Influence of grassland management on the abundance of the endophytic fungi Neotyphodium

The endophytic fungus Neotyphodium spec. Glenn, Bacon and Hanlin is common in many temperate grass species. This fungus is known to enhance the resistance of its host to adverse environmental conditions and to promote its growth. In addition, Neotyphodium spec. produces a wide range of alkaloids and can therefore be toxic to livestock. Many studies dealing with this topic were conducted in the intensively managed grasslands of the USA and New Zealand. However, we still lack knowledge about the abundance of this fungus in cultivated grasslands in Europe under different management regimes. This information could contribute to a better understanding of the fungal ecology and help to assess the risk of toxicoses in European grasslands. Hence, the objective of this study was to investigate the impact of grassland management on the incidence of Neotyphodium spec. in different grass species. 1) In the rural district of Northeim (central Germany) the impact of land use intensity and type of grassland use on the incidence of Neotyphodium lolii (Latch, Christensen and Samuels), Glenn, Bacon and Hanlin in perennial ryegrass (Lolium perenne L.) was investigated in 90 managed grasslands. - The mean infection rate of L. perenne with N. lolii was low. - The intensity of land use (extensive, intermediate, intensive) and the type of grassland (pasture, mown pasture, meadow) had no influence on the abundance of the fungus. - Geology significantly influenced the incidence of N. lolii: L. perenne plants collected from sites with limestone bedrock had higher infection rates than plants from other soils. 2) The influence of fertilization, cutting frequency and sward composition on the abundance of Neotyphodium spec. in four grass species was tested in an experimental grassland in the Solling Uplands, central Germany. - Cutting frequency and sward composition did not influence the incidence of Neotyphodium spec. - Fertilization significantly increased the infection rate of the tested grass species compared to non-fertilized plots. 3) It was tested how grazing of cattle, sheep and co-grazing of cattle and sheep under different diversity regimes (high and low sward diversity) influences the abundance of Neotyphodium spec. in L. perenne and Festuca pratensis Huds. (meadow fescue). This investigation was carried out in an experimental pasture in the Solling Uplands, central Germany. - In the high diversity treatment grazing and co-grazing of cattle and sheep had no influence on the abundance of the fungus in both grass species. - In the low diversity treatment the incidence of Neotyphodium unicatum (Gams, Petrini and Schmidt) Glenn, Bacon, Price and Hanlin in F. pratensis was significantly higher in plots co-grazed by cattle and sheep compared to plots grazed by sheep only. In contrast, there was no difference in the abundance of N. lolii in L. perenne between the grazing treatmen

