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'.

No description available.

This thesis is composed of two topics both concerning diverse and obligatory trypanosomatid parasites. First part deals with identification of new Trypanosoma species identified in blood meal of tsetse flies caught in Dzanga-Sangha Protected Areas, Central African Republic, and identification of feeding preferences of tsetse flies. The second part concerns extraordinary monoxenous trypanosomatid Paratrypanosoma confusum which constitutes the most basal branch between free-living Bodo saltans and parasitic trypanosomatids. This thesis helped to elucidate morphology and biology of this deep branching trypanosomatid. Using genome and transcriptome sequencing and comparative bioinformatics approaches enabled search for ancestral genes shared with free-living bodonids and confirmed genome streamlining in trypanosomatids.

<p> The rhizoplast (flagellar rootlet) of the amebaflagellate Naegleria gruberi has been studied in sectioned cells and in the isolated state. Since the organelle arises, as do the other components of the flagellar apparatus, through a de novo assembly, and possibly a de novo synthesis during the ameba-to-flagellate transformation, the characterization of the rhizoplast's nature may be of importance in the study of a differentiation process in a eukaryotic cell. </p> <p> Structurally, the organelle is a periodically-banded, longitudinally fibrous structure arising in the basal body area of the cell and tapering towards its end in the cytoplasm adjacent to the nucleus. The attachment of the organelle to the basal bodies is mediated through the interbasal body connector and the rhizoplast-associated microtubules. Attachment to the nucleus is unlikely as it has never been unequivocally demonstrated in electron-microscope studies. </p> <p> Rhizoplasts exhibit a distinct periodicity composed of alternating electron-opaque and electron-transparent bands. Variations in the width of both bands has been observed and is discussed in terms of the possible role of the organelle as an anchor and stapilizing structure for the flagellar complex, with contractility and elasticity being discussed as possible mechanisms of this variation. </p> <p> An isolation and partial purification of the rhizoplast has been achieved and is described with reference to its possible use as a tool in the biochemical analysis of the rhizoplast. An aggregation phenomenon of dissolved rhizoplast material by divalent cations has been observed and is discussed, keeping in mind the similar phenomena exhibited by the contractile proteins paramyosin and tropomyosino Collagen, which exhibits a reaggregation from solution, is discussed and tentatively discarded as a possibility for rhizoplast material due to its tendency towards solubilization rather than reaggregation in solutions containing divalent cations. </p> / Thesis / Master of Science (MSc)

