Dissertations / Theses on the topic 'Flagellati'

2012/2013
Heterotrophic nanoflagellate (HNF) grazing is one of the major source of prokaryotic mortality in marine ecosystems, acting as a strong selection pressure on communities. Protozoans may thus affect prokaryotic abundance and alter the diversity and the taxonomic composition of the prey community, as individual prokaryotes can develop distinct grazing-resistant mechanisms. Moreover, the microbial loop is well known to regulate carbon fluxes in surface marine environments but few studies have quantified the impact of HNF predation on prokaryotes in the dark ocean. The present work was aimed to: (1) quantify the impact of HNF predation on the deep prokaryotes biomass; (2) investigate if and how prey diversity varies in response to different predation pressure; (3) define taxonomic community composition in studied areas and identify most affected prokaryotic phylotypes by HNF grazing (4) evaluate the effects of small HNF (<3 µm), which are known to dominate nano-sized compartment and represent the main bacterivores in aquatic ecosystems, being an important link between bacteria and larger protists; (5) evidence differences in community sensitivity to grazing between surface and mesopelagic ecosystems (6) identify the main environmental drivers shaping microbial community diversity. Predation experiments were performed with surface and mesopelagic water samples collected from the Southern Adriatic and Northern Ionian basins. An additional predation experiment was set up in the North-eastern Adriatic Sea. We coupled the traditional ‘dilution method’ with high-throughput molecular analysis (ARISA and Ion Torrent/454 sequencing) to provide a quantitatively and qualitatively evaluation of the grazing process occurring in marine microbial communities. The present work is structured by four manuscripts in preparation and one manuscript already submitted. 1. Heterotrophic nanoflagellate grazing on picoplankton in deep waters (manuscript in preparation) 2. Effects of heterotrophic flagellate predation on bacterial community diversity (manuscript in preparation) 3. HNF grazing impact on taxonomic composition of marine prokaryotic community (manuscript in preparation) 4. Environmental drivers structuring surface and deep bacterial communities in Adriatic and Ionian Seas (manuscript in preparation) 5. Biodiversity changes of bacterial community under predation pressure analyzed by 16S rRNA pyrosequencing (manuscript submitted) My PhD research led to important progresses in the comprehension of microbial dynamics regulating carbon cycles and bacterial diversity in the Adriatic and Ionian basins. Prokaryotic abundance and biomass were one order of magnitude higher in the photic than in the aphotic layers of Southern Adriatic and Ionian Seas (surface biomass 1.68 ± 1.76 µC L-1, deep biomass 9.00 ± 2.11 µC L-1). The Northern Adriatic community presented the highest biomass value (57.46 µC L-1), according to its richer trophic status. All in situ communities displayed the same evenness, being dominated by rare phylotypes. Rare taxa were confirmed to represent the major contributors of microbial communities, with only a few phylotypes dominant. Mesopelagic bacterial communities were as rich and variable as surface assemblages, despite the significant biomass decrease along the water column. Natural archaeal assemblages were characterized by very low richness as we recovered only two genera (Cenarchaeum and Nitrosopumilus), while in situ bacterial communities were composed by the six major marine phyla (Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Deinococcus-Thermus), whose contribution varied according to sampling depth. Flagellates were demonstrated to efficiently control their preys (ingestion rates: 7.86-22.26 µg C L-1 in surface experiments, 0.53-10.61 µg C L-1 in deep experiments), causing important losses in the potentially produced prokaryotic biomass. Despite picoplankton and HNF abundance reduction with depth contrasts with the hypothesis that at least 108 picoplanktonic cells L-1 are necessary to sustain HNF community, our data confirm that also in mesopelagic waters prey and predator concentrations are sufficient to sustain efficient microbial food webs. HNF grazing modified bacterial community diversity in both surface and deep marine systems but with different strength. Mesopelagic communities were more sensitive to grazing impact, evidencing a bell-shaped response to the increasing ingestion rates. Moderate-high top-down control preserved or enhanced bacterial diversity, that fell at low predation. In upper communities grazing did not induce wide variations of bacterial richness and evenness, revealing to be more stable. Small HNF (<3 µm) were the dominant size fraction within flagellate communities and likely constituted the main bacterivores. After the removal of large HNF, a higher fraction of prokaryotic phylotypes was affected. Larger protists partially reduced small flagellate impact on their preys. Larger HNF had a more important role in photic systems compared to mesopelagic waters. The fraction of bacterial taxa favored or affected by predation when small HNF were the only predators more markedly varied in surface experiments, while few phylotypes changes their behavior between the two size treatments in deep experiments. Some taxa were consumed mainly by larger HNF (3-10 µm), while others were grazed by smaller ones (<3 µm). Over 50% of the predated phylotypes belonged to the rare biosphere, mainly in the surface experiments. Rare bacteria are thus not only a dormant ‘seed bank’ but constitute a fundamental component of microbial food webs and actively vector the carbon transfer toward higher trophic levels, being as important as dominant organisms. Although general patterns applicable to all communities were not found, trends of selectivity over different phylotypes were highlighted within sampling layer along the water column and between different systems. While the majority of predator-prey interactions were characteristic to specific environments, some can be considered common to different systems (e.g. Burkholderiaceae and Pseudomonadaceae were exclusively selected in all mesopelagic sites, Bacterivoracaceae were subjected to small HNF predation independently from sampling site or depth). The Southern Adriatic and Ionian basins were significantly distinguished by both the physicochemical water characteristics and the prokaryotes and protists abundance distributions. Cluster analysis based on Jaccard and Bray-Curtis metrics evidenced that depth and geographical location of sampling sites influenced bacterial community similarity. The Southern Adriatic Sea was clearly distinguished from the Ionian Sea. The Northern Adriatic samples were always separated from the others, coherently with different biotic and abiotic characteristics of the sub-basin. Additionally, temperature, chl a and O2 concentration represented important environmental drivers shaping biodiversity of bacterial communities that inhabit Adriatic and Ionian basins. In conclusion, we evidenced that heterotrophic flagellates control bacterial biomass and select certain taxa among all possible preys, grazing also on the rare ones. HNF predation thus shapes bacterial community structures, which in turn influence the ecosystem functioning. Despite the cell abundance decrease of both predators and preys reduces encounter probabilities, the dark ocean hosts complex microbial food webs, structured around three trophic levels (i.e. prokaryotes, small and large heterotrophic flagellates).
I nanoflagellati eterotrofi (HNF) costituiscono una delle principali cause di mortalità dei procarioti in ambiente marino, esercitando una forte selezione sulle comunità predate. Possono modificarne l’abbondanza cellulare e alterarne la diversità e la composizione tassonomica, in quanto le diverse specie procariotiche possono sviluppare distintivi meccanismi di resistenza alla predazione. Mentre l’impatto degli HNF sui procarioti degli acque marine superficiali è ben noto, pochi studi si sono focalizzati sullo studio degli ambienti profondi. Il presenta lavoro di dottorato è stato finalizzato a: (1) quantificare l’impatto della predazione da parte degli HNF sulla biomassa procariotica profonda; (2) capire se e come la biodiversità della comunità predata vari in risposta alla diversa pressione di predazione; (3) definire la composizione tassonomica delle comunità presenti nell’area di studio e identificare i filotipi maggiormente colpiti dalla predazione da parte degli HNF; (4) valutare il contributo dei piccolo flagellati (<3 µm), i quali costituiscono la più abbondante frazione nanoplanctonica e rappresentano i principali organismi batterivori negli ambienti acquatici; (5) evidenziare possibili differenze nella risposta alla predazione tra comunità procariotiche che vivono in acque superficiali e profonde; (6) identificare i principali fattori ambientali che modulano la diversità delle comunità microbiche. Esperimenti di predazione sono stati condotti su campioni di acqua superficiale e mesopelagica raccolti nel Mar Adriatico meridionale e nel Mar Ionio settentrionale. Un ulteriore esperimento è stato condotto nel Mar Adriatico nord-orientale. Il tradizionale metodo delle diluizioni è stato abbinato ad analisi molecolari quali elettroforesi capillare (ARISA) e sequenziamento (Ion Torrent e 454) per consentire una valutazione quali-quantitativa degli effetti della predazione sulle comunità microbiche marine. La presente tesi è costituita da quattro articoli in preparazione e un articolo già sottomesso: 1. Heterotrophic nanoflagellate grazing on picoplankton in deep waters (articolo in preparazione) 2. Effects of heterotrophic flagellate predation on bacterial community diversity (articolo in preparazione) 3. HNF grazing impact on taxonomic composition of marine prokaryotic community (articolo in preparazione) 4. Environmental drivers structuring surface and deep bacterial communities in Adriatic and Ionian Seas (articolo in preparazione) 5. Biodiversity changes of bacterial community under predation pressure analyzed by 16S rRNA pyrosequencing (articolo sottomesso) La ricerca condotta durante il mio dottorato ha portato a interessanti progressi nella comprensione delle dinamiche microbiche che regolano i cicli del carbonio e la diversità batterica nei bacini adriatico e ionico. L’abbondanza e la biomassa delle comunità procariotiche superficiali è risultata un ordine di grandezza superiore rispetto alle comunità profonde in Mar Adriatico meridionale e Mar Ionio (biomassa superficiale 9.00 ± 2.11 µC L-1, biomassa profonda 1.68 ± 1.76 µC L-1). La comunità descritta nel Mar Adriatico settentrionale è caratterizzata dai valori più elevati di biomassa (57.46 µC L-1), coerentemente con l’eutrofia del bacino. I flagellati eterotrofi hanno causando perdite significative nella biomassa procariotica in tutti gli esperimenti condotti, con tassi di ingestione pari a 7.86-22.26 µgC L-1 negli esperimenti superficiali e 0.53-10.61 µgC L-1 negli esperimenti profondi. Un’abbondanza picoplanctonica di 108 cellule L-1 è stata ipotizzata come necessaria per sostenere la comunità degli flagellati. Nonostante l’aumento della profondità comporti una riduzione dell’abbondanza del picoplancton tale da non raggiungere questa soglia, i nostri dati confermano che anche negli ambienti profondi si instaurano interazione preda-predatore sufficienti a sostenere le reti trofiche microbiche. Tutte le comunità in situ hanno mostrato la medesima distribuzione, con prevalenza di filotipi rari e pochi gruppi dominanti. Le comunità mesopelagiche presentano diversità e variabilità analoghe a quelle superficiali, nonostante il decremento in biomassa lungo la colonna d’acqua. Una bassa diversità è stata osservata nelle comunità naturali di Archea, dove sono stati rilevati due soli generi (Cenarchaeum e Nitrosopumilus), mentre le comunità batteriche sono composte dai sei principali phyla marini (Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, Firmicutes e Deinococcus-Thermus), la cui frequenza varia in base alla profondità di campionamento. La predazione esercitata dagli HNF ha modificato la diversità delle comunità sia superficiali che profonde ma con diversi effetti. Le comunità profonde si sono dimostrate più suscettibili alla diversa intensità della predazione. Un controllo top-down medio-alto ha preservato o incrementato la diversità batterica, che invece è risultata fortemente ridotta con bassa pressione di predazione. Al contrario, le comunità superficiali hanno subito solo leggere variazioni nella biodiversità batterica in risposta ai diversi tassi di ingestione, dimostrandosi più stabili. I piccoli flagellati (<3 µm) costituiscono la frazione dominante delle comunità nanoplanctoniche. In seguito alla rimozione dei predatori >3 µm, variazione significative dell’abbondanza sono state riscontrate in una maggiore percentuale di filotipi procariotici. Flagellati di maggiori dimensioni possono quindi mitigare l’impatto dei piccoli predatori sulle prede, con una maggior influenza nei sistemi fotici. Alcuni taxa batterici sono stati consumati prevalentemente dal grandi HNF (3-10 µm), mentre altri sono stati selezionati dai piccoli flagellati (<3 µm). Oltre il 50% dei filotipi predati apparteneva alla biosfera rara, soprattutto negli esperimenti condotti in superficie. I batteri rari (0.1-1% dell’abbondanza totale) non rappresentano quindi una frazione ‘dormiente’ il cui contributo varia in seguito a cambiamenti delle condizioni ambientali, come inizialmente ipotizzato. Costituiscono invece una componente fondamentale delle reti trofiche microbiche e contribuiscono attivamente al trasferimento di carbonio verso i livelli trofici superiori, così come gli organismi dominanti. Nonostante ciascuna comunità risponda in maniera distintiva alla predazione, in funzione della composizione tassonomica delle comunità stesse e dello stato trofico del sistema, alcuni indizi di selettività sono stati individuati. Alcune interazioni preda-predatore si sono rivelate tipiche delle comunità profonde o superficiali, mentre altre erano comuni ad entrambi i sistemi (es. Burkholderiaceae e Pseudomonadaceae sono stati selezionati sono in ambiente pelagico, Bacterivoracaceae sono stati sottoposti a predazione da parte di piccolo flagellati in tutti gli esperimenti, indipendentemente dalla profondità e dal sito di campionamento). I bacini Adriatico meridionale e Ionio settentrionale sono significativamente distinti sia per le caratteristiche chimico-fisiche della colonna d’acqua, sia per l’abbondanza di pico- e nanoplancton. La cluster analisi basata sugli indici di Jaccard e Bray-Curtis ha evidenziato che profondità di campionamento e localizzazione geografica sono i principali fattori che determinano la similarità tra le comunità batteriche. Il Mar Adriatico settentrionale è risultato sempre separato dagli altri campioni, coerentemente con le diverse caratteristiche biotiche e abiotiche del bacino. Oltre a profondità e sito geografico, temperatura, concentrazione di chl a e ossigeno contribuiscono a determinare la biodiversità batterica adriatica e ionica. In conclusione, il presente lavoro ha evidenziato come i flagellati eterotrofi controllino la biomassa procariotica e mostrino preferenza per determinati taxa, selezionando anche quelli rari. La predazione influenza la struttura delle comunità e di conseguenza il funzionamento degli ecosistemi. Anche gli ambienti marini profondi ospitano complesse reti trofiche, strutturate attorno a tre livelli principali (procarioti, piccoli e grandi flagellati eterotrofi) così come le acque superficiali.
XXVI Ciclo
1986

