我們研究趨磁螺菌 AMB-1局限在 100微米厚的空間內的運動，細菌濃度約為每立方厘米 10⁹個。整個過程以一台安裝在顯微鏡上的攝像機，以暗場摸式觀察及拍攝。在地球磁場下，我們可以觀察到細菌聚集成環紋，並開始擴大。擴大的初始速度與細菌的游泳速度接近。半小時後，環紋擴大至離液滴邊緣毫米左右，然後停止擴大。環寬約 130微米，比大腸桿菌的趨化環結構小 100倍。我們對這個現象作出了一系列的實驗，研究其特性。 / 我們測試了不同化學成份的實驗緩衝液對環紋的影響。發現當緩衝液缺少琥珀酸時，環紋不會出現；另一方面，當使用琥珀酸作為緩衝液的唯一化學成份時，環紋能清楚地被觀測。這表明琥珀酸是環紋形成的關鍵成份。 / 實驗環境的氧氣含量能按不同比例混合氮和氧來控制。當環境改變為純氮時，環紋進一步擴大；當環境氧氣含量提高時，環紋縮小。實驗結果與微好氧細菌的特性相同。 / 在施加外加的磁場後，環紋被拉成長橢球形，證明細菌的擴散在環紋的形中有重要的作用。在更大的外加磁場下（ 0.3mT，十倍地球磁場），細胞聚集在液滴的兩端，隨後在這些位置長出環紋。該現象證明了環紋會在高細菌濃度的條件下形成， AMB-1有可能存在群體感應機制。 / We study the motion of Magnetospirillum magneticum strain AMB-1 in solution of concentration around 10⁹ cells cm⁻³, which was conned between two glasses with separation 100 μm. The motion was imaged with a EMCCD camera attached to a microscope in darkeld mode and growing ring pattern was observed. Under the earth magnetic eld, the ring migrated under the velocity close to the bacteria swimming velocity. After about half an hour, the ring had expanded to around 1 mm from the edge of droplet. The ring width is about 130 μm, which is 2 orders of magnitude smaller than that of similar ring structure found in E. Coli. A series of experiments were conducted to study the properties of such ring. / In studying the chemical composition of the buffer uid, different compositions were tested. No ring was obseved when succinic acid was absent; on the other hand, ring pattern was observed when using succinic acid alone as a buffer, which suggests that succinic acid is one of the key components of ring formation. / Oxygen level was controlled by mixing nitrogen and oxygen in dierent ratios. Ring further expanded to the edge of droplet when pure nitrogen was pumped in; and shrank when oxygen level was high. The results are consistent with the property of micro-aerophilic band in all micro-aerophilic bacteria. / With an applied magnetic eld, the swarm ring elongated to ellip¬soidal shape, which suggests that the diusion of bacteria plays an important role in the formation of ring. Under even larger magnetic eld (10 times earth magnetic eld), cells aggregated at opposite ends of the droplet, and rings formed at these positions afterwards, which suggests that ring grows at high cell concentrations. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chan, Siu Kit = 趨磁螺菌AMB-1的環紋觀測 / 陳兆傑. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 48-50). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Chan, Siu Kit = Qu ci luo jun AMB-1 de huan wen guan ce / Chen Zhaojie. / Abstract --- p.i / Acknowledgement --- p.iii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- History --- p.1 / Chapter 1.2 --- General Properties of MTB --- p.1 / Chapter 1.2.1 --- Microaerophilic --- p.2 / Chapter 1.2.2 --- Magnetotaxis and Magnetosome --- p.2 / Chapter 1.3 --- Motivation --- p.6 / Chapter 1.3.1 --- Observation of Ring Pattern --- p.6 / Chapter 2 --- Experimental Setup --- p.8 / Chapter 2.1 --- Cell Culturing --- p.8 / Chapter 2.1.1 --- Incubation --- p.9 / Chapter 2.1.2 --- Culture Characterization --- p.11 / Chapter 2.1.3 --- Storage --- p.11 / Chapter 2.1.4 --- Strain Maintenance --- p.11 / Chapter 2.2 --- Bacteria Tracking --- p.14 / Chapter 2.2.1 --- Darkfield microscopy --- p.14 / Chapter 2.2.2 --- Concentration Measurement --- p.17 / Chapter 2.2.3 --- Darkfield image and cell density --- p.18 / Chapter 2.3 --- Design of Experiment --- p.19 / Chapter 2.3.1 --- Magnetic Field --- p.19 / Chapter 2.3.2 --- Chemicals --- p.20 / Chapter 2.3.3 --- Air Chamber --- p.20 / Chapter 3 --- Experimental Result and Analysis --- p.22 / Chapter 3.1 --- Ring properties --- p.22 / Chapter 3.2 --- Chemotactic Property --- p.24 / Chapter 3.3 --- Oxygen Concentration Control --- p.27 / Chapter 3.3.1 --- Micro-aerophilic property --- p.27 / Chapter 3.4 --- Response to Magnetic Field --- p.28 / Chapter 3.4.1 --- Ring under constant magnetic field --- p.32 / Chapter 3.4.2 --- Analysis on the change of shape --- p.35 / Chapter 4 --- Conclusion and Discussion --- p.41 / Chapter 4.1 --- Discussion --- p.41 / Chapter 4.1.1 --- Formation of ring --- p.41 / Chapter 4.1.2 --- Band Property --- p.43 / Chapter 4.2 --- Suggested Focus --- p.44 / Chapter A --- MSGM Content --- p.45 / Chapter B --- Shrinking of Ring --- p.47 / Bibliography --- p.48

