Recombinant biomineralizing polypeptides PXSY : structure and dynamics upon the interaction with silica

Diatomeen bilden Zellhüllen aus Kieselsäure, wobei sie die Morphologie des abgelagerten Silikates auf der Nano- bis Mikroebene kontrollieren. Sie nutzen organische Verbindungen, wie Peptide, Kohlenhydrate und Poly(alkylamin), um die Silikat-Bildung zeitlich und räumlich zu steuern. Da die Silikat-Fällung in Diatomeen unter chemisch milden, physiologischen Bedingungen abläuft, ist deren Biomineraisations-Strategie interessant für Umsetzungen in technischen Applikationen. Insbesondere Polypeptide bieten breite Anwendungsmöglichkeiten, da sie in ihrer Sequenz variiert werden können und somit eine vielseitige Klasse von Biomineralisations-Agenzien darstellen. In dieser Arbeit wurden synthetische, Silikat-biomineralisierende Polypeptide mit hoher kationischer Nettoladung, genannt PXSY, bakteriell exprimiert und in Lösung, als auch während der Interaktion mit Silikat strukturell untersucht. Die PXSY Aminosäure-Komposition ist ähnlich jener der Silaffine, kationischen Peptiden aus Diatomeen welche die in vitro Silikat-Bildung induzieren. Die bakterielle Synthese der Polypeptide gelang als Fusion mit einer hydrophoben Pro-Sequenz, die die Ablagerung in Einschlusskörpern bewirkt und dadurch schädliche Interaktionen der kationischen Peptide mit Zellbestandteilen (Nukleinsäuren, Membranen) unterband. Nach chemischer Spaltung der Fusion wurden für das längste Polypeptid der PXSY-Serie (P5S3, 50 Aminosäuren, 20 kationische Ladungen) Ausbeuten bis zu 15 µmol pro Liter Expressionskultur erreicht. In Lösung nehmen die Polypeptide eine gestreckte Konformation mit hohem Anteil an Polyprolin-II-Sekundärstruktur ein. Sie assemblieren, obgleich die kationischen Ladungen eine Abstoßung hervorrufen sollten. Die Assemblierung geht vermutlich zurück auf eine Interaktion der kationischen Aminosäure-Seitenketten mit den Amiden der Peptid-Bindung, die in der gestreckten Konformation exponiert sind. Diese Interaktion ist unter anderem bekannt bei der Bindung von Peptid-Liganden. In Anwesenheit von P5S3 wird die Bildung von Silikat aus übersättigter Kieselsäure-Lösung (30 mmol/L) unterstützt. Demgegenüber ist die Kieselsäure-Polymerisation in schwach übersättigter Lösung (8,3 mmol/L) bei niedrig mikromolarer P5S3-Konzentration verlangsamt. Die Auflösung von Silikat in ungesättigter Umgebung (1 mmol/L) wird bei niedriger P5S3-Konzentration ebenfalls verzögert. Die Interaktion mit Silikat verringert in P5S3 den Anteil der gestreckten Konformation, ersichtlich aus Änderungen im Zirkulardichroismus(CD)-Spektrum. Während der Silikat-Auflösung, bei der durchgängig Kieselsäure frei wird, werden im CD zwei Phasen offenbar: zunächst eine zunehmend stärker werdende P5S3-Silikat Interaktion, gefolgt von der P5S3-Freisetzung. Dies belegt erstmalig die dreifache Wirkung eines biomineralisierenden Polypeptides in der i) induzierten Silikat-Bildung, ii) verzögerten Kieselsäure-Polymerisation und iii) verlangsamten Silikat-Auflösung. Die CD-spektroskopische Untersuchung zeigt, dass diese Wirkungen auf die gleiche Peptid-Silikat Interaktion zurückgehen und von der jeweiligen Kieselsäure- und Peptid-Konzentration bedingt werden.Diatoms are unicellular algae which deposit silica to build mineralized cell walls. They exert spatial and temporal control during the formation of silica, thereby guiding the morphology of the deposited silica on the nano- to microscale. To induce the