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.

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    Extended-spectrum and AmpC B-lactamase-producing Escherichia coli in broilers and peoplelivingand/or working on broiler farms: prevalence, risk factors and molecular characteristics
    Huijbers, P.M.C. ; Graat, E.A.M. ; Haenen, A.P.J. ; Santen, M.G. van; Essen-Zandbergen, A. van; Mevius, D.J. ; Duijkeren, E. van; Hoek, A.H.A.M. van - \ 2014
    Journal of Antimicrobial Chemotherapy 69 (2014)10. - ISSN 0305-7453 - p. 2669 - 2675.
    livestock-associated mrsa - enterobacteriaceae - netherlands - humans - identification - plasmids - poultry - genes - meat - pcr
    OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.
    Chromosomal location of bla ctx-m genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK
    Rodriguez, I. ; Thomas, K.M. ; Essen-Zandbergen, A. van; Schink, A.K. ; Day, M. ; Chattaway, M. ; Wu, G. ; Mevius, D.J. ; Helmuth, R. ; Guerra, B. - \ 2014
    International Journal of Antimicrobial Agents 43 (2014)6. - ISSN 0924-8579 - p. 553 - 557.
    extended-spectrum - beta-lactamases - coli - plasmids - ctx-m-15
    This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n = 356) obtained in Germany, The Netherlands and the UK (2005–2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. ß-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 ß-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.
    Characteristics of Cefotaxime-Resistant Escherichia coli from Wild Birds in The Netherlands
    Veldman, K.T. ; Tulden, P. ; Kant, A. ; Testerink, J.J. ; Mevius, D.J. - \ 2013
    Applied and Environmental Microbiology 79 (2013)24. - ISSN 0099-2240 - p. 7556 - 7561.
    spectrum-beta-lactamase - extended-spectrum - antibiotic-resistance - natural reserve - ctx-m - salmonella - plasmids - gulls - enterobacteriaceae - environments
    Cloacal swabs from carcasses of Dutch wild birds obtained in 2010 and 2011 were selectively cultured on media with cefotaxime to screen for the presence of extended-spectrum beta-lactamase (ESBL)/AmpC-producing Escherichia coli. Subsequently, all cefotaxime-resistant E. coli isolates were tested by broth microdilution and microarray. The presence of ESBL/AmpC and coexisting plasmid-mediated quinolone resistance (PMQR) genes was confirmed by PCR and sequencing. To determine the size of plasmids and the location of ESBL and PMQR genes, S1 pulsed-field gel electrophoresis (PFGE) was performed on transformants, followed by Southern blot hybridization. The study included 414 cloacal swabs originating from 55 different bird species. Cefotaxime-resistant E. coli isolates were identified in 65 birds (15.7%) from 21 different species. In all, 65 cefotaxime-resistant E. coli ESBL/AmpC genes were detected, mainly comprising variants of bla(CTX-M) and bla(CMY-2). Furthermore, PMQR genes [aac(6')-lb-cr, qnrB1, and qnrS1] coincided in seven cefotaxime-resistant E. coli isolates. Overall, replicon typing of the ESBL/AmpC-carrying plasmids demonstrated the predominant presence of IncI1 (n = 31) and variants of IncF (n = 18). Our results indicate a wide dissemination of ESBL and AmpC genes in wild birds from The Netherlands, especially among aquatic-associated species (waterfowl, gulls, and waders). The identified genes and plasmids reflect the genes found predominantly in livestock animals as well as in humans.
    Multifactorial diversity sustains microbial community stability
    Erkus, O. ; Jager, V.C.L. de; Spus, M. ; Alen-Boerrigter, I.J. van; Rijswijck, I.M.H. van; Hazelwood, L. ; Janssen, P.W. ; Hijum, S.A.F.T. van; Kleerebezem, M. ; Smid, E.J. - \ 2013
    ISME Journal 7 (2013)11. - ISSN 1751-7362 - p. 2126 - 2136.
    lactic-acid bacteria - complete genome sequence - lactococcus-lactis - dairy environment - subsp lactis - raw-milk - cremoris - plasmids - cheese - identification
    Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty
    The public health risks of enterobacterial isolates producing extended-spectrum Beta-lactamases (ESBL) or AmpC Beta-lactamases in food and food-producing animals: An EU perspective of epidemiology, analytical methods, risk factors and control options
    Liebana, E. ; Carattoli, A. ; Coque, T.M. ; Hasman, H. ; Magiorakos, A.P. ; Mevius, D.J. ; Peixe, L. ; Schuepbach-Regula, G. ; Torneke, K. ; Torren-Edo, J. ; Torres, C. ; Threlfall, J. - \ 2013
    Clinical infectious diseases 56 (2013)7. - ISSN 1058-4838 - p. 1030 - 1037.
    escherichia-coli - salmonella-enterica - ctx-m - antimicrobial resistance - plasmids - poultry - strains - farms - spain - identification
    blaESBL and blaAmpC in Enterobacteriaceae are spread by plasmid-mediated integrons, insertion sequences and transposons, some of which are homologous in bacteria from food-animals, foods and humans. These genes have been frequently identified in Escherichia coli and Salmonella from food-animals; the most common genes being blaCTX-M-1, blaCTX-M-14, and blaCMY-2. Identification of risk factors for their occurrence in food-animals is complex. As well as generic antimicrobial use, cephalosporin usage is an important risk factor for selection and spread of these genes. Extensive international trade of animals is a further risk factor. There are no data on the effectiveness of individual control options in reducing public health risks. A highly effective option would be to stop or restrict cephalosporin usage in food-animals. Decreasing total antimicrobial use is also of high priority. Implementation of measures to limit strain dissemination (increasing farm biosecurity, controls in animal trade, and other general post-harvest controls) are also important.
    Extended-spectrum-ß-lactamase- and AmpC-ß-lactamase-producing Escherichia coli in Dutch broilers and broiler farms
    Dierikx, C.M. ; Goot, J.A. van der; Fabri, T. ; Essen-Zandbergen, A. van; Smith, H.E. ; Mevius, D.J. - \ 2013
    Journal of Antimicrobial Chemotherapy 68 (2013)1. - ISSN 0305-7453 - p. 60 - 67.
    salmonella-enterica - resistance genes - plasmids - humans - food - animals - poultry - chicken - enterobacteriaceae - identification
    Objectives The aim of this study was to establish the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli at Dutch broiler farms and in farmers and to compare ESBL/AmpC-producing isolates from farmers and their animals. Methods Twenty-five to 41 cloacal swabs collected from broilers at each of 26 farms and 18 faecal samples from 18 broiler farmers were analysed for determination of the presence of ESBL/AmpC-producing E. coli. ESBL/AmpC genes were characterized by microarray, PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing or restriction fragment length polymorphism. E. coli genotypes were determined by multilocus sequence typing. Results Birds from all farms were positive for ESBL/AmpC-producing E. coli, and on 22/26 farms the within-farm prevalence was =80%. Six of 18 farmers carried isolates containing ESBL/AmpC genes blaCTX-M-1, blaCMY-2 and/or blaSHV-12, which were also present in the samples from their animals. In five of these isolates, the genes were located on identical plasmid families [IncI1 (n¿=¿3), IncK (n¿=¿1) or IncN (n¿=¿1)], and in isolates from two farmers the genes were carried on identical plasmid subtypes (IncI1 ST12 and IncN ST1, where ST stands for sequence type) as in the isolates from their animals. Conclusions This study shows a high prevalence of birds carrying ESBL/AmpC-producing E. coli at Dutch broiler farms and a high prevalence of ESBL/AmpC-producing E. coli in farmers. This is undesirable due to the risk this poses to human health. Future research should focus on identification of the source of these isolates in the broiler production chain to make interventions resulting in reduction of these isolates possible.
    Correlated evolution of senescence and ephemeral substrate use in the Sordariomycetes
    Geydan, T.D. ; Debets, A.J.M. ; Verkleij, G.J. ; Diepeningen, A.D. van - \ 2012
    Molecular Ecology 21 (2012)11. - ISSN 0962-1083 - p. 2816 - 2828.
    podospora-anserina - metarhizium-anisopliae - colony sectorization - filamentous fungi - phylogenies - plasmids - genes - dna - populations - ascomycota
    Evolutionary theory predicts that senescence--a decline in reproduction and survival with increasing age--can evolve as a trade-off between investment in reproduction on one side and in somatic maintenance and repair on the other. The ecology of a species is crucial because it provides the external causes of death that determine the statistical limit to a species' lifespan. Filamentous fungi are generally believed to be nonsenescent, and there are indeed spectacular examples of very old fungal individuals in nature. However, some fungi utilize ephemeral resources, and therefore, senescence is expected to have evolved, like in the coprophilic Podospora anserina, the only well-studied filamentous fungus with intrinsic senescence. Here, we hypothesize that rapid senescence is more common in fungi than generally believed and that the phylogenetic distribution of senescence correlates with ecology. We collected lifespan data for a set of Sordariomycetes and constructed phylogenies based on several nuclear sequences. Several of the strains were from the CBS culture collection, originally isolated from various substrates, some of which ephemeral. In addition, we isolated new strains from short-lived substrates. Senescence was observed throughout the phylogeny. Correlation tests support the hypothesis that in the Sordariomycetes, senescence is a trait that has arisen in response to ephemeral substrates, and that it has evolved repeatedly and independently along the phylogeny
