Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Airborne microorganisms utilize these proteins to enhance their survival by reducing their atmospheric residence times. INpro also have critical environmental effects including impacts on the atmospheric water cycle, through their role in cloud and precipitation formation, as well as frost damage on crops. INpro are ubiquitously present in the atmosphere where they are emitted from diverse terrestrial and marine environments. Even though bacterial genes encoding INpro have been discovered and sequenced decades ago, the details of how the INpro molecular structure and oligomerization foster their unique ice-nucleation activity remain elusive. Using machine-learning based software AlphaFold 2 and trRosetta, we obtained and analysed the first ab initio structural models of full length and truncated versions of bacterial INpro. The modeling revealed a novel beta-helix structure of the INpro central repeat domain responsible for ice nucleation activity. This domain consists of repeated stacks of two beta strands connected by two sharp turns. One beta-strand is decorated with a TxT amino acid sequence motif and the other strand has an SxL[T/I] motif. The core formed between the stacked beta helix-pairs is unusually polar and very distinct from previous INpro models. Using synchrotron radiation circular dichroism, we validated the β-strand content of the central repeat domain in the model. Combining the structural model with functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. Using computational docking of the new INpro model based on rigid-body algorithms we could reproduce a previously proposed homodimer structure of the INpro CRD with an interface along a highly conserved tyrosine ladder and show that the dimer model agrees with our functional data. The parallel dimer structure creates a surface where the TxT motif of one monomer aligns with the SxL[T/I] motif of the other monomer widening the surface that interacts with water molecules and therefore enhancing the ice nucleation activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity.

The Arctic is a hot spot for climate change with potentially large consequences on a global scale. Aerosols, including bioaerosols, are important players in regulating the heat balance through direct interaction with sunlight and indirectly, through inducing cloud formation. Airborne bacteria are the major bioaerosols with some species producing the most potent ice nucleating compounds known, which are implicated in the formation of ice in clouds. Little is known about the numbers and dynamics of airborne bacteria in the Arctic and even less about their seasonal variability. We collected aerosol samples and wet deposition samples in spring 2015 and summer 2016, at the Villum Research Station in Northeast Greenland. We used amplicon sequencing and qPCR targeting the 16S rRNA genes to assess the quantities and composition of the DNA and cDNA-level bacterial community. We found a clear seasonal variation in the atmospheric bacterial community, which is likely due to variable sources and meteorology. In early spring, the atmospheric bacterial community was dominated by taxa originating from temperate and Subarctic regions and arriving at the sampling site through long-range transport. We observed an efficient washout of the aerosolized bacterial cells during a snowstorm, which was followed by very low concentrations of bacteria in the atmosphere during the consecutive 4 weeks. We suggest that this is because in late spring, the long-range transport ceased, and the local sources which comprised only of ice and snow surfaces were weak resulting in low bacterial concentrations. This was supported by observed changes in the chemical composition of aerosols. In summer, the air bacterial community was confined to local sources such as soil, plant material and melting sea-ice. Aerosolized and deposited Cyanobacteria in spring had a high activity potential, implying their activity in the atmosphere or in surface snow. Overall, we show how the composition of bacterial aerosols in the high Arctic varies on a seasonal scale, identify their potential sources, demonstrate how their community sizes varies in time, investigate their diversity and determine their activity potential during and post Arctic haze.

Microbially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Their critical role in rain formation and frost damage of crops together with their diverse commercial applications warrant an in-depth understanding of their inherent ice nucleation mechanism. We used the machine-learning based software AlphaFold to develop the first ab initio structural model of a bacterial INpro which is a novel beta-helix structure consisting of repeated stacks of two beta strands connected by two sharp turns. Using the synchrotron radiation circular dichroism, we validated the β-strand content of the model. Combining functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity and the basis for investigating the mechanistic role of INpro-induced ice formation in the atmosphere, and for commercial design and production of ice-nucleating particles for industrial applications.

<p>The Arctic is a region that is particularly vulnerable to the impacts of climate change, as it is warming at a faster rate than the rest of the world. This warming causes a decline in multiyear sea ice cover, which results in an increasing open-ocean surface with a much lower albedo therefore leading to positive feedback and enhanced warming. Another factor that plays a role in regulating the temperature in the Arctic is the type and extent of cloud cover. Aerosols that can serve as cloud condensation nuclei or ice nucleating particles (INPs) are key for cloud formation. Some microorganisms are known to produce INPs, but it is not well understood which microorganisms are responsible, which environments they inhabit, and how active they are. In this study, we set out to investigate the partitioning of INPs between the Arctic marine and atmospheric environment by combining in situ measurements with laboratory experiments.</p> <p>First, we wanted to determine if sea ice acts as a reservoir for INPs and, if so, whether the INPs are partitioned into the sea ice during its formation or produced by microorganisms within the sea ice. We used a modified ice-finger to grow sea ice using natural samples from West Greenland and found that INPs concentrate into the ice fraction during sea-ice formation, and that these INPs typically are associated with microorganisms.</p> <p>Next, we wanted to understand the temporal and spatial dynamics of INPs in Arctic sea ice. We collected sea ice cores from the Arctic before and during the spring sea ice phytoplankton bloom and analysed them using cold-stage INP measurements, flow-cytometry, and amplicon sequencing. The results showed that there are between <10<sup>5</sup> · L<sup>-1</sup> (at the top) and >10<sup>6</sup> · L<sup>-1</sup>  (at the bottom of the sea ice) INP<sub>-10</sub> present in the Arctic sea ice.  </p> <p>Finally, we wanted to determine the potential contribution of sea ice to the atmospheric INP pool in the Arctic. We introduced natural samples of bulk water and sea ice from Nuuk and Station Nord into a temperature-controlled sea spray simulation chamber and quantified the microorganisms and INPs present in the bulk water, surface microlayer and air before and after aerosolization. The results showed that the highly active INPs are efficiently aerosolized into the atmosphere during bubble-bursting where they may contribute to the formation of ice in clouds.</p> <p>Overall, this study provides new insight into the role of Arctic sea ice as a reservoir for INPs and the microorganisms that produce them, as well as the mechanisms by which INPs are released into the Arctic atmosphere. This information is important for understanding the impact of climate change on the Arctic region and the potential consequences for the rest of the world.</p>

<p>With raising temperatures in the Arctic, the extent of sea ice is decreasing dramatically resulting in a larger fraction of the Arctic ocean surface being exposed to the atmosphere. Therefore, the ice-free ocean and in particular the sea surface microlayer (SML), which represents the upper 1 mm of the water column is becoming of increasing interest as a source of bioaerosols with ice nucleating properties. These biological ice nucleating particles (INPs) can be aerosolized by wave breaking and bubble bursting. In the atmosphere, they may trigger the freezing of cloud droplets and thus affect the lifetime of clouds as well as their radiative properties. Recent studies proposed a link between biological ice nucleating aerosols in the Arctic sea water and phytoplanktonic blooms.</p> <p>Thus, we examined the concentration and characteristics of INPs in both, the sea bulk water, and the surface microlayer for two locations in southwest Greenland throughout a phytoplankton bloom. Further, we investigated possible links between INP concentrations in the sea water, the abundance and community composition of bacteria and algae, as well as the phytoplanktonic growth season derived from satellite data and in-situ chlorophyll concentrations. Preliminary results will be presented.</p> <p> </p>