silica-formation, diatoms employ organic compounds such as peptides, carbohydrates and poly(alkylamines). By this, they are able to deposit silica under chemically benign, i.e. physiological conditions, a feature which renders their biomineralization-strategy interesting for technical applications. Silica-precipitating polypeptides in particular are interesting for biomimetic approaches since they can easily be varied in their amino acid sequence and are therefore a versatile class of biomineralizing agents. In this thesis, synthetic, silica-biomineralizing polypeptides with the general name PXSY were investigated. These peptides bear a high net cationic charge and are similar to silaffins, cationic peptides from diatoms which facilitate the in vitro deposition of silica, in terms of their amino acid sequence. The production of the PXSY polypeptides was achieved by recombinant expression in Escherichia coli, and their structure and dynamics in solution and upon the interaction with silica were assessed. The bacterial overexpression of the PXSY-polypeptides was achieved as fusion with a hydrophobic pro-sequence which mediates the deposition of the fusion in inclusion bodies, thereby preventing possible detrimental interactions of the cationic polypeptide with anionic cellular compounds such as nucleic acids or membrane lipids. After chemical cleavage for removal of the fusion, yields for the longest polypeptide of the PXSY-series (P5S3, 50 amino acids of which 20 are cationic) were in the range of 15 µmol per liter bacterial culture. In solution, the polypeptides adopt an extended structure with a considerable fraction of the polyproline II conformation. They self-assemble, even though their cationic charge should lead to electrostatic repulsion. This assembly presumably results from an interaction of cationic amino acid side chains with the peptide bond amides, the latter being exposed to the solvent in the extended conformation. Comparable interactions are known in folded proteins and in the interaction of proteins with peptide-ligands. P5S3 facilitates the formation of silica from supersaturated 30 mmol/L solutions of silicic acid. By contrast, at lower, but still supersaturated silicic acid concentration of 8.3 mmol/L, P5S3 at low micromolar concentration retards the polycondensation of silicic acid. Furthermore, the dissolution of silica at globally undersaturated 1 mmol/L silicic acid concentration is slowed down in the presence of P5S3 at low micromolar concentration. The extended conformation of P5S3 is reduced upon the interaction with silica, as revealed by circular dichroism (CD) spctroscopy. During the dissolution of silica, two phases are seen in CD spectroscopy during which silicic acid is constantly released: In the first phase, CD indicates a successively more intimate P5S3-silica association with a loss of the extended conformation, whereas in the second phase the spectra reveal a succesive liberation of P5S3 until the silica is dissolved and the peptide is again in the extended conformation it adopts in solution. This analysis for the first time reveals a triple-fold impact of a biomineralizing polypeptide in the i) induced silica-formation, ii) retarded silicic acid polymerisation and iii) retarded silica-dissolution. The CD spectroscopic analysis shows that these different effects are outcomes of the same peptide-silica interaction, though occur depending on the respective concentrations of silicic acid and the polypeptide.VI, 186 Seite