. / We report the work we lead on magneto tactic bacteria, from the point of view of active matter. The ability of this bacterium to swim at 100µm/s directed by the magnetic field makes it a good candidate to study driven active matter. Indeed, in this configuration, the self-propelled system is not dragged by an external force, and its directed motion comes from its biased orientation. We choose the strain MC-1 for our study, for the robustness of its individual behavior and its swimming speed. We studied the individual behavior, confirming previous results where the bacteria passively aligns on the magnetic field being disoriented solely by the magnetic field, but also succeeded in triggering activity in their reorientation, suspending it in different chemical environments, or directing them against a solid interface, where this bacteria could tumble. This tumbling behavior, very common amongst non-magnetic bacteria, was not reported for Mangetotactic bacteria. These new results leaded us to develop a model of Run and Tumble under a magnetic field. We studied their behavior when densely concentrated in a micro-channel, in jammed configuration, using standard microfluidics tools. We observed their motion in hour glass shaped micro-channels, without any flow, and characterized the chronology of the jamming process. We investigated the interaction of their swim with a shear, in a counter flow experiments, where MC-1 would be directed against a Poiseuille flow. Due to equilibrium between the magnetic torque and the hydrodynamic shear, bacteria would focus instantly in the middle of the channel. We studied this phenomenon theoretically, and checked our model with the experiments. We discovered a instability of a new kind in the same configuration, for high magnetic fields. Indeed, beyond a threshold the focused suspension would become wavy to end up in segregated droplets of bacteria. We characterized experimentally this phenomenon which reminds us of Rayleigh-Plateau and Kelvin-Helmholtz instabilities, varying the flow rate, the Magnetic field and the density of the suspension. Recirculation in the droplets is observed and explained. We interpret these convection droplets as the source of the instability of the focused suspension

