the unsung heroes of the rhizosphere. This chapter addresses some aspects of the taxonomical and functional microbial diversity of the rhizosphere. Bacteria, Archea, viruses and Fungi will be at the heart of our discussion, while other rootassociated eukaryotes are the subjects of other chapters
Population Dynamics of a Single-Stage Sulfidogenic Bioreactor Treating Synthetic Zinc-Containing Waste Streams
Dar, S.A. ; Bijmans, M.F.M. ; Dinkla, I.J.T. ; Geurkink, B. ; Lens, P.N.L. ; Dopson, M. - \ 2009
Microbial Ecology 58 (2009)3. - ISSN 0095-3628 - p. 529 - 537.
sulfate-reducing bacteria - gradient gel-electrophoresis - polymerase-chain-reaction - scatologenes strain sl1 - acid-mine drainage - environmental-samples - microbial diversity - sulfide toxicity - ribosomal-rna - sp-nov
Waste streams from industrial processes such as metal smelting or mining contain high concentrations of sulfate and metals with low pH. Dissimilatory sulfate reduction carried out by sulfate-reducing bacteria (SRB) at low pH can combine sulfate reduction with metal-sulfide precipitation and thus open possibilities for selective metal recovery. This study investigates the microbial diversity and population changes of a single-stage sulfidogenic gas-lift bioreactor treating synthetic zinc-rich waste water at pH 5.5 by denaturing gradient gel electrophoresis of 16S rRNA gene fragments and quantitative polymerase chain reaction. The results indicate the presence of a diverse range of phylogenetic groups with the predominant microbial populations belonging to the Desulfovibrionaceae from delta-Proteobacteria. Desulfovibrio desulfuricans-like populations were the most abundant among the SRB during the three stable phases of varying sulfide and zinc concentrations and increased from 13% to 54% of the total bacterial populations over time. The second largest group was Desulfovibrio marrakechensis-like SRB that increased from 1% to about 10% with decreasing sulfide concentrations. Desulfovibrio aminophilus-like populations were the only SRB to decrease in numbers with decreasing sulfide concentrations. However, their population was <1% of the total bacterial population in the reactor at all analyzed time points. The number of dissimilatory sulfate reductase (DsrA) gene copies per number of SRB cells decreased from 3.5 to 2 DsrA copies when the sulfide concentration was reduced, suggesting that the cells' sulfate-reducing capacity was also lowered. This study has identified the species present in a single-stage sulfidogenic bioreactor treating zinc-rich wastewater at low pH and provides insights into the microbial ecology of this biotechnological process.
A mixture of grass and clover combines the positive effects of both plant species on selected soil biota
Eekeren, N.J.M. van; Liere, D. ; Vries, F.T. de; Rutgers, M. ; Goede, R.G.M. de; Brussaard, L. - \ 2009
Applied Soil Ecology 42 (2009)3. - ISSN 0929-1393 - p. 254 - 263.
ryegrass lolium-perenne - white clover - nematode communities - microbial diversity - trifolium-repens - organic-matter - maturity index - earthworms - nitrogen - biodiversity
The introduction of N2-fixing white clover (Trifolium repens) in grassland is a management measure that may contribute to sustainable grassland systems by making them less dependent on inorganic fertilizers. However, little is known about the impact of this measure on soil biota and ecosystem services. We investigated earthworms, nematodes, bacteria and fungi in an experiment in which white clover-only and a mixture of grass and white clover without fertilization were compared with grass-only with and without fertilization. In comparison with grass-only, white clover-only had a lower total root biomass and a lower C/N-ratio in the above- and below-ground plant biomass. These plant characteristics resulted in a lower bacterial biomass, a lower fungal biomass, a higher proportion of bacterivorous nematode dauerlarvae, a lesser proportion of herbivorous nematodes and a greater abundance of earthworms in clover-only. The quantity and quality (C/N-ratio) of the above- and below-ground plant biomass in the mixture of grass and white clover (20–30% clover in the DM) was comparable with grass fertilized with 150 kg N ha-1 of inorganic fertilizer. Differences between these treatments might show specific clover effects in the grass–clover mixture on soil biota other than quantity and C/N-ratio of the litter. However, the only differences were a higher proportion of bacterivorous nematode dauerlarvae and a different nematode community composition in grass–clover. The soil structure in white clover-only showed a higher proportion of angular blocky elements, a lower penetration resistance, a higher number of earthworm burrows, a higher potential N-mineralization and respiration than the soil in grass-only. This suggests that clover stimulates the ecosystem services of water infiltration and supply of nutrients, but is less conducive to soil structure maintenance. The grass–clover mixture differed from grass-only in a higher respiration and from clover-only in a higher percentage of soil crumbs. We suggest that when clover is introduced in grassland to reduce the reliance on inorganic fertilizer, the mixture of grass and clover maintains the positive impact of grass roots on soil structure and increases the supply of nutrients via the soil food web. Thus, a grass–clover mixture combines the agronomic benefits of the two plant types