Chromera velia was discovered a decade ago associated with stony corals in the Great Barrier Reef and Sydney Harbour, Australia. This unicellular alveolate possesses housekeeping genes and ultrastructural features typical of apicomplexans, and has active photosynthetic pathways related to dinoflagellates. This unique connection suggests that C. velia can be used to understand the evolution of apicomplexan parasites from their algal ancestors. C. velia exists in either an immotile coccoid state or an active motile flagellated form. The life cycle is not well understood and little is known about what triggers the transformation between states, and how this translates to ecological conditions. With its structural similarity to both apicomplexans and dinoflagellates, the flagellated form of C. velia represents a relatively unexplored part of the life cycle that is critical for understanding the ecological significance of this novel organism. The presence of a flagellated state may enhance competitive fitness of the organism under certain environmental conditions. This thesis investigated the flagellated form of C. velia and explored environmental factors that induce or suppress flagellation. Optimised conditions for flagellation were then used to test the flagellated form as a screening platform to test anti-apicomplexan drugs. In addition, the ecology and biodiversity of C. velia was explored by screening freshly procured coral samples for new chromerid cultures. To explore the flagellated state of C. velia cells were cultured in a range of light, temperature and physical and chemical stress conditions. Light wavelength had a significant effect on flagellation, with different spectra causing the induction or suppression of flagellation. The highest levels of flagellation were seen when C. velia was cultured in blue light or the dark. C. velia exhibited a temporal cycle of motility over 24 hours similar to that of the coral symbiont Symbiodinium, which exhibits motility rhythms in a light-dark environmental cycle. However, in C. velia this pattern continued when the cells were grown in constant dark, suggesting that the cycle is not completely light dependent. High cell density completely suppressed flagellation, and no flagellates were seen in cultures of 106 cells/mL. Non-optimal temperatures and a range of other stressors either killed cells or suppressed flagellation, but did not induce it. A negative correlation was found between light conditions inducing cell multiplication and flagellation, and this has been explored in a proposed new amendment to the life cycle model. Culturing techniques adapted by Moore (2006) were used to screen coral samples for novel chromerids to gain a better understanding of the biodiversity and ecological niche of C. velia. A new strain of C. velia was isolated from L. purpurea present in the Heron Island lagoon, and this additional strain was used in developing a multilocus sequence typing (MLST) system with eight genetic loci that was able to differentiate the three strains of C. velia that are now available. The three strains had considerable diversity at the genetic level, and attempts were made to distinguish them phenotypically by studying growth, flagellation and B vitamin synthesis. The three strains did not require the addition of B vitamins for growth, and possessed a typical algal B7 synthesis pathway, no synthesis or requirement for B12, and an unusual B1 synthesis pathway involving a mosaic of genes with closest homologs of bacterial and fungal origin. New insights on flagellation in C. velia were used to design a flagellation assay to screen compounds for apicomplexan drug development. The Malaria Box, a set of 400 compounds with anti-malarial activity, was provided by the Medicines for Malaria Venture and screened using this assay, finding a number of positive hits. These positive hits were used by structural chemist collaborators at SPECS to synthesise an additional 80 structurally similar compounds that were also screened. A high proportion of compounds found to inhibit flagellation in each assay belong to the diaminopyrimidine class of compounds that are known to affect the folate biosynthesis pathway. A bioinformatic study of C. velia folate synthesis revealed a pathway similar to that of Plasmodium, demonstrating that C. velia has biochemical pathways similar to the parasite and screening may pick up compounds that can be used as starting points for therapeutics. C. velia is closely related, phylogenetically speaking to apicomplexans and the flagellated state is the most structurally similar and also the active form, capable of sensing changes in the environment and moving the cell in response to these changes. To date most studies have only looked at the structure of this form and little has been done to explore its role in the life cycle of C. velia. This study has provided new insights into the ecology and biodiversity of C. velia by identifying triggers for flagellation, producing an amended life cycle model, identifying a new strain of C. velia and designing a set of molecular markers for future population studies. The findings were used to design a flagellation assay able to identify compounds that may be useful starting points for anti-apicomplexan therapeutics.

