Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation

Probing the Subcellular Localization of Hopanoid Lipids in Bacteria Using NanoSIMS

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary ^2H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes

SIGMA: Bulletin of European statistics No 2-3 1994. Statistics of services

We present the fabrication and characterization of an aluminum transmon qubit on a silicon-on-insulator substrate. Key to the qubit fabrication is the use of an anhydrous hydrofluoric vapor process which selectively removes the lossy silicon oxide buried underneath the silicon device layer. For a 5.6 GHz qubit measured dispersively by a 7.1 GHz resonator, we find T_1 = 3.5 μs and T_2* = 2.2 μs. This process in principle permits the co-fabrication of silicon photonic and mechanical elements, providing a route towards chip-scale integration of electro-opto-mechanical transducers for quantum networking of superconducting microwave quantum circuits. The additional processing steps are compatible with established fabrication techniques for aluminum transmon qubits on silicon

Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

Combined systems approaches reveal highly plastic responses to antimicrobial peptide challenge in Escherichia coli

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies

Broad neutralization of SARS-related viruses by human monoclonal antibodies

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines

Biothermodynamic studies of adsorption of monoclonal antibodies on chromatographic materials

Monoklonale Antikörper sind therapeutische Proteine mit großem Potential in der modernen Medizin. Für ihre kostenoptimierte Produktion und ein rationales Design der technischen Trennprozesse ist ein Verständnis der Zusammenhänge zwischen den Prozessparametern, den Vorgängen in der Proteinlösung und dem Adsorptionsvorgang notwendig. In der vorliegenden Arbeit wurden systematisch thermodynamische Untersuchungen zum Adsorptionsvorgang zweier monoklonaler Antikörper an CEC-Trennmaterialien sowie einige erste Untersuchungen an HIC-Trennmaterialien durchgeführt. Für die CEC-Trennmaterialien wurden in Batch-Experimenten Gleichgewichts-Adsorptionsisothermen bei unterschiedlichen pH-Werten, Salzkonzentrationen und unterschiedlichen Adsorbermaterialien aufgenommen. An diese Gleichgewichtsdaten wurden Modelle aus der Literatur angepasst. Zur reinen Korrelation der Meßwerte wurde sowohl das Langmuir-Modell als auch das Langmuir-Freundlich-Modell verwendet. Beide Modelle wurden verglichen und bewertet. Das Langmuir-Freundlich-Modell liefert erwartungsgemäß eine bessere Anpassung der Daten. Für die thermodynamische Betrachtung ist es jedoch aufgrund unphysikalischer Aussagen über den Zustand unendlicher Verdünnung ungeeignet. Zur Beschreibung der Abhängigkeit der Proteinadsorption von der Salzkonzentration wurden das steric mass-action (SMA) und das available area Modell (AA) bewertet. Beide liefern ähnliche Ergebnisse. Bei pH-Werten nahe dem isoelektrischen Punkt der Antikörper konnte die Abhängigkeit der Adsorption von der Salzkonzentration mit diesen Modellen sehr gut beschrieben werden. Die aus der Anpassung erstellten Modellparameter lassen sich physikalisch sinnvoll interpretieren. Die Antikörpernettoladung ist hier gering. Bei hohen Antikörperladungen, d.h. pH-Werte, die deutlich vom pI abweichen, treffen wesentliche Annahmen beider Modelle nicht mehr zu, sodass sich keine sinnvollen Korrelationen mehr erzielen lassen. Zusätzlich wurden in der Arbeit unterschiedliche Methoden zur Bestimmung der SMA- Modellparameter vorgestellt und untersucht. Einerseits können die Parameter durch Anpassung an Gleichgewichtsisothermen, andererseits direkt aus Chromatographieläufen bestimmt werden. Es zeigte sich eine sehr gute Übereinstimmung. Eine weitere Methode, um die thermodynamischen Grundlagen des Adsorptionsmechanismus zu untersuchen, ist die Mikrokalorimetrie. Durch Experimente mit einem isothermen Titrationskalorimeter konnte in Verbindung mit den Ergebnissen der Adsorptionsisothermen die spezifische Adsorptionsenthalpie als wichtige thermodynamische Größe bestimmt werden. hasd ist sowohl vom Beladungszustand des Adsorbers, als auch vom pH-Wert der Pufferlösung abhängig. Entlang der Isotherme ist hads nicht konstant und nimmt betragsmäßig ab. In Abhängigkeit der Adsorberbeladung adsorbieren die Antikörper auf unterschiedliche Art und Weise und nehmen andere Konformationen bzw. Orientierungen ein. Während bei neutralen pH-Werten hads negativ ist, ist die Adsorption im Sauren dagegen endotherm und entropisch getrieben, verursacht durch den großen Einfluss der Ionenwolke und den bei der Adsorption im Sauren verursachten Entropiegewinn. Die Kombination aus kalorimetrischen Experimenten und Adsorptionsisothermen ermöglicht eine Berechnung der thermodynamischen Parameter der Adsorption gads, hads,sads und Keq . Im Rahmen dieser Arbeit dienten statische Laserlichtstreumessungen zur Untersuchung der Wechselwirkungen der gelösten Antikörper. Dabei wurde der zweite osmotische Virialkoeffizient A2 sowie die massengemittelte Molmasse Mw ermittelt. Bei den Messungen wurden der pH-Wert, die Salzkonzentration und die Salzart variiert. Mit steigender Salzkonzentration nimmt der A2 ab und für hohe Salzkonzentrationen wird er negativ, da starke anziehende Wechselwirkungen überwiegen. Die Lichtstreumessungen wurden nach zwei unterschiedlichen Methoden durchgeführt. Zum einen im Batch-Modus (SLS-Batch-Modus) und zum anderen im online Modus bei dem ein Laser-Detektor in ein HPLC-System mit SEC-Säulen