Bio-sensing using toroidal microresonators & theoretical cavity optomechanics

In this thesis we report on two matters, (i) time-resolved single particle bio-sensing using a cavity enhanced refractive index sensor with unmatched sensitivity, and (ii) the theoretical analysis of parametric normal mode splitting in cavity optomechanics, as well as the quantum limit of a displacement transducer that relies on multiple cavity modes. It is the unifying element of these studies that they rely on a high-Q optical cavity transducer and amount to a precision measurement of an optical frequency. In the first part, we describe an experiment where a high-Q toroidal microcavity is used as a refractive index sensor for single particle studies. The resonator supports whispering gallery modes (WGM) that feature an evanescent fraction, probing the environment close to the toroid's surface. When a particle with a refractive index, different from its environment, enters the evanescent field of the WGM, the resonance frequency shifts. Here, we monitor the shift with a frequency resolution of df/f=7.7e-11 at a time resolution of 100µs , which constitutes a x10 improvement of the sensitivity and a x100 improvement in time resolution, compared to the state of the art. This unprecedented sensitivity is the key to real-time resolution of single lipid vesicles with 25nm radius adsorbing onto the surface. Moreover -- for the first time within one distinct measurement -- a record number of up to 200 identifiable events was recorded, which provides the foundation for a meaningful statistical analysis. Strikingly, the large number of recorded events and the high precision revealed a disagreement with the theoretical model for the single particle frequency shift. A correction factor that fully accounts for the polarizability of the particle, and thus corrects the deviation, was introduced and establishes a quantitative understanding of the binding events. Directed towards biological application, we introduce an elegant method to cover the resonator surface with a single lipid bilayer, which creates a universal, biomimetic interface for specific functionalization with lipid bound receptors or membrane proteins. Quantitative binding of streptavidin to biotinylated lipids is demonstrated. Moving beyond the detection limit, we provide evidence that the presence of single IgG proteins (that cannot be resolved individually) manifests in the frequency noise spectrum. The theoretical analysis of the thermo-refractive noise floor yields a fundamental limit of the sensors resolution. The second part of the thesis deals with the theoretical analysis of the coupling between an optical cavity mode and a mechanical mode of much lower frequency. Despite the vastly different resonance frequencies, a regime of strong coupling between the mechanics and the light field can be achieved, which manifests as a hybridization of the modes and as a mode splitting in the spectrum of the quadrature fluctuations. The regime is a precondition for coherent energy exchange between the mechanical oscillator and the light field. Experimental observation of optomechanical mode splitting was reported shortly after publication of our results [cf. Gröblacher et al., Nature 460, 724--727]. Dynamical backaction cooling of the mechanical mode can be achieved, when the optical mode is driven red-detuned from resonance. We use a perturbation and a covariance approach to calculate both, the power dependence of the mechanical occupation number and the influence of excess noise in the optical drive that is used for cooling. The result was one to one applied for data analysis in a seminal article on ground state cooling of a mechanical oscillator [cf. Teufel et al., Nature 475, 359--363]. In addition we investigate a setting, where multiple optical cavity modes are coupled to a single mechanical degree of freedom. Resonant build-up of the motional sidebands amplifies the mechanical displacement signal, such that the standard quantum limit for linear position detection can be reached at significantly lower input power.In dieser Dissertation werden zwei Themen behandelt. Im ersten Teil widmen wir uns experimentell der zeitaufgelösten Messung von Liposomen mit Hilfe eines Nahfeld-Brechungsindex-Sensors. Der zweite Teil handelt von der theoretischen Beschreibung des Regimes der starken Kopplung zwischen einem mechanischen Oszillator und dem Feld eines optischen Resonators. Des Weiteren erörtern wir ein Messschema, das es erlaubt eine mechanische Bewegung, mit Hilfe von