Analysis of diversity and function of the human small intestinal microbiota
Booijink, C.C.G.M. - \ 2009
Wageningen University. Promotor(en): Willem de Vos; Michiel Kleerebezem; Erwin Zoetendal. - [S.l. : S.n. - ISBN 9789085853626 - 138
darmmicro-organismen - dunne darm - genexpressie - ileostomie - koolhydraatmetabolisme - metabolisme - microbiële diversiteit - functionele biodiversiteit - functionele genomica - genexpressieanalyse - intestinal microorganisms - small intestine - gene expression - ileostomy - carbohydrate metabolism - metabolism - microbial diversity - functional biodiversity - functional genomics - genomics
The gastrointestinal (GI) tract is the main site where the conversion and absorption of food components takes place in humans. As the small intestine is the first site of interaction between the microbiota and ingested food, knowledge about the microbial composition as well as functionality is essential for a complete understanding of the symbiotic interactions and to the potential modulation of metabolically important groups. Subjects carrying an ileostomy were chosen as model system and ileostomy effluent samples were collected over time. The diversity as well as activity of the inhabiting microbiota was analysed in ileostomy effluent samples of five healthy individuals, collected in the morning and afternoon over a period of 28 days. This revealed that the diversity of the ileostomy effluent microbiota was different from that in the faeces, mainly concerning the lower complexity and stability over time. In terms of composition the relative abundance of species belonging to the genera Streptococcus and Veillonella was higher, whereas a lower relative abundance of species related to the Ruminococcus obeum, R. gnavus and Bacteroides plebeius-like organisms was observed in ileostomy effluent samples. Marked differences in microbiota composition between the five subjects with an ileostomy were found, indicative for a highly personal ileal microbiota profile. Differences in microbiota composition profiles were observed over time, even visible within one day, although the overall fluctuations were around a relatively large stable core group, consisting of species belonging to three streptococci-related groups (S. bovis, S. intermedius and S. smitis), Clostridium cluster I, Enterococcus, Veillonella and Oxalobacter formigenes. Overall, the data presented in this thesis indicated that the genus Streptococcus is not only numerically abundant, but also predominates randomly generated metabolic activity profiles of the microbial ecosystem of the ileostomy effluent microbiota. Predominant functions exerted were related to metabolism, especially carbohydrate metabolism and transport. The fast transit of the ileal contents appears to generate an environment in which the capacity to rapidly metabolise the available carbohydrates is an important selective advantage.
Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity
Tzeneva, V.A. ; Salles, J.F. ; Naumova, N. ; Vos, W.M. de; Kuikman, P.J. ; Dolfing, J. ; Smidt, H. - \ 2009
Research in Microbiology 160 (2009)2. - ISSN 0923-2508 - p. 89 - 98.
gradient gel-electrophoresis - land-use history - microbial diversity - community structure - microorganisms - genes - dna - identification - crop
Archived soil samples are a valuable source for retrospective ecological studies, and their recent analysis using molecular ecological approaches has drawn significant attention within the scientific community. However, the possibility of addressing ecological questions regarding detectable microbiota in dried and extensively stored soils has not yet been fully evaluated. To achieve this, soil samples collected from two long-term grassland experiments in the United Kingdom and The Netherlands were subjected to air-drying at 40-42 degrees C and stored at room temperature. Total bacterial, Bacillus benzoevorans-related and eukaryotic communities associated with these samples were analyzed by DGGE-fingerprinting of PCR-amplified ribosomal RNA gene fragments. Changes in microbial community structure due to drying and storage were evaluated by multivariate analysis in relation to changes caused by other environmental conditions, such as soil pH, type of fertilizer and vegetation. Soil drying and storage affected the detectable community structure, but did not materially impair our capacity to identify the effect of soil parameters studied in long-term grassland experiments. Although, in some cases, the amplitude of the influence of a given parameter changed due to sample preservation, analyses revealed that pH, fertilization and soil type significantly influenced microbial community structure in the analyzed samples
Effect of sulfate on methanogenic communities that degrade unsaturated and saturated long-chain fatty acids (LCFA)
Sousa, D. de; Alves, J.I. ; Alves, M.M. ; Smidt, H. ; Stams, A.J.M. - \ 2009
Environmental Microbiology 11 (2009)1. - ISSN 1462-2912 - p. 68 - 80.