Chemotaxis is the ability of an organism to sense its environment and move towards attractants and away from repellents. The two-component system controlling chemotaxis in bacteria contains a histidine kinase CheA, which is autophosphorylated in response to a signal from a ligand-bound transmembrane methyl-accepting chemotaxis protein. CheA transfers the phosphate group to its cognate response regulator which modulates flagellar rotation. Signal termination by dephosphorylation of the response regulator is necessary for the organism to react rapidly to changes in the environment. The phosphorylated response regulator CheY in <i>Escherichia coli</i> is dephosphorylated by CheZ, a phosphatase; certain organisms, such as <i>Sinorhizobium meliloti</i>, that lack a CheZ homolog have developed alternate methods of signal termination. The signaling chain of S. meliloti contains two response regulators, CheY1 and CheY2, in which CheY2 modulates flagellar rotation and CheY1 causes signal termination by acting as a phosphate sink. In addition to known chemotaxis components, the second gene in the chemotaxis operon of <i>S. meliloti</i> codes a 97 amino acid protein, called CheS. The phenotype of a cheS deletion strain is similar to that of a cheY1 deletion strain. Therefore, the possibility that CheS causes signal termination was explored in this work. The derived amino acid sequence of CheS showed similarities with its orthologs from other °-proteobacteria. Sequence conservation was highest at the centrally located °4 and °5 helices. Earlier observations that CheS localizes at the polar chemotaxis cluster in a CheA-dependent manner were confirmed, and the co-localization of CheS with CheA was demonstrated by fluorescence microscopy. The stable expression of CheS in the presence of CheA was confirmed by immunoblot. The same approach was used to establish the stable expression of CheS only in the presence of the P2 domain of CheA, but not with the P1 or P345 domains. Limited proteolysis followed by mass spectrometry defined CheA_163-256 as the CheS binding domain, and this domain overlapped the previously defined CheY2-binding domain, CheA_174-316. The role of CheS in the phosphate flux in S. meliloti chemotaxis was analyzed by assays using radio-labeled [?-?°P]ATP. CheS does not play a role in the autophosphorylation of CheA. However, CheS accelerated the rate of CheY1~P dephosphorylation by almost two-fold, but did not affect the rate of CheY2~P dephosphorylation. CheS also does not seem to affect phosphate flow in the retrophosphorylation from CheY2~P to CheA using acetyl [?°P]phosphate as phosphodonor. Since CheS increases the rate of CheY1 dephosphorylation, it can be envisioned that it either increases the association of CheY1 to CheA, increasing the flow of phosphate from CheA to CheY1, or directly accelerates the dephosphorylation of CheY1~P. The presence of a STAS domain and a conserved serine residue in CheS also raises the possibility that CheS may be phosphorylated by a yet unknown kinase, in a mechanism similar to the phosphorylation of <i>Bacillus subtilis</i> SpoIIAA by its cognate kinase SpoIIAB. Phosphorylated CheS may then switch CheA between a kinase or phosphotransferase ON/OFF state or activated CheS may directly interact with CheY1. Further studies are needed to determine the association of CheY1 with CheS to elucidate the mechanism of CheY1 dephosphorylation. This work has confirmed the <i>in vitro</i> association of CheS with CheA, determined the CheS binding domain on CheA, and indicated that CheS accelerates the dephosphorylation of CheY1~P. This has advanced our understanding of the role of CheS in the chemotaxis signaling chain of <i>S. meliloti</i>. / Master of Science

No description available.

The bacterial flagellar motor is one of the few rotary motors found in nature, and an excellent example of a complex molecular machine. Flagellar motors in the model organism Escherichia coli are products of the coordinated expression of ∼50 different genes. The E. coli flagellar motor is powered by the proton-motive force (pmf), an electrochemical gradient across the cell membrane. Motor torque is gen- erated by proton flow through membrane-embedded stator units which bind to the basal body of the motor. This thesis aimed to investigate the relationship between the pmf and the flag- ellar motor. A novel pmf control system was developed, based on the light-driven proton pump proteorhodopsin (pR). This system enabled pmf -dependent changes in motor behaviour to be precisely monitored in vivo. Expression of pR in E. coli was shown to be sufficient to drive the flagellar motor at wild-type speeds. Using the pR-based pmf control system, the motor was shown to respond to changes in pmf on a timescale of milliseconds. Surprisingly, motor speed increase was observed when pmf was increased above the physiological norm. Reduction of pmf to low levels enabled individual steps in motor rotation to be observed. Motor response to loss of pmf was investigated. Motors were shown to exhibit a two-stage speed decrease after disruption of pmf , with motor speed falling to ∼20 % of its initial value within milliseconds, reaching a complete stop after 1 s. Extended periods of pmf loss was shown to lead to disengagement of stators from the motor, with motor speed increasing in a stepwise fashion after pmf restoration. The integrity of the motor at different pmf levels was investigated by using TIRF microscopy to directly image positioning of fluorescently tagged motor components. The stator protein MotB was shown to physically leave and rejoin the motor after pmf disruption and restoration, with MotB dispersal following motor stop.