The propulsion of low Reynolds number swimmers has been widely studied, from the swimming sheet models of Taylor (1951), which were analogous to swimming spermatozoa, to more recent studies by Smith (2010) who coupled the boundary element method and method of regularised Stokeslets to look at cilia and flagella driven flow. While the majority of studies have investigated the propulsion and hydrodynamics of spermatozoa and bacteria, very little has been researched on bi-flagellate green algae. Employing an immersed boundary algorithm and a flexible beat pattern Fauci~(1993) constructed a model of a free-swimming algal cell. However, the two-dimensional representation tended to over-estimate the swimming speed of the cell. Jones~\etal~(1994) developed a three-dimensional model for an idealised bi-flagellate to study the gyrotactic behaviour of bottom-heavy swimmers. However, the un-realistic cell geometry and use of resistive force theory only offered order of magnitude accuracy. In this thesis we, investigate the hydrodynamics of swimming bi-flagellates via the application of the method of regularised Stokeslets, and obtain improved estimates for swimming speed and behaviour. Furthermore, we consider three-dimensional models for bi-flagellate cells with realistic cell geometries and flagellar beats. We investigate the behaviour of force- and torque-free swimmers with bottom-heavy spheroidal bodies and two flagella located at the anterior end of the cell body, which beat in a breast stroke motion. The cells exhibit gravitactic and gyrotactic behaviour, which result in cells swimming upwards on average in an ambient fluid and also towards regions of locally down-welling fluid, respectively. In order to compare how important the intricacies of the flagellar beat are to a cell's swimming dynamics we consider various beat patterns taken from experimental observations of the green alga \Rein~and idealised approaches from the literature. We present models for the bi-flagellate swimmers as mobility problems, which can be solved to obtain estimates for the instantaneous translational and angular velocities of the cell. The mobility problem is formulated by coupling the method of regularised Stokeslets with the conditions that there is no-slip on the surface of the body and flagella of the cell and that there exists a balance between external and fluid forces and torques. The method of regularised Stokeslets is an approach to computing Stokes flow, where the solutions of Stokes equations are desingularised. Furthermore, by modelling the cells as self-propelled spheroids we outline an approach to estimate the mean effective behaviour of cells in shear flows. We first investigate bi-flagellate swimming in a quiescent fluid to obtain estimates for the mean swimming speed of cells, and demonstrate that results for the three-dimensional model are consistent with estimates obtained from experimental observations. Moreover, we explore the various mechanisms that cells may use to re-orientate and conclude that gyrotactic and gravitactic re-orientation is due to a combination of shape and mass asymmetry, with each being equally important and complimentary. Next, we compare the flow fields generated by our simulations with some recent experimental observations of the velocity fields generated by free-swimming \rein, highlighting that simulations capture the same characteristics of the flow found in the experimental work. We also present our own experiments for \rein~and \Dunny~detailing the trajectories and instantaneous swimming speeds for free-swimming cells, and flow fields for trapped cells. Furthermore, we construct flagellar beats based upon experimental observations of \dunny~and \textit{D. bioculata}, which have different body shapes and flagellar beats than \chlamy. We then compare the estimates for swimming speed and re-orientation time with \rein, highlighting that, in general, \Dun~achieve greater swimming speeds, but take longer to re-orientate. The behaviour of cells in a shear flow is also investigated showing that for sufficiently large shear, vorticity dominates and cells simply tumble. Moreover, we obtain estimates for the effective cell eccentricity, which, contrary to previous hypotheses, shows that cells with realistic beat patterns swim as self-propelled spheres rather than self-propelled spheroids. We also present a technique for computing the effective eccentricity that reduces computational time and storage costs, as well as being applicable to unordered image data. Finally, we examine what effects interactions with boundaries, other cells, and obstacles have on a free-swimming cell. Here, we find that there are various factors which affect a cell's swimming speed, orientation and trajectory. The most important aspect is the distance between the interacting objects, but initial orientation and the flagella beat are also important. Free-swimming cells in an unbounded fluid typically behave as force-dipoles in the far field, and we find that for cell-to-cell and cell-to-obstacle interactions the far field behaviour is similar. However, swimming in the proximity of a boundary results in the flow field decaying faster. This implies that hydrodynamic interactions close to solid no-slip surfaces will be weaker than in an infinite fluid.