propionate-oxidizing bacterium - methane-producing bacteria - anaerobic granular sludge - nov sp-nov - gen-nov - reducing bacterium - uasb reactors - microbial diversity - pure culture - waste-water
Anaerobic bacteria involved in the degradation of long-chain fatty acids (LCFA), in the presence of sulfate as electron acceptor, were studied by combined cultivation-dependent and molecular techniques. The bacterial diversity in four mesophilic sulfate-reducing enrichment cultures, growing on oleate (C(18:1), unsaturated LCFA) or palmitate (C(16:0), saturated LCFA), was studied by denaturing gradient gel electrophoresis (DGGE) profiling of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. These enrichment cultures were started using methanogenic inocula in order to assess the competition between methanogenic communities and sulfate-reducing bacteria. Phylogenetic affiliation of rRNA gene sequences corresponding to predominant DGGE bands demonstrated that members of the Syntrophomonadaceae, together with sulfate reducers mainly belonging to the Desulfovibrionales and Syntrophobacteraceae groups, were present in the sulfate-reducing enrichment cultures. Subculturing of LCFA-degrading methanogenic cultures in the presence of sulfate resulted in the inhibition of methanogenesis and, after several transfers, archaea could no longer be detected by real-time PCR. Competition for hydrogen and acetate was therefore won by sulfate reducers, but acetogenic syntrophic bacteria were the only known LCFA-degrading organisms present after subculturing with sulfate. Principal component analysis of the DGGE profiles from methanogenic and sulfate-reducing oleate- and palmitate-enrichment cultures showed a greater influence of the substrate than the presence or absence of sulfate, indicating that the bacterial communities degrading LCFA in the absence/presence of sulfate are rather stable
The mucin degrader Akkermansia muciniphila is an abundant resident of the human intestinal tract
Derrien, M.M.N. ; Collado, M.C. ; Ben-Amor, K. ; Salminen, S. ; Vos, W.M. de - \ 2008
Applied and Environmental Microbiology 74 (2008)5. - ISSN 0099-2240 - p. 1646 - 1648.
targeted oligonucleotide probes - 16s ribosomal-rna - microbial diversity - bacteria - hybridization - microflora - database - reveals - samples - nov.
A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract
Experimental ecology and evolution of microbial diversity : the role of spatial structure
Habets, M.G.J.L. - \ 2008
Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser. - S.l. : s.n. - ISBN 9789085048619 - 102
micro-organismen - diversiteit - biodiversiteit - evolutie - ecologie - adaptatie - heterogeniteit - mutanten - microbiële diversiteit - microorganisms - diversity - biodiversity - evolution - ecology - adaptation - heterogeneity - mutants - microbial diversity
In the light of the competitive exclusion principle, which states that complete competitors cannot coexist, many explanations have been sought to explain the high diversity found in nature. The most common explanation is the niche differentiation hypothesis: coexistence is obtained through differentiation of species in ecological niches. Spatial structure is thought to be a factor capable of providing opportunities for niche differentiation. We have focused on four aspects of spatial structure enabling genetic diversity to emerge and /or to be maintained.
First of all, population fragmentation, resulting from growth in spatially structured habitats, can increase diversity, because the resulting smaller subpopulations, due to their smaller population size, are more likely to adaptively diverge. By allowing small and large populations of E. coli to evolve for 500 generations in two different nutrient environments, we test this hypothesis. The results demonstrate higher variance in fitness among small populations, and consequently more heterogeneous adaptive trajectories for small populations, some of which surprisingly lead to higher fitness peaks than reached by even the best adapted large population.