    Letter tot the editor: Characterization of qnr-positive Escherichia coli isolates from food-producing animals in the Netherlands
    Veldman, K.T. ; Essen-Zandbergen, A. van; Kant, A. ; Mevius, D.J. - \ 2012
    Journal of Antimicrobial Chemotherapy 67 (2012)1. - ISSN 0305-7453 - p. 239 - 240.
    mediated quinolone resistance - salmonella-enterica - beta-lactamases - plasmids - sequence
    D-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum
    Siemerink, M.A.J. ; Kuit, W. ; Lopez Contreras, A.M. ; Eggink, G. ; Oost, J. van der; Kengen, S.W.M. - \ 2011
    Applied and Environmental Microbiology 77 (2011)8. - ISSN 0099-2240 - p. 2582 - 2588.
    reductase/2,3-butanediol dehydrogenase - transcriptional program - butanol fermentation - solvent production - atcc 824 - gene - biofuels - metabolism - tolerance - plasmids
    Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions Clostridium acetobutylicum ATCC 824 (and derived strains) generates both D- and L-stereoisomers of acetoin, but due to the lack of an ACR enzyme, does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89-90% of the natively produced acetoin was converted to 20-22 mM D-2,3-butanediol. Addition of a racemic mixture of acetoin did lead to the production of both, D-2,3-butanediol and meso-2,3-butanediol. A metabolic network is proposed that is in agreement with the experimental data. Native 2,3-butanediol production is a first step towards a potential homo-fermentative 2-butanol producing strain of C. acetobutylicum.
    Characterization of multidrug-resistant, qnrB2-positive and extended-spectrum-ß-lactamase-producing Salmonella Concord and Salmonella Senftenberg isolates
    Veldman, K.T. ; Dierikx, C.M. ; Essen-Zandbergen, A. van; Pelt, W. van; Mevius, D. - \ 2010
    Journal of Antimicrobial Chemotherapy 65 (2010). - ISSN 0305-7453 - p. 872 - 875.
    mediated quinolone resistance - escherichia-coli - united-states - gene - plasmids - enterica - identification - netherlands - prevalence - children
    Objectives To characterize plasmids and resistance genes of multidrug-resistant (MDR) Salmonella Senftenberg and Salmonella Concord isolated from patients in the Netherlands. Methods The resistance genes of four MDR Salmonella isolates (three Salmonella Concord and one Salmonella Senftenberg) were identified by miniaturized microarray, PCR and sequencing. Plasmids were characterized by S1 nuclease-PFGE and PCR-based replicon typing (PBRT). Linkage between plasmids and genes was determined by conjugation experiments and microarray analysis. The genetic relationship between the three Salmonella Concord isolates was determined by XbaI-PFGE. Results A large variety of resistance genes was detected, including qnrB2 and the ß-lactamase genes blaTEM-1 and blaSHV-12 in all isolates; moreover all Salmonella Concord isolates also harboured blaCTX-M-15. Salmonella Senftenberg harboured a large IncHI2 plasmid. The three Salmonella Concord isolates harboured two large plasmids typed as IncHI2 and IncA/C. Conclusions We detected the first plasmid-mediated MDR Salmonella isolates in the Netherlands harbouring both qnr and extended-spectrum ß-lactamase (ESBL) genes. In Salmonella Senftenberg one large plasmid (IncHI2) and in Salmonella Concord two large plasmids (IncHI2 and IncA/C) were responsible for the multidrug resistance.
    Inhibition of Binding of the AB5-Type Enterotoxins LT-I and Cholera Toxin to Ganglioside GM1 by Galactose-Rich Dietary Components
    Becker, P.M. ; Widjaja-Greefkes, H.C.A. ; Wikselaar, P.G. van - \ 2010
    Foodborne Pathogens and Disease 7 (2010)3. - ISSN 1535-3141 - p. 225 - 233.
    heat-labile enterotoxin - escherichia-coli - receptor antagonists - crystal-structure - b-pentamer - human-milk - oligosaccharides - plasmids - identification - strains
    Cholera, travelers' diarrhea, or colibacillosis in pigs can possibly be prevented or attenuated by dietary provision of competitive inhibitors that react with the GM1-binding sites of the enterotoxins cholera toxin (CT), human Escherichia coli heat-labile enterotoxin of serogroup I (LTh-I), and porcine LT-I (LTp-I). The interfering efficiency of natural substances with binding of the toxins to the gangliosid receptor GM1 was tested using a specially adapted GM1-coated-microtiter-well enzyme-linked immunosorbent assay. The substances tested for their GM1 displacing capacity were galactose-containing or -related saccharides from bovine milk, skim milk powder, galactan from gum arabic, food stabilizers as well as ground fenugreek seed and soy bean constituents that contain galactomannans, the galactopolysaccharides agar and agarose, and larch wood and other plant materials that contain arabinogalactans. Skim milk powder, compared with the pure milk saccharides tested, interfered to a higher extent with LTh-I (65–66% inhibition at 5mg test substance/mL) and CT binding (63–67% inhibition at 5mg test substance/mL) when supplied before or simultaneously with the toxins in the GM1-enzyme-linked immunosorbent assay. Ground fenugreek seed counteracted GM1 binding of 5ng LTh-I/mL as well as 5ng and 1µg LTp-I/mL (43–65% inhibition at 5mg test substance/mL), and 4ng CT/mL (61–92% inhibition at 5mg test substance/mL) very efficiently when supplied before the toxin–GM1 complex had formed. With 50mg/mL fenugreek seed, inhibition percentages of even 92–99% were reached for LTh-I and CT binding. Efforts to resolve already bound toxin from GM1 with the test substances were less effective than preincubations and concurrent incubations.
    On the natural and laboratory evolution of an antibiotic resistance gene
    Salverda, M.L.M. - \ 2008
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser; John van der Oost. - [S.l.] : S.n. - ISBN 9789085049999 - 144
    evolutie - bèta-lactamase - plasmiden - recombinatie - bacteriën - fenotypen - mutagenese - fylogenie - moleculaire genetica - selectie - antibioticaresistentie - evolution - beta-lactamase - plasmids - recombination - bacteria - phenotypes - mutagenesis - phylogeny - molecular genetics - selection - antibiotic resistance
    TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified.
    Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype.
    In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well.
    A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.
    PCR detection of oxytetracycline resistance genes from diverse habitats in total community DNA and in streptomycete isolates.
    Nikolakopoulou, T.L. ; Egan, S. ; Overbeek, L.S. van; Guillaume, G. ; Heuer, H. ; Wellington, E.M.H. ; Elsas, J.D. van; Collard, J.M. ; Smalla, K. ; Karagouni, A.D. - \ 2005
    Current Microbiology 51 (2005)4. - ISSN 0343-8651 - p. 211 - 216.
    environmental bacteria - efflux protein - mycobacterium - prevalence - validation - plasmids - clusters - rimosus - primers - soil
    A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (TcR) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of TcR streptomycetes originated from bulk and rhizosphere soil. Fewer TcR streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene¿carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which TcR streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common
    On the ecology and evolution of fungal senescence
    Maas, M.F.P.M. - \ 2005
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - [S.l.] : S.n. - ISBN 9789085042846 - 118
    pezizomycotina - neurospora - veroudering - verouderen - ecologie - evolutie - mitochondria - plasmiden - celbiologie - mutaties - genetica - pezizomycotina - neurospora - senescence - aging - ecology - evolution - mitochondria - plasmids - cellular biology - mutations - genetics
    Aging evolves in the shadow of natural selection: Since the efficiency of natural selection declines with age, organisms will over the course of generations accumulate intrinsic, genetic factors that have a negative effect only late in life. This is generally known as the 'mutation accumulation' theory of aging. Should these factors additionally have apleiotropic, positive effect early on in life, for example on fertility, they could even befavoredby natural selection. This is known as the 'antagonisticpleiotropy' theory of aging. Aging is thus expected to be a multi-causal process resulting from intrinsic factors with negative effects late in life and possibly additional, positive effects early in life. It can be seen as the result of a lack of investment in somatic maintenance, a legacy of an organism's evolutionary past.