La maladie du sommeil ou trypanosomiase africaine humaine fait partie des maladies tropicales négligées sévissant en Afrique sub-saharienne. Elle est causée par le parasite mono-flagellé, Trypanosoma brucei, véhiculé par la mouche tsé-tsé (Glossina spp.). Le flagelle de ce parasite prend naissance au niveau du corps basal et émerge de la cellule en traversant une structure appelée poche flagellaire (FP). Cette poche est formée par l’invagination de la membrane plasmique autour de la base proximale du flagelle. Elle est essentielle à la survie du parasite, car elle constitue l’unique site d’endo- et d’exocytose de la cellule. Cette structure est maintenue autour du flagelle via un constituant du cytosquelette appelé, collier de la poche flagellaire (FPC). Ce collier décrit une structure en anneau ou en fer-à-cheval à la zone de sortie du flagelle. Le premier composant identifié au niveau du FPC est une protéine appelée BILBO1. BILBO1 est essentielle et nécessaire à la biogenèse du FPC et de la FP. Une analyse protéomique et un crible en double-hybride réalisé contre une banque génomique de T. brucei ont permis d’identifier plusieurs partenaires potentiels de BILBO1. Nous avons pu identifier et caractériser de nouvelles protéines du FPC, localisées comme BILBO1 dans une structure en anneau. Nous avons étudié leur fonction chez le parasite, en caractérisant les effets de la surexpression de ces protéines ou de leur ARN interférence sur la croissance et la morphologie cellulaire. / The Human African Trypanosomiasis is a Sub-Saharan Neglected Tropical Disease, caused by Trypanosoma brucei, a mono-flagellate protozoan transmitted by the tsetse fly (Glossina spp.). The T. brucei flagellum originates from a cytoplasmic basal body then grows, to emerge from the cell, by traversing an unusual and essential structure called the Flagellar Pocket (FP). This pocket is an invagination of the pellicular membrane at the base of the flagellum. The FP is essential for the survival of the parasite, because it is the unique site for endo- and exocytosis. The Flagellar Pocket Collar (FPC) is a cytoskeletal component of the FP, and is located at the neck of the FP where it maintains a ring/horseshoe structure at the exit site of the flagellum. The FPC contains numerous uncharacterised proteins, including the first protein identified as FPC component - BILBO1. BILBO1 is essential and required for FPC and FP biogenesis. A proteomic analysis and a private two-hybrid genomic screen experiment on T. brucei have revealed a number of potential BILBO1 partners. We found several proteins localize to the FPC like BILBO1 in a ring-like structure. We characterise these new FPC proteins and their function in the parasite. We have characterised the effects of the GFP fusion protein over-expression and RNAi on cell growth and morphology in T. brucei.

The control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques. High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively. Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively). To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad). Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units.

Rotary molecular motors are protein complexes which convert chemical or electrochemical energy from the environment into mechanical work in the form of rotary motion. The work in this thesis examines two of these motors: the F₁ portion of F₁F_O- ATP synthase, which is responsible for ATP production in bacteria and eukaryotes, and the bacterial flagellar motor (BFM), which rotates the flagella of a bacterium, enabling locomotion. The aim of these investigations was to measure the stepping dynamics of these motors, in order to further elucidate details of the stepping mechanism, the mechanism of rotation, and the mechanochemical cycle. A back-scattering laser dark field microscope of unprecedented resolution was designed and constructed to observe the rotation of gold nanoparticles attached to fixed motors. This micro- scope is capable of sub-nanometer and 20μs resolution. The protocols and algorithms to collect and analyze high resolution rotational data developed for these experiments have yielded novel discoveries for both F₁ and the BFM. While most of the previous single-molecule work has been done on F₁ from the thermophilic Bacilus PS3 (TF₁), only mitochondrial F₁ has been well characterized by high-resolution crystal structures, and single-molecule studies of mesophilic F₁ are lacking. This thesis presents evidence that mesophilic F₁ from E. coli and wild type yeast F₁ from S. cerevisiae are governed by the same mechanism as TF₁ under laboratory conditions. Experiments with yeast F₁ mutants allow a direct comparison between single-molecule rotation studies and high resolution crystal structures. A data set of unprecedented size and resolution was acquired of high speed, low load BFM rotation, enabling the first observation of steps in the BFM under physiological conditions. Preliminary results from this analysis question previously published results of the dependence of speed on stator number at low load and provide novel hypotheses necessitating new models of BFM rotation.