Molecular and behavioral analysis of magneto-aerotaxis in Magnetospirillum gryphiswaldense

Magnetotactic bacteria (MTB) contain nanometer-sized crystals of a magnetic iron mineral enabling directed swimming along geomagnetic field lines. However, although this unique behavior was discovered already 40 years ago, it still has remained poorly understood at the cellular level and the molecular mechanisms responsible for sensing environmental stimuli and transducing signals to the flagellar motors have been unknown. Therefore, the major goal of this thesis was to investigate the swimming behavior of Magnetospirillum gryphiswaldense both at the behavioral and molecular level. Individual motors of tethered M. gryphiswaldense cells were found to rotate both clockwise and counterclockwise with equal speed. Cells swam at speeds of up to 60 µm s-1 and commonly displayed runs of several hundred µm in length. In striking contrast to E. coli, which reorients the cell body between run intervals at random angles, motor switching events caused swimming reversals with reorientation angles close to 180°. The sensory repertoire of M. gryphiswaldense was analyzed by classical macroscopic chemotaxis assays, and aerotaxis was found to be the dominant behavior. In addition to the strong microaerophilic response in oxygen gradients, I observed tactic bands also under anoxic conditions within gradients of the alternative electron acceptor nitrate, suggesting that aerotaxis is part of a general redox or energy taxis mechanism. The aerotactic response of M. gryphiswaldense was furthermore analyzed by recording and tracking single cells under controlled atmospheric conditions in a gas perfusion chamber. Compared to other well-studied bacteria, M. gryphiswaldense displayed unusually low swimming reversal rates (<0.1 s-1) under equilibrium conditions. Abruptly shifting oxygen levels from 2% to 0% only slightly increased reversal rates, whereas a reverse shift from 0% to 2% caused a transient threefold increase in reversal rates that was directly followed by an extraordinarily sustained smooth-swimming phase without return to pre-stimulus levels. Apart from 56 putative genes encoding chemoreceptors that might be involved in magnetotaxis, four putative chemotaxis operons (cheOp1-4) were identified in the genome of M. gryphiswaldense, containing genes commonly involved in signal transduction from chemoreceptors to the flagellar motors. Single or combined deletions of cheOp2-4 did not have any pronounced effect on motility or aerotaxis. In striking contrast, deletion of cheOp1, which comprises only the canonical set of chemotaxis genes (cheAWYBR), caused individual cells to swim straight without reversing, resulting in a complete loss of aerotaxis. When analyzed under oxic conditions, most MTB possess a clear directional preference corresponding to downward movement in their natural habitat, referred to as “polar magneto-aerotaxis”. Although cultivated strains of magnetotactic spirilla were previously assumed to lack any directional preference, in this work polar swimming behavior could be restored in M. gryphiswaldense through repeated cultivation of cells in magnetic fields superimposed on oxygen gradients. Individual cells displayed a gradual bias of swimming runs with one of the cell poles leading that depended on ambient oxygen levels. In anoxic microdroplets, addition of 2% oxygen rapidly reversed the overall swimming direction of the entire population. However, in the absence of CheOp1 swimming polarity could be no longer selected and no reversal of swimming bias was observed. These findings for the first time show that there is a direct molecular link between aerotactic sensing and the determination of magnetotactic polarity, through the sensory pathway CheOp1. In a joint project in the last part of this thesis, I demonstrated how magnetotactic behavior can be manipulated through artificial recruitment of polarly localized CheW1-GFP fusion proteins to midcell anchors. GFP-labelled proteins were trapped by expressing GFP-binding nanobodies on the magnetosome membrane surface (referred to as “nanotrap”). By varying the expression level of the nanobody, a gradual knockdown of magneto-aerotaxis was achieved.Magnetotaktische Bakterien (MTB) enthalten