Interrogating metabolism as an electron flow system

Metabolism is generally considered as a neatly organised system of modular pathways, shaped by evolution under selection for optimal cellular growth. This view falls short of explaining and predicting a number of key observations about the structure and dynamics of metabolism. We highlight these limitations of a pathway-centric view on metabolism and summarise studies suggesting how these could be overcome by viewing metabolism as a thermodynamically and kinetically constrained, dynamical flow system. Such a systems-level, first-principles based view of metabolism can open up new avenues of metabolic engineering and cures for metabolic diseases and allow better insights to a myriad of physiological processes that are ultimately linked to metabolism. Towards further developing this view, we call for a closer interaction among physical and biological disciplines and an increased use of electrochemical and biophysical approaches to interrogate cellular metabolism together with the microenvironment in which it exists

Manganese oxide biomineralization is a social trait protecting

Manganese bio-mineralization is a widespread process among bacteria and fungi. To date there is no conclusive experimental evidence for, how and if this process impacts microbial fitness in the environment. Here we show how a model organism for manganese oxidation is growth-inhibited by nitrite, and that this inhibition is mitigated in presence of manganese. We show that such manganese-mediated mitigation of nitrite-inhibition is dependent on the culture inoculum size and that manganese oxide (MnOX) forms granular precipitates in the culture, rather than sheaths around individual cells. We provide evidence that MnOX protection involves both its ability to catalyze nitrite oxidation into (non-toxic) nitrate under physiological conditions, and its potential role in influencing processes involving reactive oxygen species (ROS). Taken together, these results demonstrate improved microbial fitness through MnOX deposition in an ecological setting, i.e. mitigation of nitrite toxicity, and point to a key role of MnOX in handling stresses arising from ROS

Impact of spatial organization on a novel auxotrophic interaction among soil microbes

A key prerequisite to achieve a deeper understanding of microbial communities and to engineer synthetic ones is to identify the individual metabolic interactions among key species and how these interactions are affected by different environmental factors. Deciphering the physiological basis of species–species and species–environment interactions in spatially organized environments requires reductionist approaches using ecologically and functionally relevant species. To this end, we focus here on a defined system to study the metabolic interactions in a spatial context among the plant-beneficial endophytic fungus Serendipita indica, and the soil-dwelling model bacterium Bacillus subtilis. Focusing on the growth dynamics of S. indica under defined conditions, we identified an auxotrophy in this organism for thiamine, which is a key co-factor for essential reactions in the central carbon metabolism. We found that S. indica growth is restored in thiamine-free media, when co-cultured with B. subtilis. The success of this auxotrophic interaction, however, was dependent on the spatial and temporal organization of the system; the beneficial impact of B. subtilis was only visible when its inoculation was separated from that of S. indica either in time or space. These findings describe a key auxotrophic interaction in the soil among organisms that are shown to be important for plant ecosystem functioning, and point to the potential importance of spatial and temporal organization for the success of auxotrophic interactions. These points can be particularly important for engineering of minimal functional synthetic communities as plant seed treatments and for vertical farming under defined conditions

Recombinant biomineralizing polypeptides PXSY : structure and dynamics upon the interaction with silica

