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

Magnetotactic bacteria are a group of prokaryotes that share the ability to align with external magnetic fields, due to the presence within their cytoplasm of one or several chains of nanometer-sized magnetic crystals called the magnetosomes. The orientation of the chain within the cell is critical for magnetotaxis, which allows these bacteria to swim along the geomagnetic field lines. To do so, the magnetic moment and thus the chain need to lie parallel to the swimming direction which, for elongated bacteria such as AMB-1, is roughly parallel to the long axis of the cell. In most studies, the alignment between the magnetic moment and the cell axis is taken for granted, however no precise measurement has been performed to confirm this. In this thesis, experiments performed to test this assumption are presented, and the results show that for most studied bacteria the alignment is not perfect. The effect on the orientation distributions is discussed and accounted for in the analysis performed to measure the magnetic moment of individual bacteria. A second project presented in this thesis is focused on the biomineralization process in AMB-1. Magnetotactic bacteria synthesize crystals characterized by a well-controlled morphology and a high chemical purity, which makes them interesting for biomedical applications. To study how these crystals are produced, we used scanning trans- mission X-ray microscopy, and preliminary results show that this tool is suitable for studying this complex process. The methods developed and improved during this MSc to perform these experiments are presented, and the first results show an evolution in the spectroscopy of the magnetosomes as they grow. / Thesis / Master of Science (MSc)

>Magister Scientiae - MSc / Magnetosomes, produced by magnetotactic bacteria (MTB), are the most attractive alternative source of non-toxic biocompatible magnetic nanoparticles (MNPs). A magnetosome contains Fe2O4 magnetite with properties superior to MNPs synthesized by the traditional chemical route. However, synthesis of magnetosomes on large scale has not been achieved yet because magnetotactic bacteria are fastidious to grow. In addition, magnetosomes are generally “soft” magnetic materials which can only be used for some applications, while other applications require “hard” magnetic materials. Here at the Institute of Microbial Biotechnology and Metagenomic (IMBM), a study is being conducted on cloning and expression of the magnetosome gene island (MIA), the genetic machinery for magnetosome formation, in an easy to culture E. coli strain. The magnetic properties of the magnetosome can be manipulated by doping with divalent metals such as Ni2+ or Co2+ for a variety of applications. The specific objective of this study was to genetically engineer E. coli strains which accumulate intracellular Ni2+ or Co2+ in order to manipulate the magnetic properties of the magnetosomes. Three E. coli mutants and a wild type strain were transformed with high affinity Ni2+ or Co2+ uptake genes and evaluated for intracellular accumulation at different medium concentrations of NiCl2 or CoCl2. Cellular iron and magnesium were also evaluated because iron is the major component of the magnetosome and magnesium is important for cell growth. The wild type strain, EPI 300 habouring Ni2+ uptake permease the hoxN gene or Co2+ uptake ABC type transporter cbiKMQO operon was found to accumulate the most intracellular Ni2+ or Co2+ in medium conditions most likely to induce magnetosome formation and magnetite manipulation. This strain can be used to co-express the MIA and Ni2+ or Co2+ uptake gene for mass production of magnetosome with altered magnetic properties.

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.

Les bactéries magnétotactiques (MTB) sont des organismes capables de synthétiser des cristaux magnétiques au sein d’un organite particulier, le magnétosome. L’assemblage de ces magnétosomes leur confère des propriétés d’aimantation et d’orientation dans les champs magnétiques. Dans le contexte de l’essor des biotechnologies, nous avons procédé à la fonctionnalisation des MTB pour des applications de bioremédiation du cobalt.Nous avons ainsi développé des vecteurs adaptés aux MTB pour l’expression de machineries enzymatiques de Staphylococcus aureus et Pseudomonas aeruginosa permettant la production de métallophores analogues à la nicotianamine. Nous avons observé un phénotype double, d’augmentation de la résistance aux métaux et d’augmentation de l’accumulation du cobalt que ce soit chez Escherichia coli ou les MTB Magnetospirillum magneticum AMB-1 et Magnetospirillum gryphiswaldense MSR-1. Nous avons également observé que l’expression de systèmes d’import des métaux tel que la NiCoT perméase NxiA de Rhodopseudomonas palustris dans des souches exprimant les analogues de la nicotianamine permet d’accroître encore l’accumulation des métaux.De plus, nous avons montré que la production de ces analogues permet un enrichissement en cobalt des magnétosomes, mais ne conduit pas à de modification de la spéciation de ce métal chez les MTB.Nous proposons donc ici l’utilisation des MTB comme châssis cellulaire pour de nouvelles applications biotechnologiques. / Magnetotactic bacteria (MTB) are organisms able to synthesize magnetic crystals within a specific organelle, the magnetosome. The assembly of these magnetosomes gives them magnetization and orientation properties in magnetic fields. In the context of the development of biotechnology, we have performed the functionalization of MTBs for cobalt bioremediation applications.We have thus developed vectors suitable for MTB for the expression of enzymatic machineries of Staphylococcus aureus and Pseudomonas aeruginosa allowing the production of metallophores analogous to nicotianamine. We observed a double phenotype, increased resistance toward metals and increased cobalt accumulation in Escherichia coli or MTBs Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. We have also observed that the expression of metal import systems such as Rhodopseudomonas palustris NiCoT permease NxiA in strains expressing nicotianamine analogs further increases the accumulation of metals.Moreover, we have shown that the production of these analogs allows a cobalt enrichment of the magnetosomes, but does not lead to a modification of the speciation of this metal in MTB.We introduce here the use of MTBs as cellular chassis for new biotechnological applications.