In der vorliegenden Arbeit wurden 212 Kotproben von Chamäleons auf Parasitenstadien und 75 Tierkörper pathologisch sowie bei einem nachgewiesenen Parasitenbefall histopathologisch untersucht. Ziel war es, anhand dieser Untersuchungen das Vorkommen und die Schadwirkungen von Endoparasiten unter Berücksichtigung der Herkunft, des Alters, des Geschlechts und der Chamäleonart zu beschreiben. Von 212 Kotproben wiesen 55,2% Endoparasitenstadien auf. Bei 54,7% der 64 sezierten und auswertbaren Tiere wurden Endoparasiten nachgewiesen. Der Anteil positiver Proben zeigt zwischen Nachzuchten (55,5%) und Wildfängen (54,1%) keinen wesentlichen Unterschied. In Wildfängen konnten häufiger Endoparasiten mit einem indirekten Lebenszyklus ermittelt werden, Nachzuchten beherbergten dagegen öfter Parasiten mit einem direkten Entwicklungszyklus. In den untersuchten Chamäleons konnten regelmäßig Kokzidien der Gattung Isospora und Oxyuriden nachgewiesen werden. Zestoden konnten in der koproskopischen Untersuchung gar nicht aufgefunden werden, wohingegen sie in der pathologischen Untersuchung sporadisch im Darm diagnostiziert werden konnten. Die Häufigkeit des koproskopischen Nachweises von Parasitenstadien bezogen auf die Gesamtzahl der untersuchten Chamäleons stellte sich wie folgt dar: in 30,4% wurden Protozoon gefunden, 21,7% der Tiere waren mit Kokzidien infiziert (davon 78,3% Isospora spp., 13,0% Choleoeimeria spp., 6,5% Eimeria spp., 2,2% Mischinfektion Isospora spp./Choleoeimeria spp.) und 8,5% mit Flagellaten oder Ziliaten. Bei 83,3% der Tiere mit gastrointestinalen Symptomen konnte ein Befall mit Kokzidien der Gattung Isospora nachgewiesen werden. In 38,7% der koproskopischen Untersuchungen konnten Nematoden (65,9% Oxyuriden, 19,5% Askariden/ Heterakiden, 1,4% Rhabdias sp., 2,8% Strongyloides sp., je 0,5 % Spirurida, Heterakiden/Filarien, Oxyuriden/Strongyloiden) und in 2,8% Trematoden (Digenea) aufgefunden werden. Anamnestisch konnten in 35,8% aller Tiere klinische Symptome beobachtet werden, wovon bei 88,2% der erkrankten Tiere ein Endoparasitenbefall nachzuweisen war. Insgesamt 64,1% der sezierten Chamäleons waren mit Endoparasiten befallen, wovon 68,3% Mono- und 31,7% Mischinfektionen beherbergten. In 31,3% der sezierten Chamäleons wurden Nematoden gefunden und der Befall wurde in 55,0% dieser Fälle als hochgradig eingestuft. Es wurden Befallsraten von 25,0% für Strongyloides sp., 23,4% für Askariden/ Heterakiden,15,0% für Filarien, 5,0% für Rhabdias sp., 9,4% für Zestoden, 10,9% für Digenea registriert. In 11,3% der Fälle lagen Mischinfektionen vor. Damit ist ein Endoparasitenbefall bei Chamäleons häufig und kann zu Erkrankungen führen. Die Exposition unterscheidet sich bei Wildfängen und Nachzuchten aufgrund der unterschiedlichen Umgebungsbedingungen. Auch klinisch unauffällige Tiere waren zu 27,8% mit Parasiten befallen, so dass eine klinische Symptomatik nicht zwingend aus einem Parasitenbefall resultiert. Insgesamt betrachtet verdient der Endoparasitenbefall von Chamäleons das Augenmerk von Tierärzten und Tierhaltern und sollte bei augenscheinlich hohem Infektionsdruck zu Gegenmaßnahmen, insbesondere auch einer verbesserten Hygiene, Anlaß geben
In the present study 212 chameleon fecal samples were examined for parasite stages and 75 carcasses were examined histopathologically and pathologically in a proven case of a parasite infestation. The basis of this study was to describe the occurrence and harmful effects of internal parasites considering the origin, age and sex of the chameleons. Of the 212 fecal samples 55.2% showed stages of endoparasites. Parasites were detected at 54.7% of 64 evaluated and dissected animals. The proportion of positive samples shows no significant difference between offspring (55.5%) and wild specimens (54.1%). In wild specimens common internal parasites could be determined with an indirect life cycle, however offspring harbored more parasites with a direct life cycle. In the studied chameleons coccidia as the genus Isospora and Oxyurids were regularly detected. In the coprological study Cestodes could not be found, while in the pathological examination they could be diagnosed sporadically in the intestine. Based on the total number of investigated chameleons the frequency of detection of parasite stages are presented as follows: Protozoa were found in 30.4%, 21.7% of the animals were infected with coccidia (of which 78,3% Isospora spp, 13,0% Choleoeimeria spp., 6.5% Eimeria spp., 2.2% polyinfections between Isospora spp./ Choleoeimeria spp.) and 8.5% with flagellates or ciliates. At 83.3% of the animals with gastrointestinal symptoms coccidia of the genus Isospora were detected. In 38.7% of the fecal examination nematodes were determined (65.9% Oxyurids, 19.5% Ascarids/ Heterakis, 1.4% Rhabdias sp., 2.8% Strongyloides sp., 0.5% Spirurida, Heterakids/ Filariae, Oxyurids/ Strongyloides sp.) and Trematodes in 2.8% (Digenea) were found. The anamnesis showed that clinical symptoms could be observed in 35.8% of all of the animals, whereas endoparasite infestation could be detected inn 88.2% of the affected animals. Overall, 64.1% of the dissected chameleons were infested with parasites, of which 68.3% harbored mono- and 31.7% polyinfections. In 31.3% of the dissected chameleons nematode infestations were found and 55.0% of these cases were classified as severe. Prevalences were registered: 25.0% for Strongyloides spp., 23.4% for Ascarids/ Heterakids, 15.0% for Filaria, 5.0% for Rhabdias sp., 9.4% for Cestodes, 10.9% for Digenea. In 11.3% of the cases mixed infections were reported. Thus, endoparasite infestation is common among chameleons and can lead to diseases. Exposure differs from wild-specimens and captive-bred due to the different environmental conditions. Also, 27.8% of clinically healthy animals were also infested with parasites, which means that clinical symptoms are not necessarily the result of a parasitic infestation. Overall, chameleon endoparasites deserve the attention of veterinarians and pet owners and should be treated promptly when there is a high likelihood of infection or hygiene is of concern