mehreren optischen Resonatormoden genauer auszulesen. Die Gemeinsamkeit beider Arbeiten besteht darin, dass es sich jeweils um eine Präzisionsmessung einer optischen Frequenz handelt. Im experimentellen Teil benutzen wir Toroid-Mikroresonatoren mit extrem hoher optischer Güte als Biosensoren. Dabei handelt es sich um eine ringförmige Glasstruktur, entlang welcher Licht im Kreis geleitet wird. Dazu muss eine Resonanzbedingung erfüllt sein, die besagt, dass der (effektive) Umfang des Rings einem ganzzahligen Vielfachen der optischen Wellenlänge entspricht. Ein Teil des zirkulierenden Lichts ist als evaneszente Welle empfänglich für Brechungsindexänderungen nahe der Oberfläche des Resonators. Ein Partikel, dessen Brechungsindex sich von dem der Umgebung unterscheidet, induziert beim Eintritt in das evaneszente Feld eine Frequenzverschiebung der optischen Resonanz. Im Rahmen dieser Arbeit lösen wir relative Frequenzverschiebungen mit einer Genauigkeit von df/f=7.7e-11 und einer Zeitkonstante von 100µs auf. Dies stellt eine Verbesserung des derzeitigen Stands der Technik um einen Faktor x10 in der Frequenz und einen Faktor x100 in der Zeit dar. Diese bisher unerreichte Empfindlichkeit der Messmethode ist der Schlüssel zur Echtzeitdetektion einzelner Lipidvesikel mit einem Radius von 25nm . Zudem gelingt es uns innerhalb einer Messung, bis zu 200 Einzelteilchenereignisse aufzunehmen, welche die Basis für eine aussagekräftige Statistik liefern. Bemerkenswerterweise konnten wir Dank der außerordentlichen Präzision und der Vielzahl der Ereignisse eine Abweichung zur bis dato akzeptierten und angewandten Theorie feststellen. Wir ergänzen das Model um einen Korrekturfaktor, der die Polarisierbarkeit des Teilchens vollständig berücksichtigt und erlangen dadurch ein umfassendes und quantitatives Verständnis der Messergebnisse. Im Hinblick auf biologisch relevante Fragestellungen zeigen wir eine elegante Methode auf, die es erlaubt, den Resonator mit einer einzelnen Lipidmembran zu beschichten. Wir kreieren somit eine biomimetische Schnittstelle, welche das Grundgerüst für eine spezifische Funktionalisierung mit lipidgebundenen Rezeptoren, Antikörpern oder Membranproteinen darstellt. Des Weiteren zeigen wir, dass der Empfindlichkeit eine fundamentale Grenze durch thermische Brechungsindexfluktuationen gesetzt ist. Hierzu wird ein theoretisches Modell speziell für den relevanten niederfrequenten Bereich errechnet. Im zweiten Teil der Arbeit beschäftigen wir uns mit der theoretischen Beschreibung eines optischen Resonators, dessen Lichtfeld an eine mechanische Schwingung gekoppelt ist. Obwohl sich die Resonanzfrequenzen der Optik und der Mechanik typischerweise um mehrere Größenordnungen unterscheiden, existiert ein Regime der starken Kopplung, in dem die Fluktuationen des Lichts und die mechanischen Vibrationen hybridisieren. Dies offenbart sich zum Beispiel im Phasenspektrum, wo sich das ursprüngliche Maximum der Resonanz in zwei Maxima aufspaltet. Die starke Kopplung stellt die Grundlage für kohärenten Energie- und Informationsaustausch zwischen Licht und Mechanik dar und ist daher von besonderem technischen und wissenschaftlichen Interesse. Es ist anzumerken, dass die starke Kopplung und die einhergehende Aufspaltung der Resonanz bereits kurz nach Veröffentlichung unserer theoretischen Beschreibung im Experiment beobachtet wurde [vgl. Gröblacher et al., Nature 460, 724--727]. Wenn der optische Resonator (zur längeren Wellenlänge hin) verstimmt von der Resonanz angeregt wird, kann über dynamische Rückkopplung eine effektive Kühlung der mechanischen Schwingung erreicht werden. Wir berechnen die thermische Besetzungszahl der mechanischen Mode (und somit die Temperatur) mit Hilfe eines störungstheoretischen und eines Kovarianzansatzes. Dabei berücksichtigen wir sowohl ein klassisches Rauschen des optischen Feldes als auch den Einfluss der optomechanischen Kopplung auf die Grenztemperatur. Der hergeleitete Ausdruck für die finale Besetzungszahl wurde eins zu eins für die Datenanalyse in dem wegweisenden Artikel über das Kühlen eines mechanischen Oszillators in den Quantengrundzustand verwendet [vgl. Teufel et al., Nature 475, 359--363]. Abschließend betrachten wir ein Schema, bei dem die Lichtfelder mehrerer optischer Resonanzen an eine mechanischen Schwingung gekoppelt sind. Die resonante Verstärkung der Information über die mechanische Bewegung in den optischen Seitenbändern ermöglicht es, eine durch das Standard Quantenlimit begrenzte Empfindlichkeit bei signifikant niedriger Eingangsleistung zu erreichen