    In contrast to unitary organisms like most animals, modular organisms like plants, fungi and colonial invertebrates should not be subject to aging: In these organisms, there is no clear distinction between germ line and soma. Because the germ line should be immortal, in modular organisms aging or senescence is generally not expected, though parts or modules may be subject to aging or senescence. Though this is rare, there are striking examples oforganismalsenescence in fungi and plants, in which all parts of an individual die at the same time.

    This thesis deals with aging in two genera of filamentous fungi:NeurosporaandPodospora. It deals with the question whether there are similarities, both at the proximate or mechanistic level and at the ultimate or evolutionary level, between aging processes in fungi and aging processes as we know them from animals. It is shown that, at least in the pseudo-homothallic filamentousascomycetePodosporaanserina, aging is an intrinsic andmulticausalprocess as may be expected. An analysis of natural variation in life span shows that the main source of variation in life span corresponds to the presence or absence of mitochondrial plasmids, molecular parasites that interfere with respiration. Variation that arises spontaneously in the laboratory often corresponds to mitochondrial mutations in the electron transport chain. The latter mutations are all associated with the induction of alternative; nuclear encoded respiratory pathways and this leads via a yet unknown route to a stabilization of the otherwise unstable mitochondrial genome, a reduced level of reactive oxygen species as well as a reduced energy level. These mutations hence confer longevity at the cost of fertility. In addition to spontaneous mutations and chemical modifications of the electron transport chain, a dietary  reduction in the amount of glucose can extend life span in fungi. The latter effect is strongly reduced by the presence of a type of mitochondrial plasmid that interferes with respiration, which indicates that it is strongly dependent on properly functioning mitochondria. The latter underlines the critical role of mitochondria in the fungal senescence
    Genomic conflicts in Podospora anserina = Genomische conflicten in Podospora anserina
    Gaag, M. van der - \ 2005
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - s.l. : S.n. - ISBN 9789085042556 - 152
    pezizomycotina - schimmels - genomen - genetica - plasmiden - meiotic drive - meiose - uitkruisen - pezizomycotina - fungi - genomes - genetics - plasmids - meiotic drive - meiosis - outcrossing
    This thesis deals with genomic conflicts raised by selfish elements in the ascomycete fungus Podospora anserina .Genomic conflicts arise when the effects of the selfish elements are opposite to the interests of the other parts of the genome. Two types of selfish elements are studied as well as certain characteristics of Podospora involved in the population dynamics of these elements, such as vegetative and sexual incompatibility, senescence and outcrossing.The natural habitat of Podospora anserina is dung of herbivores where it has an optimum growth temperature of 27 °C. The fungus can only reproduce sexually and the ascospores are the products of meiosis as well as the next generation of the fungus. Perithecia or fruiting bodies contain asci with four linearly arranged ascospores, which provide unique opportunities to analyse abnormal segregation and makes this fungus one of the genetic model organisms. Most ascospores are capable of completing the lifecycle of the fungus, as they contain two nuclei, each with one of the two mating types. This fungal trait is called pseudo or secondairy homothallism, and it allows sexual offspring to be produced by either selfing or outcrossing. Sometimes smaller single mating type ascospores are formed containing one nucleus and less cytoplasmic content and mitochondria. Colonies from these spores must outcross with another isolate to produce offspring. The fungal isolates used in this thesis were sampled from dung around Wageningen, theNetherlandsduring 1990-1997, but also some older French isolates dating from 1937 were used.