Bacterial flagellar filaments are long cell surface appendages that generate propulsion for movement. They also play key roles in surface attachment and host-bacterial interactions. The filament is made from a single protein species flagellin. Approximately 45 % of annotated flagellar systems possess multiple flagellin genes. We have investigated the ability of Caulobacter crescentus to build a flagellar filament using six flagellins: FljJ, FljK, FljL, FljM, FljN and FljO. Our analysis showed that this flagellar system exhibits extensive structural redundancy, in that one species of flagellin is sufficient to sustain motility. However, when that flagellin is FljJ cells are non motile. Distinct flagellar assembly checkpoints are utilised by bacteria in order to coordinate and couple flagellar gene expression to the assembly pathway. One of these checkpoints, hook-basal-body completion, is sensed and overcome by the secretion of a flagellar-associated secretion substrate. This mechanism results in a system switch to the export of proteins, which are needed for filament assembly. In C. crescentus the post-transcriptional regulators FlbT and FlaF have been implicated to function at this switch. As the flagellins themselves are being secreted we asked the logical question: are the flagellins involved in subunit feedback control of the regulation of filament assembly? In a bacterial two-hybrid assay, FljJ was the only flagellin able to interact with FlbT and FlaF. Furthermore, FljJ along with FlbT was found to interact with flagellin mRNA. In light of our data we a propose model for the regulation of flagellar filament assembly in C. crescentus and discuss the implications with respect to other flagellar systems

This project investigated the Potential for using the bacterial flagellar filament as a functionalized protein nanotube. A varied approach to flagellin engineering was taken using the flagella of E. coli and R. sphaeroides, with engineered flagellar flilaments from E. coli proving successful. A variety ofepitopes were inserted into the E. coli flagellin, resulting in filaments which displayed adhesive peptides or cleaveable sites. His-tagged filaments allowed the binding of metal ions and nanoparticles, and the biotin-avidin system was empl~yed to allow more specific binding of ligands. Cys-tagged filaments were 'made to order' for a collaborator to allow improved binding of gold to flagellar filaments. The TEV protease recognition site was used to allow specific cleavage of the surface displayed portion of the flagellin within the intact filament structure. Other proteases allowed further digestion ofthis surface displayed region. By combining the adhesive epitope with flanking TEV protease sites selective excision of the tag was possible, allowing the potential for directed whole filament removal from a surface or scaffold in applications such as nanolithography. An in trans expression system was developed to allow assembly of different flagellins into the same filament, with expression levels varied to result in specific ratios of each functionalised monomer. This allowed the creation of bi-functional protein nanotubes, which could bind more than one ligand.

Flagellin was demonstrated to initiate signalling responses in intestinal epithelial cells resulting in the induction of interleukin-8 (IL-8) and human beta defensin-2 (hBD-2) genes.  Intestinal epithelial cell responses to flagellin isolated from both these organisms and signalling responses to whole bacteria and their mutants was examined. Studies with S. enteritidis mutants confirmed the importance of flagellin in signalling responses in Caco-2 intestinal epithelial cells.  Flagellin was a requirement for the nuclear locations of the transcription factor NF-κB.  IL-8 and hBD-2 gene transcription in response to S. enteritidis was found to be dependent on bacterial cell density.  At high densities there was a down-regulation in the induction of these genes.  This down-regulatory signal was isolated in bacterial cell-free conditioned media.  Experiments with R. intestinalis in a novel anaerobic/aerobic co-culture system also suggested that this organism may regulate aspects of the intestinal epithelial cell response to flagellin.  Proteomic methods were employed to characterise proteins in bacterial cell-free conditioned media produced by S. enteritidis, and also to examine density-dependent changes in the S. enteritidis proteome.  These techniques were also employed to investigate epithelial cell-induced changes in the proteome of flagellate commensal Roseburia hominis. In summary this study confirmed previous published evidence that flagellin is important in signalling responses in Caco-2 cells challenged with S. enteritidis. Novel experiments with commensal R. intestinalis derived flagellin (fliC gene sequenced in this project) showed that it also induced the transcription of IL-8 and hBD-2 genes.

The "microbial loop" of aquatic systems consists of picoplankton (heterotrophic and photosynthetic), nanoflagellates (heterotrophic and mixotrophic forms), and ciliates. Heterotrophic flagellates (HNAN) are considered to be the principal grazers of both photosynthetic picoplankton (PICO) and heterotrophic bacteria (HBAC). The factors controlling PICO and HBAC abundances have been studied extensively. However, the factors which regulate HNAN remain less clear. An experimental study was conducted to examine the effects of nutrients and planktivorous fish additions on HNAN in freshwater systems using large enclosures (8m in diameter) installed at both 4 m and 11 m depths in a small oligotrophic lake. In addition to obligate heterotrophs, mixotrophic flagellates (MIXO) were also examined as were their prey, HBAC and PICO. Experimental results did not demonstrate that increases in nutrient levels would lead to increases in HNAN as current empirical models imply. The relationship between HBAC and HNAN was therefore examined within the range of trophy manipulated in the enclosures. A lake survey was conducted in dimictic lakes in Ontario and Western Quebec. (Abstract shortened by UMI.)

Aquatic microbial food webs are in general size structured. Phytoplankton, which constitute the base of the food web, are grazed by protozoa and mesozooplankton, which in turn are consumed by planktivorous fish. Food web efficiency (FWE) is a measure of how efficiently energy is transported up the food web. FWE is low if the phytoplankton is inedible by the grazers, while FWE is higher if the phytoplankton community is dominated by edible phytoplankton. Recently, the presence of microfungi in aquatic food webs have been suggested to facilitate energy transfer up the food web, via the “mycoloop”. The aim of the study was to set-up a model system of phytoplankton – zooplankton food chains, relevant to the Baltic Sea, and to test FWE in diatom and flagellate-based food webs. Further, I wanted to introduce microfungi in the system and observe their impact on FWE. After many phytoplankton and zooplankton species tests, I decided to perform grazing experiments using one grazer, the ciliate Tetrahymena pyriformis, and two phytoplankton species: a diatom (Skeletonema marinoi) and a flagellate (Rhodomonas baltica). I hypothesized that T. pyriformis would more efficiently feed on flagellates than on diatoms. I performed a grazing experiment where the increase in ciliate abundance was measured, the consumption of the phytoplankton monitored and the FWE estimated. The diatom-based food web led to 14 times higher FWE than the flagellate-based food web. The variation in FWE may be explained by a difference in initial abundances introduced in the experimental treatment, which created unequal grazer:prey ratio between treatments. Further, the swimming behaviour of the flagellate might have reduced the capture efficiency by the ciliate. Microfungi were introduce in an experiment, from a natural seawater sample, but fungal infection was not observed for any of the tested phytoplankton species. Further development is needed to test the effects of microfungi on marine FWE.

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.