integriert ist (online SLS-SEC-Modus). Beide Methoden zeigen konsistente Resultate. Zur Auswertung wurden in beiden Fällen selbst entwickelte Programme eingesetzt, die auf dem Debye-Plot basieren. Die online Bestimmung des A2 war mit der kommerziell erhältlichen Software bislang nicht möglich. Das entwickelte Programm ermöglicht nun die Bestimmung aus einer einzigen Injektion in die HPLC mit nur einer Proteinkonzentration. Im Rahmen dieser Arbeit konnte gezeigt werden, dass thermodynamische Untersuchungen einen Beitrag zum besseren Verständnis der Proteinadsorption und der Vorgänge sowie der Wechselwirkungen in Proteinlösungen liefern. Dieses Wissen und die hier vorgestellten Methoden und Ergebnisse stellt eine Grundlage dar, um optimale Aufreinigungsbedingungen für ein Protein zu finden.Adsorption of therapeutic proteins on chromatographic materials is an attractive area of research which combines high academic challenges with the merits of working in an economically important and rapidly developing field. Downstream recovery of those high priced proteins is an important step in their production and high requirements on purity and aggregate content are imposed by health authorities. Protein adsorption on ion exchange (IEC) and hydrophobic interaction chromatographic (HIC) materials is extremely complex. The design of both separation methods is still based on trial and error by which the large number of process parameters like pH-value, type and quantity of salts have to be determined. Only a thorough understanding of the underlying physics and thermodynamics will allow a rational design of the technical process with its many interacting parameters. An interesting group of those therapeutic proteins are human monoclonal antibodies (hmAbs). Their importance in modern medicine is rapidly growing, because of their great potential to treat many different indications like autoimmune diseases or even cancer. For a successful design of a chromatographic protein separation with high yield and purities both the behaviour of the protein solution and the adsorption/desorption process have to be thoroughly understood. For this, the thermodynamic behaviour of the proteins under different buffer conditions is fundamental. The choice of suboptimal solution conditions can lead to less product yield, less protein solubility or even aggregates. This work extends the knowledge on thermodynamics of the adsorption of human monoclonal antibodies on cation exchange chromatographic (CEC) and HIC materials as well as the thermodynamic properties in solutions of monoclonal antibodies. The adsorption of two human monoclonal antibodies, which were supplied by an industrial partner, was studied with isothermal titration calorimetry (ITC) and by measuring equilibrium adsorption isotherms. Furthermore, solutions of the two hmAbs and of one mouse monoclonal antibody were studied with static laser light scattering (SLS). All relevant process parameters were systematically varied in the experiments. For the adsorption experiments with three different strong cation exchange resins and one hydrophobic resin the pH-value was varied between 4.5 and 7.0, using different buffer systems with different salt concentrations at 25°C. The equilibrium data were modelled using adsorption models from literature, like for example the Langmuir-model. Particular attention was paid to the modelling of the salt concentration which is a very important parameter for CEC. Therefore the steric mass-action (SMA) and the available area (AA) models were used and compared. Calorimetric techniques are useful for acquiring thermodynamic information on biological systems. ITC is the most important technique in that field. The advantage of ITC in the present application is its ability to directly monitor the adsorption process by detecting the heat released or taken up upon binding. The ITC data and the equilibrium adsorption isotherms allow determining the specific enthalpy of adsorption of the antibody hads. At pH-values near 7.0, where the antibodies are only weakly charged, the adsorption is exothermal. At small loadings the absolute number of hads is large and almost constant but it significantly decreases at higher loadings. This shows that the arrangement of antibody molecules on the absorber material depends on the loading and is less favourable at higher loadings. Despite the high positive charge of the antibody at pH-values of about 5.0 the value of hads is almost zero along the entire isotherm. Furthermore at pH 4.5 even endothermal effects were observed, although high binding capacities were found. At these conditions the adsorption process seems to be strongly influenced by the ions bound to the antibody. Their release upon adsorption explains the endothermal caloric effect. For the static laser light scattering (SLS) experiments the pH-value was varied between 4.5 and 10, as well as salt concentration of two different types of salt, namely sodium chloride and ammonium phosphate. From the results the second osmotic virial coefficient A2 and the mass-average molecular mass Mw were determined. Analysis of protein aggregates was performed with size-exclusion chromatography (SEC). At small pH-values and also small salt concentrations the A2 value is positive due to the positively charged antibodies and the resulting repulsive interactions. With increasing pH-values up to the isoelectric point a decreasing A2 was observed because of decreasing protein charge. At the isoelectric point there are attractive interactions which results in a poor solubility of the antibodies