    The first selfish elements studied in this thesis are linear plasmids with homology to pAL2-1, a 8.3 kb plasmid previously found in this fungus. Linear plasmids are parasitic autonomous replicating genetic elements. In filamentous fungi they reside in the mitochondria. Most plasmids are cryptic or have a negative fitness effect on the host. However the pAL2 plasmid found in P. anserina has been associated with a longevity phenotype, though recently also negative effects were found. Homologous linear plasmids and plasmid families were present in 21 percent of the population and detected over several years. Half of the plasmid containing isolates possesseda single plasmid and the other half a plasmid family consisting of multiple plasmid copies. The lifespan of the isolates was generally uncorrelated with the presence of the plasmid. One isolate showed an increased lifespan, but noinserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity inducing pAL2-1 plasmid. We have looked at the dynamics of plasmid transmission for these plasmids.Vertical transfer of the plasmids to the ascospores occurs only via the maternal line. This transfer is inefficient as up to 40% lose all plasmids in an outcrossing situation and up to 20% retain only the basic plasmid family member in presence of a plasmid family. No difference in efficiency of plasmid transfer to the dikaryotic and the smaller monokaryotic ascospores was detected. Plasmid transmission was also foundindependent of the genetic background of the strains.Loss of plasmids via the sexual route is compensated by horizontal transmission of the plasmid through hyphal contact between different isolates. Horizontal transfer occurs efficiently in both vegetative compatible and incompatible situations. Vegetative incompatibility can be macroscopically observed as a 'barrage', a contact zone of lysed hyphal cells. Vegetative incompatibility is thought to have evolved as a mechanism to hamper spread of mobile selfish elements and parasites. Our experiments show that vegetative incompatibility is not a perfect barrier against this type of selfish elements.

    The other selfish elements studied in this thesis are meiotic drive factors or segregation distorters. Segregation distorters are transmitted into the progeny in excess of the fair Mendelian proportion of 50 %, by actively destructing the alternative allele. Genomic conflicts arise by hitchhiking of genes with deleterious fitness effects. Meiotic drive in Podospora ischaracterized by the abortion of two of the four spores in the ascus.Seven different groups of meiotic drive elements or Spore killer types were identified and characterized. Among 99 isolates from nature, six of these meiotic drive elements occurred in our local population. All drive elements comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands and most elements can be retrieved over the years. Spore-killer type Psk-7 was also present in a French strain dating from 1937 and exists for more than 60 years. No resistance to meiotic drive was observed and all other isolates found so far are sensitive to spore killing. Each type of Spore killer differs in the percentage of asci that show killing, ranging from 50 to 95% two-spored asci, and in their mutual interactions. The aborted ascospores quickly degrade after spore wall formation, except for the Psk-3 group where they remain visible as tiny shriveled ascospores, indicating different abortion factors or timing for killing. The Psk-3 group also shows a variable percentage of two-spored asci within each perithecium. Spore-killer interactions show either mutual resistance ( i.e. no abortion is found if an allele of either Spore killer is present) or dominant epistasis ( i.e. one killer acts as a sensitive type). Genetic mapping could assign most Spore-killer types to linkage group III where they are not tightly linked to the centromere.

    Several possible models that explain the spore killing mechanism in Podospora anserina were examined. Repeatedbackcrossing of Spore killers to the same sensitive isolate produces strains with the same genetic background. Spore killers that belong to the same killer type or show mutual resistance become vegetative compatible to each other during backcrosses. On the other hand, Spore killers that show dominant epistasis, as well as the sensitive strain remained vegetative incompatible. This suggests a common mechanism for spore killing, possibly related to vegetative incompatibility, although the precise genetic nature of the correlation is not yet clear.The Podospora genome was screened for homologues of genes known to be involved in silencing in fungi. Genes were found for all silencing mechanisms (RIP, MSUD, Quelling) known in the related fungus Neurospora . However, the possible role of silencing by methylation of genes during the killing process was excluded by experiments using the drug 5-azacytidine, which both removes methylation and prevents de-novo methylation. No effect of the 5-azacytidine treatments was found on spore-killing frequency for all Spore-killer types. Also the consequences of formation of dikaryotic ascospores for expression of spore killing were examined. Crosses were used of Spore killers with a marker that increases the number of monokaryotic spores to up to eight. Spore-killer types Psk-2 , Psk-3 (Wa27) and Psk-4 produced some asci containing more than four spores in these crosses. In such asci sensitive nuclei were able to survive and resist the meiotic drive system, indicating incomplete penetrance of the spore-killing mechanism. Spore killing in the other killer types ( Psk-1, Psk-5, Psk-6 and Psk-7 ) were full penetrant; only asci with four or less spores could be detected. Here the killing mechanism works similar on all asci. Furthermore the effect of low temperatures (22 ºC) on spore killing was tested in this Chapter. Psk-2 dramatically decreased the percentage of killing at this temperature to almost zero. Other Spore-killer types were not affected by temperature. Based on all characteristics and interactions of the Spore killers we propose that spore killing in Podospora may be an example of post-segregational killing due to Toxin-Antitoxin mechanisms. A Spore Killer produces both a persisting stable toxin and a less stable antitoxin. In ascospores where the Spore Killer is absent the antitoxin disappears more quickly than the toxin, leading to abortion of the spores. Variable killing percentages can be explained by the strict balance between toxin and antitoxin and the timing of shutdown of the genes involved.

    Meiotic drive is only possible in an outcrossing situation. Podospora is in principle capable of both outcrossing and selfing. However to what extent the fungus outcrosses in nature is unknown. The likelihood of outcrossing was assessed for the secondary homothallic ascomycete Podospora anserina. We examined the extent of vegetative en sexual compatibility between wild type strains. The number of vegetative compatibility groups (VCG's) in the population was estimated based on the incompatibility reactions between isolates in our survey using accumulation curve extrapolation and the non-parametric Chao1 formula. The estimated number of VCG's compared to the maximum number of VCG's based on the currently known vegetative incompatibility genes suggest regular outcrossing in this fungus. Also the difference in sexual compatibility reactions of mating types of the same isolate assumes outcrossing takes place. Options for outcrossing in P. anserina were experimentally verified . Both single mating type monokaryotic and dikaryotic double mating type mycelial cultures proved capable of outcrossing, showing no preference for either genotype. This indicates that fertilization by selfing and outcrossing uses the same pathway. Outcrossing percentages between 1-5 percent were found in unmanipulated natural situations of ascospores on dung. The number of monokaryotic ascospores found in Spore-killer ( Psk ) strains was significantly higher than in other isolates, showing an enhancement to outcrossing in these strains.