Knowledge of the relationship between the torque generated by the bacterial flagellar motor of Escherichia coli and its rotation rate provides a valuable insight into the mechanism of the motor's function. A method was devised to measure this characteristic in the high-speed, low-load regime, so that the effect of stator number could be investigated. This i~volved the electrorotation of a polarisable particle, attached to the bacterial filament, as a handle to rotate the motor. I designed and built an upright laser position detector and fabricated an electrode flow cell and optically asymmetric, polarisable particles. The particles allow a high resolution to be achieved, so that stator numbers can be distinguished. The flow cell enables positioning of the bacterium with respect to the electrodes and exchange of the medium during the experiment. Using the novel flow cell, previous torque speed measurements for the flagellar motor of E. coli, made by rotating the cell body about its tether point, were confirmed. The method was extended to measure the torque speed curve for the polarisable particle attached to the filament. I was then able to determine a torque speed curve for varying stator numbers. Results indicate that the zero-torque speed decreases for decreasing stator numbers, contrary to previous assumptions.

What is flagellar swimming? Cilia and flagella are whip-like cell appendages that can exhibit regular bending waves. This active process emerges from the non-equilibrium dynamics of molecular motors distributed along the length of cilia and flagella. Eukaryotic cells can possess many cilia and flagella that beat in a coordinated fashion, thus transporting fluids, as in mammalian airways or the ventricular system inside the brain. Many unicellular organisms posses just one or two flagella, rendering them microswimmers that are propelled through fluids by the flagellar beat including sperm cells and the biflagellate green alga Chlamydomonas. Objectives. In this thesis in theoretical biological physics, we seek to understand the nonlinear dynamics of flagellar swimming and synchronization. We investigate the flow fields induced by beating flagella and how in turn external hydrodynamic flows change speed and shape of the flagellar beat. This flagellar load-response is a prerequisite for flagellar synchronization. We want to find the physical principals underlying stable synchronization of the two flagella of Chlamydomonas cells. Results. First, we employed realistic hydrodynamic simulations of flagellar swimming based on experimentally measured beat patterns. For this, we developed analysis tools to extract flagellar shapes from high-speed videoscopy data. Flow-signatures of flagellated swimmers are analysed and their effect on a neighboring swimmer is compared to the effect of active noise of the flagellar beat. We were able to estimate a chemomechanical energy efficiency of the flagellar beat and determine its waveform compliance by comparing findings from experiments, in which a clamped Chlamydomonas is exposed to external flow, to predictions from an effective theory that we designed. These mechanical properties have interesting consequences for the synchronization dynamics of Chlamydomonas, which are revealed by computer simulations. We propose that direct elastic coupling between the two flagella of Chlamydomonas, as suggested by recent experiments, in combination with waveform compliance is crucial to facilitate in-phase synchronization of the two flagella of Chlamydomonas.

Il fitoplancton è costituito da organismi molto importanti per l'ambiente marino e molto diversificati sia dal punto di vista morfologico che fisiologico.Questi organismi sono normalmente soggetti ai cambiamenti stagionali e alle variazioni dell'ambiente dovute sia a fenomeni naturali che all'impatto antropico, sempre più rilevante. Con questa tesi si è voluto approfondire l'effetto di erbicidi comunemente usati dall'uomo in agricoltura su delle microalghe flagellate rappresentative del Mar Adriatico. L'inquinante scelto è la Terbutilazina, sostanza utilizzata per il diserbo del mais e diffusa in tutta l'area padano-venera, come dimostrano i dati dei campionamenti ARPA, che riportano la presenza di Terbutilazina e del suo prodotto di degradazione Desetil-Terbutilazina a concentrazioni superiori al limite fissato sia nelle acque superficiali che in quelle sotterranee. Anche in mare come riportato in letteratura (Carafa et. al 2009)è stato riscontrato a concentrazioni superiori al limite previsto dalla normativa vigente in Italia. In particolare il meccanismo d'azione di questo erbicida interferisce con la fotosintesi, agendo sulle proteine di membrana dei cloroplasti, rimpiazzando il chinone accettore di elettroni QB della proteina D1 del fotosistema II. Più specie di microalghe fatte crescere in colture 'batch' sono state testate a diverse concentrazioni di erbicida in condizione di luce nutrienti costanti. Questi esperimenti sono stati inoltre condotti a due diverse temperature (20 e 25°C) per studiare l'effetto di questo inquinante in correlazione con l'attuale aumento di temperatura in atto nel pianeta. In una prima fase di screening è stato valutato l'effetto della Terbutilazina su 9 specie di flagellate rappresentative dell'Adriatico, tramite misure dell'efficienza fotosintetica. Grazie a questa prima fase sono state individuate le microalghe e le relative concentrazioni di Terbutilazina da utilizzare negli esperimenti. Per gli esperimenti, sono state scelte le due specie algali Gonyaulax spinifera e Prorocentrum minimum sulle quali si è approfondito lo studio dell'effetto dell'inquinante alle due temperature, attraverso una serie di analisi volte ad individuare le risposte in termini di crescita e di fisiologia delle alghe quali: conteggio delle cellule, torbidità, efficienza fotosintetica, consumo di nutrienti, quantità di clorofilla e di polisaccaridi extracellulari prodotti. La scelta di queste microalghe è stata dettata dal fatto che Gonyaulax spinifera si è rivelata la microalga più sensibile a concentrazioni di erbicida più vicine a quelle ambientali fra tutte le alghe valutate, mentre Prorocentrum minimum è fra le dinoflagellate più frequenti e rappresentative del Mar Adriatico (Aubry et al. 2004), anche se non particolarmente sensibile; infatti P. minimum è stato testato ad una concentrazione di erbicida maggiore rispetto a quelle normalmente ritrovate in ambiente marino. Dai risultati riportati nella tesi si è visto come l'erbicida Terbutilazina sia in grado di influenzare la crescita e la fotosintesi di microalghe flagellate dell'Adriatico anche a concentrazioni pari a quelle rilevate in ambiente, e si è evidenziato che gli effetti sono maggiori alle temperature in cui le microalghe hanno una crescita meno efficiente. Questi studi hanno messo in evidenza anche l'utilità delle misure di fotosintesi tramite fluorimetro PAM nel valutare le risposte delle microalghe agli erbicidi triazinici. Inoltre la diversa risposta delle microalghe osservata, potrebbe avere conseguenze rilevanti sulle fioriture estive di fitoplancton in caso di presenza in mare di Terbutilazina, anche a concentrazioni non particolarmente elevate. Questo lavoro si inserisce in un contesto più ampio di ricerca volto alla modellizzazione della crescita algale in presenza di inquinanti ed in concomitanza con le variazioni di temperatura. Infatti i dati ottenuti insieme a misure di carbonio cellulare, non ancora effettuate, saranno utili per la messa a punto di nuove parametrizzazioni che si avvicinino maggiormente ai dati reali, per poi simulare i possibili scenari futuri variando le condizioni ambientali e le concentrazioni dell'inquinante studiato.

This thesis is a study of the evolution and development of the flagellate green algae. The first part is a comparative study of the evolution of body size, multicellularity and segregated soma. The allometry of morphological characters, development, life history and the life cycle are also considered. The second part is an experimental test of the potential role of mutation as a determinant of the course of evolution. Mutation is directional for all the characters studied. The variances and covariances created by mutation are compared to those of 30 species of Volvocaceae; the correspondence between the two depends upon the characted examined. In the third part, the growth of germ cells grown with and without a soma is compared. The response to nutrient concentration of cells grown with an intact soma is steeper than that of cells grown without a soma. This result demonstrates a physiological advantage of soma in Volvox, attributable to a division of labour between 'source' and 'sink'.