wenige Nanometer große Kristalle magnetischer Eisenminerale, die ihnen die faszinierende Fähigkeit verleihen, sich entlang der Feldlinien des Erdmagnetfelds fortzubewegen. Obwohl diese besondere Form bakteriellen Schwimmverhaltens bereits vor nunmehr 4 Jahrzenten entdeckt wurde, ist das Verhalten einzelner Zellen, sowie die Mechanismen, die der Reizerkennung und Signaltransduktion zum Flagellenmotor zugrunde liegen, bis heute nur wenig erforscht. Das Ziel dieser Doktorarbeit war daher, die molekularen Grundlagen der Magnetotaxis im Modellorganismus Magnetospirillum gryphiswaldense zu charakterisieren und die Motilität einzelner Zellen detailliert zu untersuchen. Im ersten Teil meiner Arbeit konnte ich zeigen, dass die Flagellenmotoren von M. gryphiswaldense jeweils mit gleicher Geschwindigkeit in beide Drehrichtungen rotieren und dadurch die Zellen auf bis zu 60 µm s-1 beschleunigen. Im Gegensatz zu E. coli, das während häufiger Taumelphasen seine Ausrichtung ändert, werden die oft mehrere hundert Mikrometer langen, geraden Schwimmepisoden von M. gryphiswaldense durch Umkehrvorgänge unterbrochen, die eine Änderung der Schwimmrichtung um ca. 180° bewirken. Bei der Untersuchung des allgemeinen chemotaktischen Verhaltens in makroskopischen Tests zeigte sich, dass die mikroaerophile Antwort von M. gryphiswaldense stark dominiert. Da unter Ausschluss von Sauerstoff zudem Bandenbildung in künstlich hergestellten Nitratgradienten beobachtet wurde, kann davon ausgegangen werden, dass das dominante aerotaktische Verhalten Teil einer umfassenderen Redox- oder Energietaxis ist. Die Aerotaxis von M. gryphiswaldense wurde anschließend unter kontrollierten atmosphärischen Bedingungen auf Einzelzellebene untersucht. Im Vergleich zu anderen gut erforschten Bakterien wurden unter Gleichgewichtsbedingungen nur relativ wenige Umkehrvorgänge registriert (<0.1 s-1), und ein abruptes Absenken des Sauerstoffgehalts von 2% auf 0% führte zu einer lediglich geringen Zunahme der Umkehrvorgänge. Nach einer plötzlichen Anhebung des Sauerstoffgehalts von 0% auf 2% stieg dagegen die Umkehrfrequenz kurzzeitig um das Dreifache an. Im Anschluss hieran wurden jedoch bemerkenswerterweise über lange Zeiträume fast keine Umkehrvorgänge registriert und selbst nach 80 s lag die Umkehrfrequenz unter dem Ausgangswert. In der genomischen Sequenz von M. gryphiswaldense wurden neben 56 Chemorezeptor-Genen insbesondere vier mutmaßliche Chemotaxisoperons (cheOp1-4) identifiziert. Während die Deletion von cheOp2-4(sowohl einzeln als auch in Kombination)keinen deutlichen Einfluss auf das Schwimmverhalten hatte, wurde nach Deletion von cheOp1 ein komplett nicht-aerotaktischer Phänotyp beobachtet, gekennzeichnet durch lange, ununterbrochene Schwimmepisoden. Unter sauerstoffgesättigten Bedingungen weisen die meisten MTB eine klare Richtungspräferenz auf, sogenanntes „polares Schwimmverhalten“. Obwohl den in Reinkultur verfügbaren Magnetospirillen diese Fähigkeit traditionell abgesprochen wurde, konnte im Rahmen der vorliegenden Arbeit durch wiederholte Kultivierung von M. gryphiswaldense in Sauerstoffgradienten, die von Magnetfeldern überlagert waren, polares Verhalten erzeugt werden. Die Mehrzahl der Zellen wies eine graduelle Bevorzugung einer Schwimmrichtung auf, die sich durch Variation des Sauerstoffgehalts beeinflussen ließ. Dies wurde durch Beobachtungen an zunächst anaerobisierten Zellpopulationen untermauert, die bei Zuführung von 2% Sauerstoff kollektiv ihre Vorzugsschwimmrichtung änderten. Nach Deletion von cheOp1 wurde im Gegensatz dazu keine Wiederherstellung der Schwimmpolarität beobachtet, was den Schluss nahe legt, dass ein direkter Zusammenhang zwischen der durch cheOp1 kodierten Signaltransduktionskaskade und der molekularen Determination der magnetotaktischen Polarität existiert. Im letzten Abschnitt dieser Arbeit konnte ich im Rahmen eines Kooperationsprojekt zeigen, wie durch Expression von GFP-bindenden nanobodies auf der Magnetosomenoberfläche die native Lokalisierung von GFP-markierten Signaltransduktionskomponenten künstlich verändert wird. Durch Variation der Kopienzahl des nanobodies wurde das CheW1-GFP Fusionsprotein in unterschiedlichem Umfang zur Zellmitte verschoben, was einen graduellen Ausfall der Magneto-Aerotaxis bewirkte