    The findings in this thesis contribute to the understanding of the population dynamics and evolution of two types of selfish elements, linear plasmids and segregation distorters, in the ascomycete fungus Podospora anserina. Furthermore it increases the knowledge on genomic conflicts caused by these types of selfish elements in general
    Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans
    Vijn, I. ; Govers, F. - \ 2003
    Molecular Plant Pathology 4 (2003)6. - ISSN 1464-6722 - p. 459 - 467.
    parasitica var.-nicotianae - fluorescent protein gfp - genetic-transformation - filamentous fungi - expression - resistance - palmivora - plasmids - reporter - system
    Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blight. Binary T-DNA vectors containing neomycin phosphotransferase (npt) and beta-glucuronidase (gus) fused to oomycete transcriptional regulatory sequences were constructed. Seven days of co-cultivation followed by transfer to a selective medium containing cefotaxim to kill Agrobacterium and geneticin to select for transformants, resulted in geneticin resistant colonies. Under optimal conditions with Agrobacterium supplemented with a ternary plasmid carrying a constitutive virG gene and in the presence of acetosyringone as inducer, up to 30 transformants per 10(7) zoospores could be obtained. The majority of these transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. Using a similar protocol, geneticin resistant transformants of two other oomycetes species were obtained, Phytophthora palmivora and Pythium ultimum.
    Spore killing in the fungus Podospora anserina: a possible connection between meiotic drive and vegetative incompatibility
    Gaag, M. van der; Debets, A.J.M. ; Hoekstra, R.F. - \ 2003
    Genetica 117 (2003)1. - ISSN 0016-6707 - p. 59 - 65.
    heterokaryon incompatibility - natural-populations - neurospora - plasmids - dynamics
    Fungi in which the haploid nuclei resulting from meiosis are linearly arranged in asci provide unique opportunities to analyse abnormal segregation. Any meiotic drive system in such fungi will be observed in a cross between a driving and a sensitive strain as spore killing: the degeneration of half the ascospores in a certain proportion of the asci. In a sample of some 100 strains isolated from a single natural population we have discovered at least six different meiotic drive elements (van der Gaag et al., 2000). Here we report results of research that was aimed at elucidating a possible correlation between meiotic drive and vegetative incompatibility in eight different Spore killer strains from this population. We show that there is a strong correlation between these two phenotypes, although the precise genetic nature of the correlation is not yet clear. We discuss the implications of our results for the understanding of the population genetics of meiotic drive in Podospora
    Molecular evolution of aphids and their primary ( Buchnera sp.) and secondary endosymbionts: implications for the role of symbiosis in insect evolution.
    Sabater-Munoz, B. ; Ham, R.C.H.J. van; Martinez-Torres, D. ; Silva, F.J. ; Latorre, A. ; Moya, A. - \ 2001
    Interciencia 26 (2001)10. - ISSN 0378-1844 - p. 508 - 12.
    acyrthosiphon-pisum - leucine biosynthesis - bacteria - aphidicola - genes - plasmids - microorganisms - homoptera - sequence - system
    Aphids maintain an obligate, endosymbiotic association with Buchnera sp., a bacterium closely related to Escherichia coli. Bacteria are housed in specialized cells of organ-like structures called bacteriomes in the hemocoel of the aphid and are maternally transmitted. Phylogenetic studies have shown that the association had a single origin, dated about 200-250 million years ago, and that host and endosymbiont lineages have evolved in parallel since then. However, the pattern of deepest branching within the aphid family remains unsolved, which thereby hampers tin appraisal of, for example, the role played by horizontal gene transfer in the early evolution of Buchnera. The main role of Buchnera in this association is the biosynthesis and provisioning of essential amino acids to its aphid host. Physiological and metabolic studies have recently substantiated such nutritional role. In addition, genetic studies of Buchnera from several aphids have shown additional modifications, such as strong genome reduction, high A+T content compared to free-living bacteria, differential evolutionary rates, a relative increase in the number of non-synonymous substitutions, and gene amplification mediated by plasmids. Symbiosis is an active process in insect evolution cis revealed by the intermediate values of the previous characteristics showed by secondary symbionts compared to free-living bacteria and Buchnera.
    The transmission of cytoplasmic genes in Aspergillus nidulans
    Coenen, A. - \ 1997
    Agricultural University. Promotor(en): R.F. Hoekstra. - S.l. : Coenen - ISBN 9789054856542 - 106
    aspergillus - cytoplasmatische overerving - plasmiden - genen - genomen - eiwitsynthese - cytoplasma - aspergillus - cytoplasmic inheritance - plasmids - genes - genomes - protein synthesis - cytoplasm


    This manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces via sexual ascospores and asexual conidiospores. Cytoplasmic genes are genes that are located in the cell cytoplasm and not in the cell nucleus where most genes are situated. The cytoplasmic genes investigated in this research are the genomes of mitochondria and viruses. Selfish genes are genes that are maintained in a population despite a negative effect on the fitness of their host.

    A possibility for the spread of selfish genes is created when they can compensate their negative effect on host fitness with an enhanced transmission rate. Because cytoplasmic genes can be transmitted independently from nuclear genes they can enhance their transmission rate in ways that nuclear genes cannot. Therefore there is evolutionary selection on mechanisms that regulate the transmission of cytoplasmic genes, thus preventing the spread of selfish cytoplasmic genes. Heterokaryon incompatibility may be such a mechanism. I have investigated the genetics of heterokaryon incompatibility and the transmission of mitochondria and viruses in A. nidulans. The implications of the results for the spread of selfish cytoplasmic genes are discussed.

    Genetics of heterokaryon incompatibility

    Contact between two fungal mycelia can result in their fusion and the formation of a heterokaryon (a mycelium containing nuclei from both strains). Such mycelial fusion creates possibilities for the horizontal transmission of cytoplasmic genes (transmission between individuals of the same generation). In most fungal species heterokaryon formation is restricted by a heterokaryon incompatibility mechanism. In A.nidulans heterokaryon incompatibility is regulated by heterokaryon incompatibility genes (het genes). Strains with different alleles for one or more het genes cannot form a heterokaryon, they belong to different heterokaryon compatibility groups (hcg's).
    Strains with the same alleles for all het genes can form a heterokaryon, they belong to the same hcg.

    A sample of 24 isolates collected in England and Wales in 1992 was found to contain 20 hcg's. Only 2 of these hcg's were present in the 20 previously described hcg's (Chapter 6). Due to this large amount of variation most pairwise strain combinations will be heterokaryon incompatible.

    Genetic analysis revealed the existence of partial heterokaryons, heterokaryons that grow less vigorously than 'normal' heterokaryons (Chapter 2). At first it was thought that partial heterokaryon incompatibility was caused by partial-het genes. Later it was discovered that partial heterokaryon incompatibility between parent and progeny could be induced by sexual reproduction (Chapter 3).