Diatomeen bilden Zellhüllen aus Kieselsäure, wobei sie die Morphologie des abgelagerten Silikates auf der Nano- bis Mikroebene kontrollieren. Sie nutzen organische Verbindungen, wie Peptide, Kohlenhydrate und Poly(alkylamin), um die Silikat-Bildung zeitlich und räumlich zu steuern. Da die Silikat-Fällung in Diatomeen unter chemisch milden, physiologischen Bedingungen abläuft, ist deren Biomineraisations-Strategie interessant für Umsetzungen in technischen Applikationen. Insbesondere Polypeptide bieten breite Anwendungsmöglichkeiten, da sie in ihrer Sequenz variiert werden können und somit eine vielseitige Klasse von Biomineralisations-Agenzien darstellen. In dieser Arbeit wurden synthetische, Silikat-biomineralisierende Polypeptide mit hoher kationischer Nettoladung, genannt PXSY, bakteriell exprimiert und in Lösung, als auch während der Interaktion mit Silikat strukturell untersucht. Die PXSY Aminosäure-Komposition ist ähnlich jener der Silaffine, kationischen Peptiden aus Diatomeen welche die in vitro Silikat-Bildung induzieren. Die bakterielle Synthese der Polypeptide gelang als Fusion mit einer hydrophoben Pro-Sequenz, die die Ablagerung in Einschlusskörpern bewirkt und dadurch schädliche Interaktionen der kationischen Peptide mit Zellbestandteilen (Nukleinsäuren, Membranen) unterband. Nach chemischer Spaltung der Fusion wurden für das längste Polypeptid der PXSY-Serie (P5S3, 50 Aminosäuren, 20 kationische Ladungen) Ausbeuten bis zu 15 µmol pro Liter Expressionskultur erreicht. In Lösung nehmen die Polypeptide eine gestreckte Konformation mit hohem Anteil an Polyprolin-II-Sekundärstruktur ein. Sie assemblieren, obgleich die kationischen Ladungen eine Abstoßung hervorrufen sollten. Die Assemblierung geht vermutlich zurück auf eine Interaktion der kationischen Aminosäure-Seitenketten mit den Amiden der Peptid-Bindung, die in der gestreckten Konformation exponiert sind. Diese Interaktion ist unter anderem bekannt bei der Bindung von Peptid-Liganden. In Anwesenheit von P5S3 wird die Bildung von Silikat aus übersättigter Kieselsäure-Lösung (30 mmol/L) unterstützt. Demgegenüber ist die Kieselsäure-Polymerisation in schwach übersättigter Lösung (8,3 mmol/L) bei niedrig mikromolarer P5S3-Konzentration verlangsamt. Die Auflösung von Silikat in ungesättigter Umgebung (1 mmol/L) wird bei niedriger P5S3-Konzentration ebenfalls verzögert. Die Interaktion mit Silikat verringert in P5S3 den Anteil der gestreckten Konformation, ersichtlich aus Änderungen im Zirkulardichroismus(CD)-Spektrum. Während der Silikat-Auflösung, bei der durchgängig Kieselsäure frei wird, werden im CD zwei Phasen offenbar: zunächst eine zunehmend stärker werdende P5S3-Silikat Interaktion, gefolgt von der P5S3-Freisetzung. Dies belegt erstmalig die dreifache Wirkung eines biomineralisierenden Polypeptides in der i) induzierten Silikat-Bildung, ii) verzögerten Kieselsäure-Polymerisation und iii) verlangsamten Silikat-Auflösung. Die CD-spektroskopische Untersuchung zeigt, dass diese Wirkungen auf die gleiche Peptid-Silikat Interaktion zurückgehen und von der jeweiligen Kieselsäure- und Peptid-Konzentration bedingt werden.Diatoms are unicellular algae which deposit silica to build mineralized cell walls. They exert spatial and temporal control during the formation of silica, thereby guiding the morphology of the deposited silica on the nano- to microscale. To induce the silica-formation, diatoms employ organic compounds such as peptides, carbohydrates and poly(alkylamines). By this, they are able to deposit silica under chemically benign, i.e. physiological conditions, a feature which renders their biomineralization-strategy interesting for technical applications. Silica-precipitating polypeptides in particular are interesting for biomimetic approaches since they can easily be varied in their amino acid sequence and are therefore a versatile class of biomineralizing agents. In this thesis, synthetic, silica-biomineralizing polypeptides with the general name PXSY were investigated. These peptides bear a high net cationic charge and are similar to silaffins, cationic peptides from diatoms which facilitate the in vitro deposition of silica, in terms of their amino acid sequence. The production of the PXSY polypeptides was achieved by recombinant expression in Escherichia coli, and their structure and dynamics in solution and upon the interaction with silica were assessed. The bacterial overexpression of the PXSY-polypeptides was achieved as fusion with a hydrophobic pro-sequence which mediates the deposition of the fusion in inclusion bodies, thereby preventing possible detrimental interactions of the cationic polypeptide with anionic cellular compounds such as nucleic acids or membrane lipids. After chemical cleavage for removal of the fusion, yields for the longest polypeptide of the PXSY-series (P5S3, 50 amino acids of which 20 are cationic) were in the range of 15 µmol per liter bacterial culture. In solution, the polypeptides adopt an extended structure with a considerable fraction of the polyproline II conformation. They self-assemble, even though their cationic charge should lead to electrostatic repulsion. This assembly presumably results from an interaction of cationic amino acid side chains with the peptide bond amides, the latter being exposed to the solvent in the extended conformation. Comparable interactions are known in folded proteins and in the interaction of proteins with peptide-ligands. P5S3 facilitates the formation of silica from supersaturated 30 mmol/L solutions of silicic acid. By contrast, at lower, but still supersaturated silicic acid concentration of 8.3 mmol/L, P5S3 at low micromolar concentration retards the polycondensation of silicic acid. Furthermore, the dissolution of silica at globally undersaturated 1 mmol/L silicic acid concentration is slowed down in the presence of P5S3 at low micromolar concentration. The extended conformation of P5S3 is reduced upon the interaction with silica, as revealed by circular dichroism (CD) spctroscopy. During the dissolution of silica, two phases are seen in CD spectroscopy during which silicic acid is constantly released: In the first phase, CD indicates a successively more intimate P5S3-silica association with a loss of the extended conformation, whereas in the second phase the spectra reveal a succesive liberation of P5S3 until the silica is dissolved and the peptide is again in the extended conformation it adopts in solution. This analysis for the first time reveals a triple-fold impact of a biomineralizing polypeptide in the i) induced silica-formation, ii) retarded silicic acid polymerisation and iii) retarded silica-dissolution. The CD spectroscopic analysis shows that these different effects are outcomes of the same peptide-silica interaction, though occur depending on the respective concentrations of silicic acid and the polypeptide