Campylobacter jejuni, the cause of acute bacterial enteritis, is a Gram-negative, microaerophilic, spiral bacterium. The single polar flagellum of the organism has an important role inc ausing disease. In the published genome a nnotation of C. j ejuni some of the key regulatory flagellar genes are missing. Therefore the regulatory control of flagellar biogenesis is still not clear in the organism. FliK, the protein that controls the length of the flagellar hook and is also involved in switching of secretory pathway specificity in Salmonella, is among the regulatory proteins missing in the organism. We have identified the missingjliK gene in C. jejuni, which is Cj0041, and the homologue in its close relative Helicobacter pylori, HP0906. Although the protein encoded by Cj0041 is not readily identified by BLAST search against the major databases, a BLAST query of the C. jejuni genome database (Sanger Institute) with the FliK sequence from Aeromonas hydrophila identifies the Cj0041 gene product at E=0.056. Cj0041 knock-out mutants in two strains (NCTC11168 and 81116) 0 f C. jejuni were non-motile and showed a polyhook phenotype, which is in line with that of FliK mutants in Salmonella. SDS PAGE and mass spectrometry analysis with 'flagellar' filament proteins from Cj0041 mutants revealed increased production of FlgE hook protein in mutants compared to wild-type and indicated the synthesis of these polyhooks with mainly FlgE proteins. Microarray analysis of these mutants, verified by real-time PCR, showed overexpression of flagellar and other regulatory genes dependent on cr54 , one of the sigma factors involved in flagellar biogenesis, and provided a clear delineation of the cr54-dependent regulon. These results established the importance of Cj0041 or jliK in regulation of flagellar biogenesis in C. jejuni. We also have established by two-hybrid analysis the roles of Cj 1464 as jlgM and Cj 1465 asjlgN, two other key missing genes in the gene annotation of the organism. The roles of other flagellar genes such asjliS,jliD and Cj0848c were also studied to get a clear picture of flagellar biogenesis in C. jejuni. This study established that the organism shares most of the key regulatory flagellar genes found in model organisms like Salmonella and made it possible to define the regulation of flagellar biogenesis in C. jejuni more clearly than it has been described to date.

Motility of flagellated bacteria has been a topic of increasing scientific interest over the past decades, attracting the attention of mathematicians, physicists, biologists and engineers alike. Bacteria and other micro-organisms cause substantial damage through biofilm growth on submerged interfaces in water cooling systems, ship hulls and medical implants. This gives social and economic motivations for learning about how micro-organisms swim and behave in different environments. Fluid flows on such small scales are dominated by viscosity and therefore behave differently from the inertia-dominated flows that we are more familiar with, making bacterial motility a physically intriguing phenomenon to study as well. We use the boundary element method (BEM) to simulate the motion of singly flagellated bacteria in a viscous, Newtonian fluid. One of our main objectives is to investigate the influence of external surfaces on swimming behaviour. We show that the precise shape of the cell body and flagellum can be important for determining boundary behaviour, in particular, whether bacteria are attracted or repelled from surfaces. Furthermore, we investigate the types of motion that may arise between two parallel plates and in rectangular channels of fluid and show how these relate to the plane boundary interactions. As an extension to original models of flagellar propulsion in bacteria that assume a rotation of the rigid helical flagellum about an axis fixed relative to the cell body, we consider flexibility of the bacterial hook connecting the aforementioned parts of the swimmer. This is motivated by evidence that the hook is much more flexible than the rest of the flagellum, which we therefore treat as a rigid structure. Elastic dynamics of the hook are modelled using the equations for a Kirchhoff rod. In some regimes, the dynamics are well described by a rigid hook model but we find the possibility of additional modes of behaviour.

This thesis covers an implementation of the Boundary Element Method (BEM) to simulate an arti cial swimmer at zero Reynolds number. The swimmer has a spheroidal head, and a singular, helically shaped tail, called a agella. By imposing a rotating magnetic eld, the swimmer also rotates. This rotary motion causes the swimmer to propel forward. The implementation is inspired by a real nano robot currently being esearched by a Swiss research group 1. We explain the physical concept behind the swimmer, the governing equations and simpli cations, and the theoretical concepts behind the solution method used.

Three behavioural mutants of A. tumefaciens C58C1 (mot-l, mot-12 and fla-15) generated by transposon (Tn5) mutagenesis were studied. Analysis was initially at the molecular level, as a cosmid, pDUB1900, from a representative genomic library of C58C1 had been isolated that complemented the mutants. A region of 8624 nucleotides to which the Tn5 insertion sites of the three mutants had been mapped was sequenced completely in both directions. The comparison of this sequence with sequence databases and other computer analyses revealed six flagellar gene homologues (flgI,flgH,fliP,flaA,flaB,flaC), three open reading frames (ORFA, B and C) with no significant sequence identity to any open reading frames in the databases and the partial sequence of the flagellar gene homologue flgG. Computer analysis also showed that theflgH,flgI andfliP homologues, and ORFs A, B and C, could form the downstream region of a larger operon involved in chemotactic and motility functions. However putative transcription signals were also found within the operon. A new mutant (MANl) was created in the last gene (fliP) of the putative operon to investigate the function of possible transcription signals in the open reading frame immediately upstream of it (ORFC). The mot-12 mutant phenotype of fully synthesised but paralysed flagella is brought about by the insertion of Tn5 in ORFC. ORFC contains a possible promoter for fliP. The Tn5 insertion in ORFC should have polar effects upon the expression of fliP, unless the putative promoter can cause expression of fliP. The MANl mutant had a flagella-less phenotype. FliP in other bacteria is required early in the synthesis of flagella and the null phenotype is/7a-. Thus for flagella to be present in mot-12 suggests fliP must have a promoter. The ORFC sequence is highly conserved in R. meliloti and the overall regulation of these flagellar gene homologues may be as an operon with other regulatory signals. Evidence from other operons (including motility operons) with multiple transcription signals is discussed. The flaABC homologues were multiple copies of the gene encoding the flagellin protein of the flagellum. The mot-l phenotype of severely truncated filaments was caused by a Tn5 insertion in flaA. Analysis of the sequence showed flaABC to each have transcription signals that could lead to separate transcription. Transcription analysis by Northern blotting showed flaA to be transcribed monocistronically. Flagella were isolated from A. twnefaciens and the flagellins separated by SDS-PAGE. The migrated distances (relative to those of markers) was not as predicted from the nucleotide sequence. This anomaly could be caused by unequivalent binding of SDS or post-translational modification of FlaA. The A. tumefaciens flagellar genes were most similar to those of R. meliloti. However A. tumefaciens flagella do not exhibit the characteristic cross-hatching of the complex flagella of R. meliloti. This study also showed A. tumefaciens flagella not to be dependent on divalent cations for subunit associations unlike R. meliloti. These properties of A. tumefaciens flagella were similar to those of R. leguminosarum.The open reading frames found were isolated, radiolabelled and used as probesagainst Southern blots containing chromosomal DNA from a variety of soil bacteria, and cosmids known to contain motility genes in R. meliloti. Hybridisation revealed homologous DNA sequences in a number of these bacteria. All the A. tumefaciens open reading frames hybridised to homologous DNA in R. meliloti and are found in the same order in both species. This suggests that there are similarities at the molecular level in motility and chemotaxis functions between R. meliloti and A. tumefaciens as well as in the patterns of chemotaxis and motility observed previously.