Ces travaux de thèse s'inscrivent dans le contexte de l'imagerie moléculaire, qui vise à adapter les traitements de pathologies à la variabilité de chaque patient, grâce à l'imagerie de biomarqueurs cellulaires ou moléculaires. En particulier, l'imagerie par résonance magnétique (IRM) couplée a des nanoparticules d’oxyde de fer innovantes pourrait permettre de relever un tel défi.Cette thèse se concentre sur l'étude d'une nouvelle classe d'agents de contraste à base d'oxyde de fer pour l'IRM à haut champ magnétique. En effet, les magnétosomes sont des vésicules d’oxyde de fer produites naturellement par des bactéries appelées bactéries magnétotactiques. De telles bactéries synthétisent ces vésicules magnétiques et les alignent comme l'aiguille d'une boussole, ce qui facilite leur navigation dans les sédiments. Ces bactéries produisent donc des magnétosomes aux propriétés magnétiques exceptionnelles: 50 nm de diamètre, mono-cristallin, mono-domaine magnétique et avec une haute magnétisation à saturation. De plus, une grande variété de souches bactériennes existent dans la nature, et produisent, avec une grande stabilité, des magnétosomes dont la taille, la forme, et le contenu chimique, sont déterminés génétiquement. Enfin, les magnétosomes sont naturellement porteurs d'une membrane bi-lipidique dont le contenu est également déterminé génétiquement. Récemment, le contenu protéique de la membrane des magnétosomes a été mis à jour, ouvrant la voie à la fonctionnalisation de cette dernière par fusion des gènes codant pour des protéines présentes abondamment à la membrane avec ceux codant pour un peptide d’intérêt.Ainsi, l'utilisation de ces micro-organismes pour produire des agents de contraste innovants et fonctionnalisés pour l'imagerie moléculaire par IRM, et les applications qui en découlent, ont été étudiées pendant cette thèse. La production et l'ingénierie des magnétosomes a été réalisée par nos collègues du Laboratoire de Bioénergétique Cellulaire (LBC, CEA Cadarache), et sera présentée et discutée. Des magnétosomes sauvages ont d'abord été caractérisés en tant qu'agents de contraste pour l'IRM. De tel magnétosomes présentent des propriétés contrastantes très intéressantes pour l'IRM, ce qui a été validé à la fois in vitro puis in vivo. L'étude de faisabilité de la production d'un agent de contraste pour l'imagerie moléculaire par IRM en une seule étape, à l'aide des bactéries magnétotactiques, a été réalisée sur un modèle de souris porteur de glioblastome. Sachant par la littérature que les cellules tumorales sur-expriment les intégrines anb3, et que ces dernières peuvent être ciblées par le peptide RGD, il a été choisi de produire des magnétosomes exprimant le peptide RGD à leur membrane. L'affinité de tels magnétosomes pour les cellules tumorales U87 a été vérifiée in vitro, et démontré in vivo par IRM puis cross-validé par histologie. / This work takes place in the context of molecular imaging, which aims at tailoring medical treatments and therapies to the individual context by revealing molecular or cellular phenomenon of medical interest in the less invasive manner. In particular, it can be acheived with MRI molecular imaging using engineered iron-oxide contrast agent.This PhD thesis focuses on the study of a new class of iron-oxide contrast agent for high field MRI. Indeed, magnetosomes are natural iron-oxide vesicles produced by magnetotactic bacteria. These bacteria synthesized such magnetic vesicles and ordered them like a nano-compass in order to facilitate their navigation in sediments. This explains why magnetosomes are awarded with tremendous magnetic properties: around 50 nm, mono-crystalline, single magnetic domain and high saturation magnetization. Furthermore, a wide variety of bacterial strains exist in nature and size and shape of magnetosomes are highly stable within strain and can be very different between strains. Finally, magnetosomes are naturally coated with a bilipidic membrane whose content is genetically determined. Lately, researchers have unravelled magnetosomes membrane protein contents, opening the way to create functionnalized magnetosomes thanks to fusion of the gene coding for a protein of interest with the gene coding for an abundant protein at magnetosomes membrane.A new alternative path using living organisms to tackle the production of engineered high effciency molecular imaging probes have been investigated with magnetotactic bacteria in this PhD. The production and engineering of magnetosomes have been carried out by our partner, the Laboratoire de Bio-energétique Cellulaire (LBC, CEA Cadarache), and will be presented and discussed. We then characterized magnetosomes as contrast agent for high field MRI. We showed they present very promising contrasting properties in vitro, and assessed this observation in vivo by establishing they can be used as effcient blood pool agent after intravenous injection. Afterward, we applied the concept of producing engineered MRI molecular imaging probes in a single step by bacteria, to a mouse model of glioblastoma. Knowing that tumor cells can be actively targeted through anb3 integrins by RGD, we produced RGD functionnalized magnetosomes. We started from showing these RGD magnetosomes have a good affnity for U87 cell in vitro, prior to demonstrate it in vivo on orthotopic U87 mouse model. This in vivo affnity being fnally cross-validated with histology.