Magnetotactic bacteria (MTB) possess organelles called magnetosomes which contain magnetite (Fe_3O_4) or greigite (Fe_3S_4) nanocrystals. These particles generate a magnetic moment allowing the use of external magnetic fields to control the cell orientation. MTB use this magnetic moment to reach environments with optimal oxygen concentration, a process called magnetotaxis. There are many possible technological applications for MTB, for example, they have been used as nanorobots to push beads and they can be used to remove heavy metals and radionuclides from waste water. In order to fully understand the motion of these micron-size organisms, which takes place at very low Reynolds number where friction dominates over inertia, we set out to measure their drag coefficients. As a starting point, we used a well-studied species of MTB with a corkscrew shape, Magnetospirillum magneticum AMB-1. Simulations were done to find the best external magnetic field strength at which to observe their diffusion. We then imaged non-motile cells placed in these preferred uniform magnetic fields and used automated image analysis to determine the position and orientation of the cells in each frame. This allowed calculating orientation correlation functions and mean-squared displacements, from which rotational and translational diffusion coefficients were obtained for each individual cell. We observed that the four principal drag coefficients of these cells greatly vary as a function of cell length as predicted for cylindrical or elliptical objects with comparable radius. However, we also detecting a coupling between the rotation around and translation along the long axis of the cell only observed for chiral objects. We were able for the first time to experimentally fully characterize the friction matrix for a micron-size elongated chiral object. Continuing our work on MTB, to study live cells for long periods of time, we looked to confine them in PDMS nanowells, but found that MTB were not growing well in this environment. We then turned to a device, which incorporated a PDMS microchannel to provide continuous nutrients and a gel membrane to enable cellular growth into a 2D monolayer. Hopefully, this experimental setup combined with time-lapse microscopy can in the future be used to observe cell growth and cell division, and further to determine whether the magnetosome of the mother is passed on equally between daughter cells. / Thesis / Master of Applied Science (MASc)

Marine vibrio MV-1 is a magnetotactic bacterium capable of aligning its cell in response to the Earth’s magnetic field. This ability is due to the presence of chainlike structures comprising magnetosomes, magnetite particles enclosed in a lipid membrane with associated proteins. Strain MV-1 differs from other, bettercharacterized strains of magnetotactic bacteria as the cells produce higher amounts of biomagnetite per litre of culture and its magnetosomes are unique in shape. This study investigates the presence and organisation of a gene cluster termed a “magnetosome island” within the genome of MV-1. In other magnetotactic bacteria this genomic region has been shown to contain many of the genes associated with magnetosome formation but has not been previously investigated for MV-1. One of the conserved fragments of this region was amplified using degenerate primers followed by extension of the known sequence using inverse PCR based technique and constructing plasmid libraries. Sequencing of the genome of strain MV-1 was accomplished as a part of this study. Significant work was done on comparison of the sequence quality obtained from SOLEXA, 454 and Sanger sequencing technologies. A number of obtained contigs were joined manually and the resulting sequence was automatically annotated using RAST. The obtained genome sequence of 3.6 Mb with a G+C content of 54.3 % was preliminarily analysed and used to search for magnetosome related genes. This study also analysed proteins associated with the magnetosomes of strain MV-1 using MALDI-TOF, LC-MS and Orbitrap mass spectrometry. These approaches allowed the identification of a number of proteins in the isolated magnetosome membrane fraction. Some of these proteins have very low similarity with other characterized proteins (either in magnetotactic bacteria or in other organisms). Another significant point is that genes that code for proteins such as MamR, MamK and MmsF were found to be present in several homologous copies within the “magnetosome island” of MV-1. Interestingly, this study shows that all homologous copies of these proteins were identified in the magnetosome membrane fraction. Generation of knock-out mutants of several specific genes from the “magnetosome island” of strain MV-1 was attempted; constructs were made based on suicide plasmids carrying the cre-lox or I-SceI systems. Despite altering numerous experimental conditions it was not possible to obtain conclusive evidence of the isolation of MV-1 transconjugants containing the integrated constructs. In order to investigate the cell localization of the magnetosome associated protein CAV30779.1, an enhanced green fluorescent protein (EGFP) fusion based construct was generated and transferred into MV-1 cells. The EGFP fluorescent protein fusions within the cells were detected by microscopy. This study reveals novel information about magnetosome formation in marine vibrio MV-1. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of the complex process of magnetosome formation in bacteria.