Molecular and behavioral analysis of magneto-aerotaxis in Magnetospirillum gryphiswaldense

Magnetotactic bacteria (MTB) contain nanometer-sized crystals of a magnetic iron mineral enabling directed swimming along geomagnetic field lines. However, although this unique behavior was discovered already 40 years ago, it still has remained poorly understood at the cellular level and the molecular mechanisms responsible for sensing environmental stimuli and transducing signals to the flagellar motors have been unknown. Therefore, the major goal of this thesis was to investigate the swimming behavior of Magnetospirillum gryphiswaldense both at the behavioral and molecular level. Individual motors of tethered M. gryphiswaldense cells were found to rotate both clockwise and counterclockwise with equal speed. Cells swam at speeds of up to 60 µm s-1 and commonly displayed runs of several hundred µm in length. In striking contrast to E. coli, which reorients the cell body between run intervals at random angles, motor switching events caused swimming reversals with reorientation angles close to 180°. The sensory repertoire of M. gryphiswaldense was analyzed by classical macroscopic chemotaxis assays, and aerotaxis was found to be the dominant behavior. In addition to the strong microaerophilic response in oxygen gradients, I observed tactic bands also under anoxic conditions within gradients of the alternative electron acceptor nitrate, suggesting that aerotaxis is part of a general redox or energy taxis mechanism. The aerotactic response of M. gryphiswaldense was furthermore analyzed by recording and tracking single cells under controlled atmospheric conditions in a gas perfusion chamber. Compared to other well-studied bacteria, M. gryphiswaldense displayed unusually low swimming reversal rates (<0.1 s-1) under equilibrium conditions. Abruptly shifting oxygen levels from 2% to 0% only slightly increased reversal rates, whereas a reverse shift from 0% to 2% caused a transient threefold increase in reversal rates that was directly followed by an extraordinarily sustained smooth-swimming phase without return to pre-stimulus levels. Apart from 56 putative genes encoding chemoreceptors that might be involved in magnetotaxis, four putative chemotaxis operons (cheOp1-4) were identified in the genome of M. gryphiswaldense, containing genes commonly involved in signal transduction from chemoreceptors to the flagellar motors. Single or combined deletions of cheOp2-4 did not have any pronounced effect on motility or aerotaxis. In striking contrast, deletion of cheOp1, which comprises only the canonical set of chemotaxis genes (cheAWYBR), caused individual cells to swim straight without reversing, resulting in a complete loss of aerotaxis. When analyzed under oxic conditions, most MTB possess a clear directional preference corresponding to downward movement in their natural habitat, referred to as “polar magneto-aerotaxis”. Although cultivated strains of magnetotactic spirilla were previously assumed to lack any directional preference, in this work polar swimming behavior could be restored in M. gryphiswaldense through repeated cultivation of cells in magnetic fields superimposed on oxygen gradients. Individual cells displayed a gradual bias of swimming runs with one of the cell poles leading that depended on ambient oxygen levels. In anoxic microdroplets, addition of 2% oxygen rapidly reversed the overall swimming direction of the entire population. However, in the absence of CheOp1 swimming polarity could be no longer selected and no reversal of swimming bias was observed. These findings for the first time show that there is a direct molecular link between aerotactic sensing and the determination of magnetotactic polarity, through the sensory pathway CheOp1. In a joint project in the last part of this thesis, I demonstrated how magnetotactic behavior can be manipulated through artificial recruitment of polarly localized CheW1-GFP fusion proteins to midcell anchors. GFP-labelled proteins were trapped by expressing GFP-binding nanobodies on the magnetosome membrane surface (referred to as “nanotrap”). By varying the expression level of the nanobody, a gradual knockdown of magneto-aerotaxis was achieved.Magnetotaktische Bakterien (MTB) enthalten