Three behavioural mutants (fla-8, mot-6 and cheL) generated by transposon Tn5 mutagenesis and localised on cosmid pDUB1905 were studied. The cosmid pDUB1905, from a representative genomic library of the Agrobacterium tumefaciens C58C1 chromosome has previously been partly mapped and found to contain genes concerned with flagella. In this study a region of 5860 nucleotides from a 12 kb BamHl fragment of cosmid pDUB1905 was sequenced completely in both directions. Homology searches of this sequence with sequence databases and other computer analysis revealed two flagellar-related genes (flhA and fliR), a chemotactic gene (cheL) and four open reading frames (orfX, orfW, orfY and orfZ) with no significant sequence identity to any open reading frame in databases. A putative promoter-like sequence was also found upstream of orfZ. The FlhA and FliR are the inner members of type III flagellum-specific export apparatus which are responsible for delivering the flagellar subunits lacking a signal peptide leader to the surface of the cell. These have counterparts in the type III secretion proteins system responsible for transporting pathogen proteins to host cells. The function of CheL has not yet been identified. Three ORFs have chaperone characteristics. A mutant was created by insertion of a neomycin resistance cassette in the fliR homologue to determine the effects of the gene on motility. Phenotype analysis of the mutant showed no flagella and motility with small swarming pattern comparing to wild type, indicating that fliR is indeed a flagellar gene. In this study two more members of flagellum-specific export, a chemotactic gene, three open reading frames which could have specific chaperon activity, and an unknown open reading frame were identified in A. tumefaciens C58C'.

The flagellar motor of Escherichia coli is a bidirectional rotary nano-motor, powered by a transmembrane influx of protons. The maximum speed of rotation is about 300 Hz. The motor rotates either counterclockwise (CCW) or clockwise (CW) and the rotation direction is controlled by a chemotactic protein, CheY-P. Despite extensive structural and functional studies, precise mechanisms regarding the torque generation and the directional switching processes remain unclear. In this work, a bottom-up strategy is proposed and followed to investigate this motor. This strategy, named as a multiscale modeling approach, integrates various publicly available experimental data and ‘state-of-the-art’ molecular modeling methods to build structural models for the two most important parts of the motor, the C ring and the stator. Starting from the primary sequences of the composition proteins of these two substructures, tertiary structures are predicted by means of comparative modeling or de novo prediction when the comparative modeling is not available. Quaternary structures of these proteins’s complexes are then predicted by data-driven protein-protein docking or multiscale molecular dynamics (MD) simulations. Finally, structural models of the C ring at CCW and CW rotational states are constructed by cryo-EM aided assembling methods (constraint search that is based on the multiscale modeling tools and under the constraint of the EM images). In the case of the stator, its composition proteins, MotA and MotB, are assembled by coarse-grained MD simulations. This is the first molecular model based atomistic details for the stator. A new molecular mechanism for rotational switching is proposed based on the structural models of the C ring and the stator. The two states of the C ring display significant differences in the interfaces among the self-assembled FliMs and the orientations of FliG C-terminal domain. Based on protein docking results, a binding site of CheY-P is identified on FliM which is close to the interfaces of FliMs for self-assembling. Thereby, I propose that the CheY-P binding interferes with the interactions between neighboring FliM proteins, and thus, induces ~65° rotation of the FliGc domain with respect to FliM. Subsequently, the interactions between the stator and FliGc domains are altered and the rotation direction is changed. Furthermore, a mechanism accounting for the directed rotation of the flagellar motor is proposed based on systematic MD studies on the dynamics of FliGc. It is found that the C-terminal subdomain of FliGc is capable of rotating by ~180° with respect to the N-terminal subdomain of FliGc. If this dynamics is considered in the framework of the whole C ring, the rotation of the C-terminal subdomain of FliGc exhibits an asymmetric feature. As a result, the C ring is decorated with asymmetric teeth on the outer periphery and hence similar with Feynman’s ratchet. The preference of the motor in CCW rotation or in CW rotation is then explained based on the Feynman’s ratchet model.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

The number of flagella in Salmonella enterica serovar Typhimurium cells is closely related with the activity of FlhD4C2, a transcriptional regulator that controls the synthesis of a flagellum. In multiflagelated cells of S. enterica, FlhD4C2 activity is negatively controlled by the Type 3 Secretion chaperone, FliT. FliT interacts with the flagellar filament cap protein FliD. FliD is thus indicated as an anti-regulator in the same aspect describing FlgM inhibition of σ28 activity. Protein interactions between FliT, FliD and FlhD4C2 were explored in detail. Two independent studies, Native gel electrophoresis and gel filtration, revealed the stoichiometry of FliT:FliD interaction to be 1 to 1. In addition, Bacterial Two Hybrid (B2H) analysis showed FliT interacting with FliD In vivo. FliT was also shown to interact with FlhD4C2 by destroying the FlhD4C2 complex. FlhD4C2 activity reduced in the presence of FliT but not all FlhD4C2 activity was inhibited. Biochemical analysis of the interaction between FlhD4C2 and DNA showed that FlhD4C2 bound to DNA was insensitive to FliT regulation. Using a FliT variant, FliT94, that had the last α-helix deleted, helped show that the binding site of FliT to both FliD and FlhD4C2 possibly overlap. The negative regulation of FliT is proposed to be a flagellar specific regulatory mechanism that effectively decreases FlhD4C2 activity to a minimum level rather than inhibiting all flagellar production. FliD counteracts FliT activity to increase FlhD4C2 activity by forming the FliT:FliD complex. Importantly, FlhD4C2 bound to DNA is insensitive to FliT regulation and permits a low number of cells in a population to build a low number of flagella, even during the action of FliT. This study highlighted the importance of including the regulatory protein:DNA interaction in the working model of the regulatory circuit. This model unveils a mechanism to allow bacterial cells to remain prepared to respond quickly and efficiently to the decision to control the activity of flagellation to multiple signals generated by the external environment, internal cues and flagellar specific feedback mechanisms.

There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.

Twenty years ago the experiments of Hotani revealed that flagellar polymorphism (the ability of bacterial flagellar filaments to take on different quaternary structures, specifically helices of different handedness and pitch) can be generated by fluid stresses of the same magnitude as those that occur during natural swimming. Experimental work including the recent crystallization of flagellin, as well as theoretical studies, show how the packing properties and underlying bistability of flagellin may give rise to different static structures. Hotani's experiments showed dynamic nucleation and propagation of domains of opposing handedness on a single flagellum. Here we present the first theory to explain this phenomenon, which is of great relevance to the study of the bundling-unbundling transition in run-and-tumble behaviour of free-swimming bacteria. Our model, based entirely on measurable, physical properties of flagella, bridges the gap between protein-scale statics and cell-scale dynamics. We generate simulations of flagellar motion under fluid stress that exhibit nucleation rates and transition speeds in quantitative agreement with experiment.