In a short-term invasion experiment between a superior E. coli competitor and its inferior ancestor, we demonstrate that populations residing in structured environments experience slower invasion dynamics of beneficial mutations than well-mixed populations due to limited dispersal, and therefore local competition. Moreover, our results demonstrate a deceleration of invasion with increasing size of the invading subpopulation. This is caused by a decrease of inter specific competition relative to intra specific competition. Since inferior competitors are present in the community for a longer period of time, they can recombine with other persisting lineages or obtain new mutations, some of which might be beneficial. It is therefore possible that polymorphisms arise which would not have had the opportunity to emerge in a well-mixed environment. Even though both population fragmentation and slower competitive dynamics can increase the emergence of diversity, they do not provide a means for their maintenance.
Environmental heterogeneity on the other hand can cause maintenance of diversity. Environmental heterogeneity can be introduced by spatial structure, e.g. by providing gradients in biotic and abiotic factors, thereby increasing the number of niches. By allowing E. coli populations to evolve for 900 generations in either a well-mixed environment or two structured environments (with or without dispersal), we demonstrate stable coexistence of diversity in structured populations without dispersal. This can be attributed to negative frequency-dependent fitness interactions among niche specialists that either inhabit existing niches provided by the heterogeneous environment or new niches constructed by organisms inhabiting the environment.
In addition to examining aspects of spatial structure that provide means for populations to diversify, we examine a specific consequence of slower dynamics and environmental heterogeneity: the probability of mutators to hitchhike to fixation. Understanding the emergence of mutators is not only scientifically important, but also relevant for human health, since high frequencies of mutators have been found in bacterial populations and drug resistant mutants arise more often in mutator populations. E. coli mutator populations were introduced at different starting frequencies in a well-mixed environment and two structured environments differing in their dispersal rate. Contrary to expectations, we find an advantage in the rate of invasion for mutators in well-mixed environments. Faster competitive dynamics may allow a rapid increase of population size and hence a greater supply of mutations for subsequent adaptation. Due to a delay in mutator extinction in structured environments at low frequencies, mutators may gain from fluctuating conditions.
Finding the needles in the meta-genome haystack
Kowalchuk, G.A. ; Speksnijder, A.G.C.L. ; Zhang, K. ; Goodman, R.M. ; Veen, J.A. van - \ 2007
Microbial Ecology 53 (2007)3. - ISSN 0095-3628 - p. 475 - 485.
whole-genome amplification - ribosomal-rna genes - soil dna libraries - wide host-range - enrichment cultures - uncultured microorganisms - microbial diversity - escherichia-coli - natural-products - messenger-rna
In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth's diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and sequencing of these genomes. This approach allows microbial ecologists to access and study the full range of microbial diversity, regardless of our ability to culture organisms, and provides an unprecedented access to the breadth of natural products that these genomes encode. However, there is no way that the mere collection of sequences, no matter how expansive, can provide full coverage of the complex world of microbial metagenomes within the foreseeable future. Furthermore, although it is possible to fish out highly informative and useful genes from the sea of gene diversity in the environment, this can be a highly tedious and inefficient procedure. Microbial ecologists must be clever in their pursuit of ecologically relevant, valuable, and niche-defining genomic information within the vast haystack of microbial diversity. In this report, we seek to describe advances and prospects that will help microbial ecologists glean more knowledge from investigations into metagenomes. These include technological advances in sequencing and cloning methodologies, as well as improvements in annotation and comparative sequence analysis. More significant, however, will be ways to focus in on various subsets of the metagenome that may be of particular relevance, either by limiting the target community under study or improving the focus or speed of screening procedures. Lastly, given the cost and infrastructure necessary for large metagenome projects, and the almost inexhaustible amount of data they can produce, trends toward broader use of metagenome data across the research community coupled with the needed investment in bioinformatics infrastructure devoted to metagenomics will no doubt further increase the value of metagenomic studies in various environments.
A ribosomal RNA gene intergenic spacer based PCR and DGGE fingerprinting method for the analysis of specific rhizobial communities in soil
Oliveira, V.M. de; Manfio, G.P. ; Coutinho, H.L.D. ; Keijzer-Wolters, A.C. ; Elsas, J.D. van - \ 2006
Journal of Microbiological Methods 64 (2006)3. - ISSN 0167-7012 - p. 366 - 379.
