Membranes Are Decisive for Maximum Freezing Efficiency of Bacterial Ice Nucleators

Ice-nucleating proteins (INPs) from Pseudomonas syringae are among the most active ice nucleators known, enabling ice formation at temperatures close to the melting point of water. The working mechanisms of INPs remain elusive, but their ice nucleation activity has been proposed to depend on the ability to form large INP aggregates. Here, we provide experimental evidence that INPs alone are not sufficient to achieve maximum freezing efficiency and that intact membranes are critical. Ice nucleation measurements of phospholipids and lipopolysaccharides show that these membrane components are not part of the active nucleation site but rather enable INP assembly. Substantially improved ice nucleation by INP assemblies is observed for deuterated water, indicating stabilization of assemblies by the stronger hydrogen bonds of D2O. Together, these results show that the degree of order/disorder and the assembly size are critically important in determining the extent to which bacterial INPs can facilitate ice nucleation.We thank L. Reichelt, N. Bothen, and N. M. Kropf for technical assistance. The TOC graphic and Figures 1 and 2B were created using BioRender.com.Ye

New perspectives on evolutionary medicine: the relevance of microevolution for human health and disease

Evolutionary medicine (EM) is a growing field focusing on the evolutionary basis of human diseases and their changes through time. To date, the majority of EM studies have used pure theories of hominin macroevolution to explain the present-day state of human health. Here, we propose a different approach by addressing more empirical and health-oriented research concerning past, current and future microevolutionary changes of human structure, functions and pathologies. Studying generation-to-generation changes of human morphology that occurred in historical times, and still occur in present-day populations under the forces of evolution, helps to explain medical conditions and warns clinicians that their current practices may influence future humans. Also, analyzing historic tissue specimens such as mummies is crucial in order to address the molecular evolution of pathogens, of the human genome, and their coadaptations.Frank Jakobus Rühli and Maciej Henneber

Electrostatic Interactions Control the Functionality of Bacterial Ice Nucleators

Bacterial ice-nucleating proteins (INPs) promote heterogeneous ice nucleation more efficiently than any other material. The details of their working mechanism remain elusive, but their high activity has been shown to involve the formation of functional INP aggregates. Here we reveal the importance of electrostatic interactions for the activity of INPs from the bacterium Pseudomonas syringae by combining a high-throughput ice nucleation assay with surface-specific sum-frequency generation spectroscopy. We determined the charge state of nonviable P. syringae as a function of pH by monitoring the degree of alignment of the interfacial water molecules and the corresponding ice nucleation activity. The net charge correlates with the ice nucleation activity of the INP aggregates, which is minimal at the isoelectric point. In contrast, the activity of INP monomers is less affected by pH changes. We conclude that electrostatic interactions play an essential role in the formation of the highly efficient functionally aligned INP aggregates, providing a mechanism for promoting aggregation under conditions of stress that prompt the bacteria to nucleate ice

Interfacial Water Ordering Is Insufficient to Explain Ice-Nucleating Protein Activity

Ice-nucleating proteins (INPs) found in bacteria are the most effective ice nucleators known, enabling the crystallization of water at temperatures close to 0 °C. Although their function has been known for decades, the underlying mechanism is still under debate. Here, we show that INPs from Pseudomonas syringae in aqueous solution exhibit a defined solution structure and show no significant conformational changes upon cooling. In contrast, irreversible structural changes are observed upon heating to temperatures exceeding ∼55 °C, leading to a loss of the ice-nucleation activity. Sum-frequency generation (SFG) spectroscopy reveals that active and heat-inactivated INPs impose similar structural ordering of interfacial water molecules upon cooling. Our results demonstrate that increased water ordering is not sufficient to explain INPs' high ice-nucleation activity and confirm that intact three-dimensional protein structures are critical for bacterial ice nucleation, supporting a mechanism that depends on the INPs' supramolecular interactions

Are proteinaceous agglomerates responsible for ice nucleation activity of birch pollen?

Various biological aerosol particles such as certain pollen, fungi, and bacteria are known as icenucleating particles with high onset freezing temperatures. It came as a surprise when Pummer et al.(2012) found that solubilized macromolecules were responsible for the ice nucleation activity of tree pollen, rather than the grains themselves. More recently, ice-nucleating macromolecules (INMs) have also been found on other tree tissues (Felgitsch et al., 2018, Seifried et al., 2020). In general, INMs are present in much greater numbers than the micrometer sized pollen grains and thus the emission of INMs from the biosphere might play a more important role than previously thought (Bieber et al., 2020, Burkart et al., 2021, Seifried et al., 2020, 2021).So far, the chemical composition and structure of INMs remains largely unknown. To shine light on this, we extracted INMs from birch pollen with water and conducted various treatments, purification, and freezing experiments. For example, we detected ice nucleation activity after filtration through a 10 kDa cutoff filter. However, the concentration after 10 kDa filtration was comparatively lower than after 30 kDa or 50 kDa filtration suggesting that the INMs consist of agglomerates