wenige Nanometer große Kristalle magnetischer Eisenminerale, die ihnen die faszinierende Fähigkeit verleihen, sich entlang der Feldlinien des Erdmagnetfelds fortzubewegen. Obwohl diese besondere Form bakteriellen Schwimmverhaltens bereits vor nunmehr 4 Jahrzenten entdeckt wurde, ist das Verhalten einzelner Zellen, sowie die Mechanismen, die der Reizerkennung und Signaltransduktion zum Flagellenmotor zugrunde liegen, bis heute nur wenig erforscht. Das Ziel dieser Doktorarbeit war daher, die molekularen Grundlagen der Magnetotaxis im Modellorganismus Magnetospirillum gryphiswaldense zu charakterisieren und die Motilität einzelner Zellen detailliert zu untersuchen. Im ersten Teil meiner Arbeit konnte ich zeigen, dass die Flagellenmotoren von M. gryphiswaldense jeweils mit gleicher Geschwindigkeit in beide Drehrichtungen rotieren und dadurch die Zellen auf bis zu 60 µm s-1 beschleunigen. Im Gegensatz zu E. coli, das während häufiger Taumelphasen seine Ausrichtung ändert, werden die oft mehrere hundert Mikrometer langen, geraden Schwimmepisoden von M. gryphiswaldense durch Umkehrvorgänge unterbrochen, die eine Änderung der Schwimmrichtung um ca. 180° bewirken. Bei der Untersuchung des allgemeinen chemotaktischen Verhaltens in makroskopischen Tests zeigte sich, dass die mikroaerophile Antwort von M. gryphiswaldense stark dominiert. Da unter Ausschluss von Sauerstoff zudem Bandenbildung in künstlich hergestellten Nitratgradienten beobachtet wurde, kann davon ausgegangen werden, dass das dominante aerotaktische Verhalten Teil einer umfassenderen Redox- oder Energietaxis ist. Die Aerotaxis von M. gryphiswaldense wurde anschließend unter kontrollierten atmosphärischen Bedingungen auf Einzelzellebene untersucht. Im Vergleich zu anderen gut erforschten Bakterien wurden unter Gleichgewichtsbedingungen nur relativ wenige Umkehrvorgänge registriert (<0.1 s-1), und ein abruptes Absenken des Sauerstoffgehalts von 2% auf 0% führte zu einer lediglich geringen Zunahme der Umkehrvorgänge. Nach einer plötzlichen Anhebung des Sauerstoffgehalts von 0% auf 2% stieg dagegen die Umkehrfrequenz kurzzeitig um das Dreifache an. Im Anschluss hieran wurden jedoch bemerkenswerterweise über lange Zeiträume fast keine Umkehrvorgänge registriert und selbst nach 80 s lag die Umkehrfrequenz unter dem Ausgangswert. In der genomischen Sequenz von M. gryphiswaldense wurden neben 56 Chemorezeptor-Genen insbesondere vier mutmaßliche Chemotaxisoperons (cheOp1-4) identifiziert. Während die Deletion von cheOp2-4(sowohl einzeln als auch in Kombination)keinen deutlichen Einfluss auf das Schwimmverhalten hatte, wurde nach Deletion von cheOp1 ein komplett nicht-aerotaktischer Phänotyp beobachtet, gekennzeichnet durch lange, ununterbrochene Schwimmepisoden. Unter sauerstoffgesättigten Bedingungen weisen die meisten MTB eine klare Richtungspräferenz auf, sogenanntes „polares Schwimmverhalten“. Obwohl den in Reinkultur verfügbaren Magnetospirillen diese Fähigkeit traditionell abgesprochen wurde, konnte im Rahmen der vorliegenden Arbeit durch wiederholte Kultivierung von M. gryphiswaldense in Sauerstoffgradienten, die von Magnetfeldern überlagert waren, polares Verhalten erzeugt werden. Die Mehrzahl der Zellen wies eine graduelle Bevorzugung einer Schwimmrichtung auf, die sich durch Variation des Sauerstoffgehalts beeinflussen ließ. Dies wurde durch Beobachtungen an zunächst anaerobisierten Zellpopulationen untermauert, die bei Zuführung von 2% Sauerstoff kollektiv ihre Vorzugsschwimmrichtung änderten. Nach Deletion von cheOp1 wurde im Gegensatz dazu keine Wiederherstellung der Schwimmpolarität beobachtet, was den Schluss nahe legt, dass ein direkter Zusammenhang zwischen der durch cheOp1 kodierten Signaltransduktionskaskade und der molekularen Determination der magnetotaktischen Polarität existiert. Im letzten Abschnitt dieser Arbeit konnte ich im Rahmen eines Kooperationsprojekt zeigen, wie durch Expression von GFP-bindenden nanobodies auf der Magnetosomenoberfläche die native Lokalisierung von GFP-markierten Signaltransduktionskomponenten künstlich verändert wird. Durch Variation der Kopienzahl des nanobodies wurde das CheW1-GFP Fusionsprotein in unterschiedlichem Umfang zur Zellmitte verschoben, was einen graduellen Ausfall der Magneto-Aerotaxis bewirkte