    I attempted to isolate strains in which heterokaryon incompatibility as a result of an allelic difference for het gene A or het gene B was suppressed (Chapter 3). 1 was successful for het gene B but not for het gene A. This may be because het gene A is an essential gene. All suppressor mutations were intragenic. Suppression of het gene B results in heterokaryon compatibility with both alleles of het gene B. Strains that had switched from compatibility with allele B' of het gene B to compatibility with allele B of het gene B were also isolated. This indicates that the two alleles are highly similar.

    Mitochondrial transmission

    Biparental inheritance creates possibilities for the spread of selfish mitochondrial genes. If mitochondria are inherited biparentally the progeny contain the mitochondria from both parents. Selfish mitochondria can enhance their transmission rate by winning the ensuing competition between the maternal and the paternal mitochondria. In A. nidulans mitochondrial inheritance is strictly uniparental (Chapter 5). All of the +-* 10000 ascospores in a fruitbody are inherited from the maternal parent. An investigation of more than a hundred fruitbodies did not reveal the presence of a single paternal mitochondrion. Selfish mitochondria cannot spread through A.nidulans populations by biparental inheritance.

    The horizontal transmission of mitochondria is greatly restricted. Horizontal transmission within a kg was observed in a very low frequency and horizontal transmission between hcg's was never observed (Chapter 6). Selfish mitochondria cannot spread through A. nidulans populations by horizontal transmission.

    The combination of uniparental inheritance and horizontal transmission within a kg creates a possibility for the spread of selfish mitochondria. The recombination of het genes during sexual reproduction results in the presence of the maternal mitochondria in all the hcg's included in the progeny. Consequently a selfish mitochondrion can spread by transmission within a hcg. However due to the low transmission rate within a hcg and the presumed rarity of sexual reproduction under natural conditions the spread of selfish mitochondria will be severely restricted. It is doubtful whether such a small enhancement of the mitochondrial transmission rate will compensate for the negative effects of selfish mitochondria, on host fitness.

    Virus Transmission

    There are no reports of viruses being found in sexual aspergilli despite their ubiquity in asexual aspergilli. For this research we transferred viruses from the asexual A..niger to the sexual A..nidulans by protoplast fusion (Chapter 4). Virus infection was not observed to have any effect on the fitness of Infected fungi. This shows that the absence of viruses from A..nidulans isolates is not the result of resistance to virus infection.

    The horizontal transmission of viruses within a hcg is highly efficient. The transmission between hcg's is restricted but not prevented by heterokaryon incompatibility. Heterokaryon incompatibility in itself will not prevent the spread of viruses through A. nidulans populations.

    Viruses are excluded from ascospores (Chapter 4). This will not prevent the spread of viruses through A. nidulans populations because viruses are included in the conidiospores. However the exclusion of viruses from ascospores and the recombination of het genes during sexual reproduction does allow the formation of new virus-free hcg's. If virus-free hcg's are created faster than they are infected by horizontal transmission this will result in the exclusion of viruses from A..nidulans populations. This could explain the exclusion of viruses from populations of sexual aspergilli.

    Mobile genetic elements in Methanobacterium thermoformicicum
    Noelling, J. - \ 1993
    Agricultural University. Promotor(en): W.M. de Vos. - S.l. : Noelling - ISBN 9789054850755 - 122
    methanobacteriaceae - cytoplasmatische overerving - plasmiden - moleculaire genetica - methanobacteriaceae - cytoplasmic inheritance - plasmids - molecular genetics

    The identification of the Archaea as a third primary lineage of life and their adaptation to extreme environmental conditions have generated considerable interest in the molecular biology of these organisms. Most progress in the investigation of archaeal mobile genetic elements, i.e. viruses, plasmids and insertion sequences, has been made in the halophilic branch while only limited knowledge was available about mobile elements of methanogens (reviewed in Chapter 1). The aim of this thesis was therefore to get more insight into the molecular genetics of mobile elements from methanogens. Thermophilic species of the genus Methanobacterium were used as model organisms in this study, since they are among the best characterized methanogens.

    Phylogenetic studies of the species M.thermoformicicum and strains of M.thermoautotrophicum are described in Chapter 2. The comparison of two variable 16S rRNA regions allowed the conclusions that (i) M.thermoformicicum consists of two groups of strains, the Z-245-group (including strains Z-245, FTF, THF, FF1, FF3 and CSM3) and the CB 12-group (consisting of the strains CB12, SF-4, and HN4), which probably constitute different species, (ii) M.thermoautotrophicum Δ H is closely related to the Z-245-group, and (iii) Mthermoautotrophicum Marburg belongs to neither group and most likely represents a different species (Table 1). This classification of M.thermoformicicum and M.thermoautotrophicum strains obtained by comparative 16S rRNA analysis is in line with recently reported results derived from DNA-DNA hybridization studies. Both approaches are based on genotypic characters which generally are more reliable for the determination of phylogenetic relationships than classical phenotypic ones. Therefore, the proposed reclassification of the examined thermophilic Methanobacterium strains may reflect their phylogeny more accurately than the current classification. Moreover, some other characters of the examined methanogens including genetic fingerprints with fdh A (Chapter 2 and 4), the sensitivity to phage ΦF1 (Chapter 3) and the distribution of FR-I (Chapter 6) provide additional evidence for the proposed new taxonomy of M. thermoformicicum and M. thermoautotrophicum (Table 1). Another phylogenetically relevant aspect reported in Chapter 2 was the finding that the non-formate utilizers M.thermoautotrophicum Δ H and Marburg contain sequences similar to the fdh A and fdh B genes from M. formicicum The presence off dhAB-like sequences in strain ΔH and Marburg suggest that both M.thermoformicicum and M.thermoautotrophicum are descendants of a common formate-utilizing ancestor.

    It has recently been shown that Mthermoautotrophicum Δ H and Marburg differ clearly in their genome size (1.725 versus 1.623 kb, respectively) and the position of restriction sites in the chromosomal DNA for the endonucleases Not I, Pme I and Nhe I. These findings are likely to reflect the limited homology of both M. thermoautotrophicum strains on the genomic level. Similarly, also strain ΔH and M.thermoformicicum THF have been found to differ considerably in genome size (1.725 versus 1. 600 kb, respectively) and Not I, restriction pattern. This would contradict the results obtained by DNA-DNA similarity studies and comparative 16S rRNA sequence analysis which indicate a close relationship of these two strains (Chapter 2). However, the results of the genomic analysis of strain THF have to be interpreted with caution for two reasons. Firstly, the Not I-site (5'-GCGGCCGC) comprises the target site of the GGCC-recognizing restriction-modification system Mth TI harbored by strain THF (see Chapter 7). As the consequence, the methyltransferase of the Mth TI system probably modifies the Not I-sites contained in DNA from strain THF to yield 5'GCGG meCCGC which is resistant against cleavage by Not I . The reported two Not I, restriction fragments of 1.350 and 250 kb generated from THF DNA (7) may therefore not reflect the real number of Not I-sites in the chromosome of strain THF. Secondly, the enormous size of the large Not I, fragment probably does not allow an accurate determination of its size.