gradient gel-electrophoresis - polymerase chain-reaction - paenibacillus-azotofixans - microbial diversity - dna extraction - host plants - leguminosarum - strains - amplification - tropici
A direct molecular method for assessing the diversity of specific populations of rhizobia in soil, based on nested PCR amplification of 16S-23S ribosomal RNA gene (rDNA) intergenic spacer (IGS) sequences, was developed. Initial generic amplification of bacterial rDNA IGS sequences from soil DNA was followed by specific amplification of (1) sequences affiliated with Rhizobium leguminosarum ¿sensu lato¿ and (2) R. tropici. Using analysis of the amplified sequences in clone libraries obtained on the basis of soil DNA, this two-sided method was shown to be very specific for rhizobial subpopulations in soil. It was then further validated as a direct fingerprinting tool of the target rhizobia based on denaturing gradient gel electrophoresis (DGGE). The PCR-DGGE approach was applied to soils from fields in Brazil cultivated with common bean (Phaseolus vulgaris) under conventional or no-tillage practices. The community fingerprints obtained allowed the direct analysis of the respective rhizobial community structures in soil samples from the two contrasting agricultural practices. Data obtained with both primer sets revealed clustering of the community structures of the target rhizobial types along treatment. Moreover, the DGGE profiles obtained with the R. tropici primer set indicated that the abundance and diversity of these organisms were favoured under NT practices. These results suggest that the R. leguminosarum¿as well as R. tropici¿targeted IGS-based nested PCR and DGGE are useful tools for monitoring the effect of agricultural practices on these and related rhizobial subpopulations in soils
Impact of transgenic potatoes expressing anti-bacterial agents on bacterial endophytes is comparable with the effects of plant genotype, soil type and pathogen infection
Rasche, F. ; Velvis, H. ; Zachow, C. ; Berg, G. ; Elsas, J.D. van; Sessitsch, A. - \ 2006
Journal of Applied Ecology 43 (2006)3. - ISSN 0021-8901 - p. 555 - 566.
16s ribosomal-rna - carotovora subsp atroseptica - growth-promoting bacteria - attacin-e gene - erwinia-carotovora - t4 lysozyme - cecropin-b - hyalophora-cecropia - microbial diversity - fire blight
1. Blackleg and soft rot disease of potatoes Solanum tuberosum L., mainly caused by the bacterial pathogen Erwinia carotovora ssp. atrospetica (Eca), lead to enormous yield losses world-wide. Genetically modified (GM) potatoes producing anti-bacterial agents, such as cecropin/attacin and T4 lysozyme, may offer effective future pathogen control strategies. Because of concerns about undesirable ecological side-effects of GM crops, it is important to analyse the potential environmental impact of GM crops carefully. The objective of this study was to investigate the effect of GM potatoes with anti-bacterial activity on the diversity and functional abilities of bacteria colonizing the intercellular spaces and vascular tissues (endosphere) of potato plants. 2. A greenhouse experiment was performed to analyse the effect of GM potatoes expressing either attacin/cecropin or T4 lysozyme on endophytic bacterial communities. Endophytic bacteria colonizing the GM potato lines as well as their nearly isogenic wild types were analysed at two vegetation stages. In order to compare GM-related variations with impacts caused by changing environmental conditions, potatoes were cultivated in two different soil types, and challenged with the pathogen Eca. Endophytic diversity was assessed by 16S rRNA-based terminal-restriction fragment length polymorphism (T-RFLP) analysis. Cultivated community members were identified by 16S rRNA gene analysis and screened for a range of plant growth-promoting and plant pathogen-antagonistic abilities. 3. Both genetic transformation events induced a differentiation in the community structures of associated bacterial populations and in the related functional abilities of cultivated bacterial endophytes. In comparison with the other factors analysed, the impact of both genetic modification types was minor or comparable with the variations caused by plant genotype, vegetation stage, pathogen exposure and soil type. 4. Synthesis and applications. This study has shown that the expression of anti-bacterial proteins may affect bacterial endophytes; however, the impacts were no greater than those of other factors analysed. Future risk assessment studies of GM crops should consider different environmental factors. This study contributes to the ongoing risk assessment of GM crops and provides valuable baseline information for prospective GM crop assays.
Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes
Rasche, F. ; Marco-Noales, E. ; Velvis, H. ; Overbeek, L.S. van; Lopez, M.M. ; Elsas, J.D. van; Sessitsch, A. - \ 2006
Plant and Soil 289 (2006)1-2. - ISSN 0032-079X - p. 123 - 140.
16s ribosomal-rna - endophytic bacteria - t4 lysozyme - transgenic potatoes - soil type - microbial diversity - promoting bacteria - communities - genes - cotton
Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desirée, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desirée DL 12, with its isogenic, non-transformed parental line Desirée. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desirée lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desirée line cultivated in both field trials.