Polarity of bacterial magnetotaxis is controlled by aerotaxis through a common sensory pathway

Most motile bacteria navigate within gradients of external chemical stimuli by regulating the length of randomly oriented swimming episodes. Magnetotactic bacteria are characterized by chains of intracellular ferromagnetic nanoparticles and their ability to sense the geomagnetic field, which is believed to facilitate directed motion, but is not well understood at the behavioural and molecular level. Here, we show that cells of Magnetospirillum gryphiswaldense unexpectedly display swimming polarity that depends on aerotactic signal transduction through one of its four chemotaxis operons (cheOp1). Growth of cells in magnetic fields superimposed on oxygen gradients results in a gradual inherited bias of swimming runs with one of the cell poles leading, such that the resulting overall swimming direction of entire populations can be reversed by changes in oxygen concentration. These findings clearly show that there is a direct molecular link between aerotactic sensing and the determination of magnetotactic polarity, through the sensory pathway, CheOp1

Excessive anticoagulation identified by emergency medical service through point-of-care coagulometry

Bleeding because of excessive anticoagulation is a well-recognized complication of coumadin therapy. In cases of potentially life-threatening bleeding such as intracranial haemorrhage, reversal of anticoagulation should be carried out as soon as possible. Here we report the case of an emergency patient in whom excessive anticoagulation was diagnosed at the scene by emergency medical service personnel through the use of a point-of-care coagulometer. Following hospital admission, findings were confirmed by central laboratory assessment of prothrombin time. The time gained through the use of portable coagulometers may contribute to improved pre-hospital emergency management of anticoagulated patients

An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular "nanotraps," which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW(1)-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. IMPORTANCE Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular "nanotraps" in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks

Systematic evaluation of particle loss during handling in the percutaneous transluminal angioplasty for eight different drug-coated balloons