    Chapter 3 describes the characterization of the novel archaeal phages ΦF1 and ΦF3 that are able to infect several thermophilic strains of Methanobacterium (Table 1). Both phages differ with respect to their host range and the topology of their double-stranded DNA genomes. While phage ΦF l has a broad host range and contains a linear, approximately 85-kb genome, ΦF3 was specific for M.thermoformicicum FF3 and contained a circular, approximately 36-kb genome. No similarity was found between the genomes of ΦF1 and ΦF3 nor between both phages and genomic DNA from different Methanobacterium strains or from phage ΨM1 of M. thermoautotrophicum Marburg.

    The isolation and initial molecular analysis of three plasmids, pFV 1 (13.5 kb), pFZ 1 (11.0 kb) and pFZ2 (11.0 kb), harbored by M.thermoformicicum strains THF, Z-245 and FTF, respectively, is reported in Chapter 4. Using cloned pFZ1 DNA as hybridization probe, the plasmids were found to constitute a family of highly related elements. Whereas pFZ1 and pFZ2 are probably identical, only partial similarity was observed between pFZ1 and pFV1. Furthermore, genetic fingerprinting experiments using fdh A from M.formicicum as hybridization probe allowed a subdivision of the species M.thermoformicicum into the CB12-group and the Z-245-group, comprising among others the plasmid-harboring strains Z-245, M and THF (Table 1).

    The relatedness of the plasmid DNA was confirmed by sequence analysis of pFV I and pFZ I (Chapter 5). Comparison of the primary sequence of pFV1 (13513 bp; see also Appendix A) and pFZ I (110 14 bp; see also Appendix B) revealed a modular organization of the plasmid genomes: a backbone structure, conserved on the sequence level and in overall gene order, which is interspersed with plasmid-specific elements. The organization of the M.thermoformicicum plasmids resembles that of prokaryotic chromosomes both of which are subjected to DNA rearrangements, in particular insertions and/or deletions, thereby retaining a basic genome organization. The high sequence similarity and the comparable genetic maps suggest that pFV1 and pFZ1 (and pFZ2) have originated from a common ancestor. If so, the plasmids probably share homologous functions necessary for plasmid replication and maintenance which should be located within regions with high interplasmid similarity, i.e. the plasmid backbone. Those essential functions may include two large palindromic regions and a putative gene encoding a NTP-binding protein which are contained within the conserved backbone structure. In contrast, the plasmid-specific sequence blocks probably represent accessory elements which do not specify essential plasmid functions.

    Both plasmids pFV1 and pFZ1 harbor sequences with similarity to chromosomal DNA of different thermophilic methanogens (Chapter 6). One of those, named FR-I, represents an accessory element of pFV1 with chromosomal counterparts in several M.thermoformicicum strains and M.thermoautotrophicum Δ H (Table 1). Comparison of the plasmid-derived and chromosomal FR-I elements revealed that FR-I has a size of 1.5 kb and exists in variants which differ in the organization of two subfragments. Remarkably, the corresponding subfragments of either FR-I element were identical on the nucleotide sequence level. Although FR-I lacks terminal inverted repeats, the presence of terminal direct repeats and its occurrence in multiple copies suggest that FR-I represents a new type of archaeal insertion sequences. A second element, termed FR-II, is part of both plasmids pFV I and pFZ 1, and the chromosome of M.thermoformicicum THF, CSM3 and HN4. Sequence analysis of the two plasmid- and one chromosome-derived FR-II elements showed that they are highly similar and may code for a protein with yet unknown function. In contrast to FR-I, FR-II is present in a single chromosomal copy and does not contain terminal repeats. Its presence in plasmid- and chromosomal DNA suggests, however, that FR-II is mobile or has been mobilized.

    Each plasmid contains an accessory element encoding components of a type 11 R/M system: the GGCC-recognizing system Mth TI carried by pFV1 from strain THF (Chapter 7) and the CTAG- recognizing systems Mth ZI and Mth FI located on pFZ1 and pFZ2 from strain Z-245 and FTF, respectively (Chapter 8). These findings demonstrated for the first time plasmidencoded enzymatic activities for methanogens. The R/M systems Mth TI and Mth ZI have been characterized in detail by their cloning and expression in Escherichia coli. Strikingly, both R/M modules are located within the same plasmid backbone region suggesting that this part of the plasmids has undergone substantial genomic rearrangements: either deletion or insertion of a single R/M module from an ancestral plasmid that contained both or none of the R/M cassettes. The presence of R/M cassettes would support the concept of a modular evolution, i.e., the mobilization of functional units within the same or between different replicons. Characterization of methanogenic R/M systems with the same specificity as the plasmid-located Mth TI and Mth ZI systems may provide insight in the relationships of archaeal R/M systems and possible mechanisms of dissemination of R/M modules.

    Supporting evidence for a modular evolution is provided by the similarity between the two GGCC-recognizing R/M systems Mth TI from M.thermoformicicum THF and Ngo PII from Neisseria gonorrhoeae (Chapter 7). In contrast to other R/M systems with GGCC specificity, Mkh TI and Ngo PII comprise endonuclease- and methyltransferase genes which both were similar on the nucleotide and the deduced amino acid sequence level and exhibit an identical organization (Figure 1). These findings suggest that the Mth TI and the Ngo PII systems are homologous and have been disseminated via horizontal gene transfer. If so, gene transmission would have occurred between members of the evolutionary different domains Archaea and Bacteria.

    An interesting property of the Mkh TI system is the fact that it is present in a thermophilic organism. In contrast, all other known GGCC-recognizing, or, more general, m 5C-producing RIM systems, have been isolated from mesophilic organisms. The absence of such RIM systems in thermophiles has been attributed to an accelerated deamination rate of m 5C (m 5C _>T) at high temperatures generating G-T mismatches in double-stranded DNA which, if not corrected, would result in point mutations (1) (Figure 2). The thermophilic M.thermoformicicum THF apparently contains a mechanism which either avoids deamination of m 5C or is capable of G-T mismatch correction. The latter possibility seems to be realized in strain THF since the pFV1-located ORF10 has the capacity to code for a protein with significant similarity to E.coli DNA mismatch repair enzymes (Chapter 5; Figure 2). Similar to the organization of functionally related genes into operons, ORF10, mthTIM and mthTIR are located on the same pFV1 module (Figure 2) and may form a functional unit that specifies components of a restriction-modification-repair (RMR) system. Additionally, a putative fourth gene, ORF9, is located on the RMR module (Figure 2). Although no function could be assigned to the deduced ORF9 product, a participation in the activity of the RMR system would be plausible. Besides a possible common regulation, the observed gene clustering may also be necessary to ensure survival of a potential thermophilic recipient which acquires the RMR module via lateral gene transfer. However, if the recipient is a mesophilic organism, the DNA repair functions would probably be dispensable and may be lost after transfer. Assuming that a transfer of the entire RMR module has occurred from the thermophilic M. thermoformicicum THF to the mesophilic N.gonorrhoeae, this may explain why the similarity included the genes of the Mth TI and Ngo PII systems but did not extend beyond adjacent sequence regions.