Theoretical analysis of mechanical displacement measurement using a multiple cavity mode transducer

We present an optomechanical displacement transducer, that relies on three cavity modes parametrically coupled to a mechanical oscillator and whose frequency spacing matches the mechanical resonance frequency. The additional resonances allow to reach the standard quantum limit at substantially lower input power (compared to the case of only one resonance), as both, sensitivity and quantum backaction are enhanced. Furthermore, it is shown that in the case of multiple cavity modes, coupling between the modes is induced via reservoir interaction, e.g., enabling quantum backaction noise cancellation. Experimental implementation of the schemes is discussed in both the optical and microwave domain.Comment: 5 pages, 3 figures. Revised and amended versio

The long polar fimbriae (lpf) operon and its flanking regions in bovine Escherichia coli O157:H43 and STEC O136:H12 strains

Long polar fimbriae (Lpf) are intestinal adhesins and important virulence factors of pathogenic Escherichia coli strains. We cloned and sequenced the lpf2-1 operon (lpf2ABCD) and its flanking regions of an intimin- and Shiga toxin-negative E. coli O157:H43 strain from bovine origin, and also sequenced the lpf2-1 operon of 6 additional atypical O157 bovine Escherichia coli strains of various serotypes Nucleotide sequence comparison of these lpf operons showed sequence conservation as they contain only four polymorphic nucleotide positions. Investigation of these O157 strains as well as 13 Escherichia coli Reference Collection (ECOR) strains carrying the lpf2-1 allele revealed high degree of sequence conservation in the lpf2 flanking regions. The lpf2-1 allele is also present in a bovine Shiga toxin-producing E. coli STEC O136:H12 strain and in vitro adherence assays revealed that the absence of lpf2-1 in this strain did not affect its host cell-binding properties. Our data indicate that lpf2 loci is highly conserved in E. coli isolates, but its role in adherence might be masked by other uncharacterized adhesins

Parametric Normal-Mode Splitting in Cavity Optomechanics

Recent experimental progress in cavity optomechanics has allowed cooling of mesoscopic mechanical oscillators via dynamic backaction provided by the parametric coupling to either an optical or an electrical resonator. Here we analyze the occurrence of normal-mode splitting in backaction cooling at high input power. We find that a hybridization of the oscillator's motion with the fluctuations of the driving field occurs and leads to a splitting of the mechanical and optical fluctuation spectra. Moreover, we find that cooling experiences a classical limitation through the cavity lifetime.Comment: 4 pages, 3 figure

Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta

Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands

<p>Abstract</p> <p>Background</p> <p>A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far.</p> <p>Results</p> <p>To study mobilisation of such large genomic regions in prototypic uropathogenic <it>E. coli </it>(UPEC) strain 536, PAI II₅₃₆was supplemented with the <it>mob</it>_RP4region, an origin of replication (<it>oriV</it>_<it>R6K</it>), an origin of transfer (<it>oriT</it>_<it>RP4</it>) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II₅₃₆construct occured from strain 536 into an <it>E. coli </it>K-12 recipient. In transconjugants, PAI II₅₃₆existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the <it>leuX </it>tRNA gene. This locus is the chromosomal integration site of PAI II₅₃₆in UPEC strain 536. From the <it>E. coli </it>K-12 recipient, the chromosomal PAI II₅₃₆construct as well as the CIs could be successfully remobilised and inserted into <it>leuX </it>in a PAI II₅₃₆deletion mutant of <it>E. coli </it>536.</p> <p>Conclusions</p> <p>Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.</p

Evolution in Quantum Leaps: Multiple Combinatorial Transfers of HPI and Other Genetic Modules in Enterobacteriaceae

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains

On-line randomized construction of the upper envelope of triangles and surface patches in R3

In this paper we describe an on-line randomized algorithm for computing the upper envelope (i.e. poinwise maximum) of a set of n triangles in three dimensions. The main new feature of this algorithm is the combination of two layers of influence graphs, which were introduced. We can insert the n-th triangle in 0(log n 7A(n;r=1) F(f o(|r/2|) ; r2)) expected time, where fo(r) is the expected size of an intermediate result for r triangles. Since fo(r) = 0(r2 5 (r))., the expected time for the insertion of the last triangle is bounded in the worst case by 0(r2 5 (r)). This algorithm is easy to implement and works also nicely for surfaces and surface patches of fixed maximum degree