Paclitaxel drug coated balloons (DCBs) should provide optimal drug transfer exclusively to the target tissue. The aim of this study was to evaluate the particle loss by handling during angioplasty. A robotic arm was developed for systematic and reproducible drug abrasion experiments. The contact force on eight different commercially available DCB types was gradually increased, and high-resolution microscopic images of the deflated and inflated balloons were recorded. Three types of DCBs were classified: no abrasion of the drug in both statuses (deflated and inflated), significant abrasion only in the inflated status, and significant abrasion in both statuses. Quantitative measurements via image processing confirmed the qualitative classification and showed changes of the drug area between 2.25 and 45.73% (13.28 ± 14.29%) in the deflated status, and between 1.66 and 40.41% (21.43 ± 16.48%) in the inflated status. The structures and compositions of the DCBs are different, some are significantly more susceptible to drug loss. Particle loss by handling during angioplasty leads to different paclitaxel doses in the target regions for same DCB types. Susceptibility to involuntary drug loss may cause side effects, such as varying effective paclitaxel doses, which may explain variations in studies regarding the therapeutic outcome

The impact of fat deterioration on formation of acrylamide in fried foods

The current study investigates to what extent the reaction products of thermal degradation directly influence acrylamide formation in French fries. The frying tests at 170 and 180 °C are carried out with rapeseed oil for 32 h with 128 frying cycles. Acrylamide content in French fries is determined by LC-MS/MS. Oxidative and thermal degradation is followed by measuring total polar compounds (TPC), di- and polymerized triacylglycerols (DPTG), monomer oxidized triacylglycerols (MONOX), p-anisidine value (AnV), mono and di-acyl-glycerols (MAG and DAG), acid value (AV), epoxy fatty acids, iodine value (IV), saponification value, and fatty acid composition. During frying, the nature and degradation level of the frying medium have a direct impact on acrylamide formation. It can be shown that the pH-dependent reaction is strongly inhibited at acid values above 0.5 mg KOH g−1 oil. Acidity measured as AV or FFA is mainly caused by oxidation, and less so by hydrolysis of triacylglycerols (TAG) as assumed up to now. Obviously, acid functional groups formed by oxidation of unsaturated fatty acids bound in TAG can act not only as catalyst for dimerization of TAG but also interact with asparagine as most important precursor for acrylamide formation so that no reaction with carbonyl groups for the formation of acrylamide is necessary. Practical applications: The same acidic functional groups that are known to catalyze the formation of dimeric TAG under frying conditions (160–190 °C, access of oxygen) in a nonradical mechanism apparently can also deactivate asparagine by protonization as a potential precursor for the formation of acrylamide. It is recommended not to reduce acidity of used frying oil by active filter aids below AV ≥ 0.5 as it helps to reduce acrylamide contamination of fried food

Modeling of Fluctuations in Dynamical Optoelectronic Device Simulations within a Maxwell-Density Matrix Langevin Approach

We present a full-wave Maxwell-density matrix simulation tool including c-number stochastic noise terms for the modeling of the spatiotemporal dynamics in active photonic devices, such as quantum cascade lasers (QCLs) and quantum dot (QD) structures. The coherent light-matter interaction in such devices plays an important role in the generation of frequency combs and other nonlinear and nonclassical optical phenomena. Since the emergence of nonlinear and nonclassical features is directly linked to the noise properties, detailed simulations of the noise characteristics are required for the development of low-noise quantum optoelectronic sources. Our semiclassical simulation framework is based on the Lindblad equation for the electron dynamics, coupled with Maxwell's equations for the optical propagation in the laser waveguide. Fluctuations arising from interactions of the optical field and quantum system with their reservoirs are treated within the quantum Langevin theory. Here, the fluctuations are included by adding stochastic c-number terms to the Maxwell-density matrix equations. The implementation in the mbsolve dynamic simulation framework is publicly available.Comment: 18 pages, 5 figure