    The major function of RIM systems is proposed to be the protection of the resident DNA from contamination by for instance phage DNA (Figure 2). The same function may be supposed for the discussed R/M systems from M.thermoformicicum However, one would expect that RIM systems which generate thermostable modification products such as m 4C and m 6A would be more advantageous for a thermophilic host than those that produce m 5C since deamination of the latter methylation product increases the chance on point mutations which have to be compensated by mismatch repair. Nevertheless, the m 5C-generating Mkh TI R/M system obviously proved to be successful under thermophilic conditions since it is maintained by M.thermoformicicum THF. A possible explanation for the presence of the Mth TI system in strain THF may be that the system provides another advantage to its host, in addition to restriction of incoming phage DNA by the Mkh TI endonuclease. As discussed in Chapter 5, deamination of m 5C may serve as additional Protection mechanism against phages that contained m 5C-methylated genomes since, in contrast to the host, these phages are not be able to correct mismatches.

    Analysis of the abundance of the tetranucleotide sequences GGCC and CTAG, the targets of the Mth TI and Mth ZI R/M systems, in DNA of thermophilic Methanobacterium strains revealed some interesting results. The sequence data used for analysis comprised 48.4 kb of chromosomal and plasmid DNA, the majority of which were obtained from the EMBL and GenBank data bases (Table 2). The tetranucleotide GGCC was found 228 times within 48.4 kb, which statistically corresponds with 1 tetranucleotide per 212 bp. This value is almost equal to the expected random- frequency for a tetranucleotide which is 1 per 256 bp. In contrast, only 13 CTAG tetranucleotides were detected, corresponding with 1 per approximately 3700 bp. A similar low abundance of CTAG (1 per 2700 bp) has been reported for the E.coli genome. This finding clearly shows that the recognition sequence of the Mth ZI system, in contrast to that of the Mth TI system, is rare in DNA from thermophilic Methanobacterium strains. However, a considerable different result was obtained from analysis of the plasmid DNA harbored by M. thermoformicicum. Compared to the values observed for the genomic DNA from thermophilic members of Methanobacterium, plasmid pFZ1 exhibits a lower frequency of GGCC (1 site per 610 bp; 22 GGCC-sites per pFZ1 genome) but shows a significantly higher abundance-of CTAG (1 per 670 bp; 20 CTAG-sites per pFZ1 genome) (Table 2).

    While pFZ1 contains an comparable number of GGCC-sites (28 per plasmid genome with a frequency of 1 per 390 bp), the CTAG-frequency is substantially lower in pFZ1 from strain Z-245 (1 per approximately 1800 bp; 6 per pFZ1 genome) (Table 2). These results are interpreted as follows:

    (i) The low abundance of CTAG in the DNA of thermophilic Methanobacterium, strains and E. coli suggests that a similar mechanism is responsible for this bias. It has recently been reported that the underrepresentation of several tetranucleotides, including CTAG, in the genome of E.coli may be the result of selection against target sequences of the vsr gene product, an enzyme involved in G-T to G-C mismatch correction. Usually, those target sequences of the Vsr endonuclease are generated by deamination of m 5C in the sequence C meC(A/T)GG, the product of the dcm MTase. However, the vsr gene product displays a relative relaxed specificity since it also recognizes G-T mismatches in the sequence C T (A/T)G or T (A/T)GG where the underlined T is mismatched with G, and it does not require methylation of the unmutated strand. Consequently, T-G mismatches produced during recombination or DNA replication would, without determination which of both nucleotides is the incorrect one, sequence-specifically be corrected to C-G pairs. The tetranucleotide C T AG is one of the possible target sequences of the Vsr endonuclease and would be 'repaired' to CCAG, resulting in an elimination of CTAG-sites in time. A similar mechanism may be responsible for the low CTAG abundance in DNA of thermophilic members of Methanobacterium.

    (ii) Compared to the genomic DNA of thermophilic Methanobacteriwn strains, plasmid pFZ1 of strain THF showed a five-fold increased number of CTAG tetranucleotides. This significant difference may indicate that selection against CTAG is absent in strain THF. Alternatively, pFZ1 (and the related pFZ1 and pFZ2) does not originate from DNA of a thermophilic Methanobacterium strain. The relatively low GC content of pFZ1 (41.8%) compared to that of strain THF (49.6%) would support the latter possibility.

    (iii) Although pFZ1 of strain Z-245 is highly related to pFZ1 the abundance of the tetranucleotide CTAG is clearly lower in pFZ1 Comparison of the nucleotide sequence of plasmid pFZ1 with the one from pFZ1 showed that in particular CTAG-sites located within putative coding regions were absent in pFZ1 The lower frequency of CTAG-sites in pFZ1 correlates with the presence of the CTAG-recognizing Mth ZI system that is encoded by pFZ1 A possible explanation would be that the MTase M.Mth ZI provides only an incomplete protection against cleavage of the corresponding ENase which would result in selection against CTAG-sites. Another possibility may be that strain Z-245 contains a Vsr-like activity.

    (iv) The presence of the GGCC-recognizing Mth TI system encoded by pFZ1 seems to correlate with the underrepresentation of GGCC-sites in the pFZ1 genome. Comparison of the nucleotide sequences of pFZ1 and pFZ1 revealed that 15 GGCC-sites present in pFZ1 were changed in pFV1. Remarkably, ten of those changed GGCC-sites in pFZ1 displayed the sequence GGTC. The same base substitution would be generated by deamination of a single m 5C nucleotides within the sequence GG meCC followed by replication, i.e. without previous correction of the G-T mismatch. The observed preference for the replacement of GGCC by GGTC in pFZ1 suggests that most of the substitutions have been generated by this mechanism. It therefore seems likely that strain THF has a limited G-T mismatch repair capacity. If so, strain THF should be forced to reduce the number of GGCC-sites available for modification by the m 5C-producing MTase M. Mth TI in order to decrease the number of deamination events.

    Most advances in physiological, biochemical and genetic analysis of Methanobacteriwn have been made on M. thermoautotrophicum strains ΔH and Marburg. The present thesis focused on M.thermoformicicum and reported a genetic analysis of plasmids, phages and insertion sequences identified in this species. Since the well-studied methanogen M.Thermoautotrophicum Δ H turned out to be a non-formate utilizing relative of M.thermoformicicum, the here described mobile genetic elements may be instrumental in developing a cloning vector and a transformation system that would allow genetic engineering of strain ΔH.

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