Les bactéries magnétotactiques synthétisent des organites spécialisés appelés magnétosomes. Ils sont composés d'un cristal magnétique entouré d'une membrane et de protéines spécifiques. Arrangés en chaîne dans la bactérie, ils orientent la bactérie dans le champ magnétique, ce qui simplifierait sa recherche d’environnements microaérophiles. Dans le génome de toutes les souches magnétotactiques séquencées, l'îlot génomique de magnétotaxie contient les gènes impliqués dans la formation des magnétosomes. Nous avons procédé à l’annotation du génome de la souche magnétotactique marine QH-2 et montré que la région du génome codant les gènes de la magnétotaxie n'est, dans ce cas, pas définie comme un îlot génomique, bien qu’elle ait été acquise par transfert latéral de gènes. Dans le génome de M. magneticum AMB-1, nous avons identifié un nouvel îlot génomique de petite taille que nous avons appelé l'îlet de magnétotaxie portant 7 gènes homologues à des gènes liés à la synthèse des magnétosomes. Pour répondre à la question de la fonction biologique de cet îlet génomique, nous avons examiné le rôle de l'un des sept gènes, mamK-like. MamK-like exprimée dans E. coli forme des filaments, comme observé pour MamK. La polymérisation in vitro des deux protéines est également comparable, mais présente des différences structurales. En outre, nous démontrons que mamK-like est transcrite dans AMB-1 de type sauvage et dans le mutant ΔmamK. Par immuno-marquage, nous montrons la présence d'un filament dans le mutant ΔmamK, probablement dû à MamK-like. Nous émettons l'hypothèse que ce filament contribue à maintenir l’organisation en chaîne des magnétosomes dans la souche mutante. / Magnetotactic bacteria synthesise specialised organelles called magnetosomes. They are composed of a magnetic crystal surrounded by a lipid bilayer and specific proteins. Arranged in chains, they orient magnetotactic bacteria in the geomagnetic field, thereby simplifying their search for their microaerophilic environments. In each sequenced magnetotactic strain, the magnetotaxis genomic island contains the genes involved in magnetosomes formation. Our annotation of the newly sequenced genome of the magnetotactic strain QH-2 shows that the region coding the magnetotaxis genes is not a genomic island, though it has been acquired by lateral genes transfer. In the genome of M. magneticum AMB-1 we identified a new, small genomic island we termed the magnetotaxis islet, encoding 7 genes homologous to genes related to the magnetosomes synthesis. To assess the question of the biological function of this genomic islet, we further investigated the role of one of the seven genes, mamK-like. Filaments were observed in E. coli cells expressing MamK-like-Venus fusion by fluorescence microscopy. In vitro polymerization of both isoforms is comparable, though some differences are present at the structural level. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and ΔmamK mutant cells. Immunolabelling assay using an anti-MamK antibody reveals the presence of a filament in the ΔmamK mutant. We hypothesise that this filament is due to MamK-like and that it helps maintaining a chain-like organisation of magnetosomes in the mutant strain.

No description available.

The mechanism behind magnetoreception – the ability to sense magnetic fields for orientation and navigation – still remains one of the most difficult questions to answer in sensory biology, with fish being just one of many taxa known to possess this sense. Characterizing a magnetic sense in fish is crucial for understanding how they navigate their environment and can inform on how increasing anthropogenic sources of electromagnetic fields in aquatic environments may affect threatened fish species. This study examined the hypothesis put forth by Natan and Vortman (2017) that magnetotactic bacteria (MTB), bacteria that create their own chains of magnetic particles for navigational use, act in symbiosis with their animal host to convey magnetic information about their surroundings. Utilizing existing, publicly available datasets of raw genomic sequences, this study demonstrated the presence of MTB within a diverse array of fishes and identified differences in species diversity of MTB between freshwater and marine species of fish. Future research aimed at identifying MTB in specific fish tissues, such as the eye and other neural tissues, will be necessary to provide support for this hypothesis and to further examine the relationships that MTB may have with magnetically sensitive animals.

No description available.