Engineering microbial communities using thermodynamic principles and electrical interfaces

Microbial communities present the next research frontier. We argue here that understanding and engineering microbial communities requires a holistic view that considers not only species-species, but also species-environment interactions and feedbacks between ecological and evolutionary dynamics (eco-evo feedbacks). Due this multi-level nature of interactions, we predict that approaches aimed soley at altering specific species populations in a community (through strain enrichment or inhibition), would only have a transient impact, and species-environment and eco-evo feedbacks would eventually drive the microbial community to its original state. We propose a higher-level engineering approach that is based on thermodynamics of microbial growth, and that considers specifically microbial redox biochemistry. Within this approach the emphasis is on enforcing specific environmental conditions onto the community, that generates higher-level thermodynamic bounds onto the system, which the community structure and function can then adapt to. We believe that the resulting end-state can be ecologically and evolutionarily stable, mimicking the natural states of complex communities. Towards designing the exact nature of the environmental enforcement, thermodynamics and redox biochemistry can act as coarse-grained principles, while the use of electrodes - as electron providing or accepting redox agents - can provide implementation with spatiotemporal control

HemaCAM® - a computer assisted microscopy system for hematology

Cost and competition force modern hematology laboratories to further automate their processes. To that respect the examination and analysis of the peripheral blood is of central importance as it is relevant to a large variety of diseases while on the other hand financial reimbursement is low. Over the past eight years, the HemaCAM system has been developed by the Fraunhofer IIS, which supports the assessment of peripheral blood samples and the so-called white blood differential. Since 2010, HemaCAM has been available on the market as a certified medical product, to be more specific as an in vitro diagnostic device. This contribution provides an overview of the key components of the HemaCAM system

Impact of spatial organization on a novel auxotrophic interaction among soil microbes

A key prerequisite to achieve a deeper understanding of microbial communities and to engineer synthetic ones is to identify the individual metabolic interactions among key species and how these interactions are affected by different environmental factors. Deciphering the physiological basis of species-species and species-environment interactions in spatially organized environment requires reductionist approaches using ecologically and functionally relevant species. To this end, we focus here on a specific defined system to study the metabolic interactions in a spatial context among a plant-beneficial endophytic fungus Serendipita indica, and the soil-dwelling model bacterium Bacillus subtilis. Focusing on the growth dynamics of S. indica under defined conditions, we identified an auxotrophy in this organism for thiamine, which is a key co-factor for essential reactions in the central carbon metabolism. We found that S. indica growth is restored in thiamine-free media, when co-cultured with B. subtilis. The success of this auxotrophic interaction, however, was dependent on the spatial and temporal organization of the system; the beneficial impact of B. subtilis was only visible when its inoculation was separated from that of S. indica either in time or space. These findings describe a key auxotrophic interaction in the soil among organisms that are shown to be important for plant ecosystem functioning, and point to the potential importance of spatial and temporal organization for the success of auxotrophic interactions. These points can be particularly important for engineering of minimal functional synthetic communities as plant-seed treatments and for vertical farming under defined conditions