Les bactéries magnétotactiques (MTB) représentent un groupe de bactéries diverses sur le plan phylogénétique, morphologique et physiologique et elles ont la capacité de s'orienter grâce au champ géomagnétique terrestre afin de trouver leurs conditions optimales de développement. Ce comportement remarquable est appelé le magnétotactisme. Les connaissances actuelles de la formation des magnétosomes et du magnétotactisme sont basées principalement sur l'étude des souches magnetospirilla d'eau douce. Au cours de cette thèse, j'ai participé à l'annotation et réalisé des analyses génomiques, physiologiques et génétiques des deux MTB marines. Mes résultats ont révélé un mécanisme d'adaptation de la souche magnetospirillum QH-2 à l'habitat intertidal et des voies métaboliques (autotrophie,- fixation de l'azote, transport du fer) ainsi que des mécanismes de détection environnementaux distincts des magnétospirilla d'eau douce. La souche marine ovoïde MO-1 possède un génome composé de fortes proportions de gènes avec des origines possibles de gamma-(23,6 %), delta-(16,8 %), alpha-(13,2 %) et bêta- (9,1 %) protéobactéries. Cette constatation suggère que MO-1 est un ancêtre fossile ou une nouvelle sous-classe des Proteobacteria. J'ai caractérisé le comportement magnétoctatique de la souche MO-1 et montré que le magnétotactisme est bénéfique et même essentiel pour la croissance des MTB. Par ailleurs, j'ai caractérisé des glycoprotéines essentielles pour la structure et la fonction de l'appareil flagellaire de MO-1. L'ensemble de ces résultats contribue à notre compréhension de la diversité et l'évolution des MTB, ainsi que l'importance environnementale du magnétotactisme. / Magnetotactic bacteria (MTB) consist of a phylogenetically, morphologically and physiologically diverse group of gram-negative bacteria. They have the unique capacity of synthesizing magnetic crystal enveloped with membrane, referred to as magnetosomes, which allow the bacteria swimming along magnetic fields lines (magnetotaxis) to seek optimal oxygen concentration with maximal efficiency. Current knowledge of magnetosome formation and magnetotaxis mainly steams from the study of freshwater magnetospirillum strains. In this thesis, I participated to the expert annotation and performed genomic, physiological and genetic analyses of two marine MTB. I revealed the adaptation of marine magnetospirillum strain QH-2 to the intertidal habitat and metabolic pathways (autotrophy, N2-fixation, iron-transport) and environmental sensing mechanism distinct from those of the freshwater magnetospirilla. In addition, the genome of the marine ovoid strain MO-1 is composed of high proportions of genes with possible origins of gamma- (23.6%), delta- (16.8%), alpha- (13.2%) and beta- (9.1%) proteobacteria. This finding suggests that MO-1 is either a fossil ancestor or a new subclass of the Proteobacteria. I characterized the magnetotactic behavior of the strain MO-1 and showed that magnetotaxis is beneficial and even essential for the growth of MTB. In addition, I carried out proteomic and biochemical studies of glycol-proteins being components of the MO-1 flagellar apparatus or possibly serving as lubricants for the flagellar motor. Together these results contribute to our understanding of the diversity and evolution of MTB as well as the environmental significance of magnetotaxis.

Magnetic resonance imaging (MRI) reporter genes produce MRI signal in response to the molecular environment of the cells in which they are expressed. With an MRI scanner, the signal is detected and used to produce an image of the cells. We hypothesized that the magnetotactic bacterial gene mms6 has the potential to function as an MRI reporter gene. Magnetotactic bacteria produce magnetic iron oxide crystals in intracellular organelles called magnetosomes. mms6 encodes an iron-binding, magnetosome membrane protein Mms6, which plays a role in regulating the size and shape of the iron oxide crystals found within the magnetosomes. To test our hypothesis, several mammalian cell lines were transfected with mms6, and mms6-positive clones were genetically engineered. We then used MRI to image these clones in vitro. When the cells were incubated with iron-supplemented culture media, the mms6-positive clones produced more MRI image contrast than mms6-negative cells. Through a systematic process of elimination, the mms6-positive clone that generated the most in vitro MRI image contrast was identified. This clone, named 9L4S, was composed of mms6-positive rat glioma (9L) cells and was used for intracellular iron studies and in vivo imaging. The results of electron microscopy and optical emission spectrometry support the theory that mms6-positive clones enhance MRI image contrast due to an increase in intracellular iron. The main objective of this research was to assess the ability of mms6 to function as an in vivo MRI reporter gene, so a flank tumor animal model was created. Without any exogenous iron supplementation, tumors composed of mms6-positive cells produced greater negative contrast on an MRI image than mms6-negative cells. These results demonstrate that mms6 can be considered for use in studies requiring an MRI reporter gene.