Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity.

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 ß-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.

Corrigendum: Effect of Aerosolization and Drying on the Viability of Pseudomonas syringae Cells.

[This corrects the article DOI: 10.3389/fmicb.2018.03086.].

Effect of Aerosolization and Drying on the Viability of Pseudomonas syringae Cells.

Airborne dispersal of microorganisms influences their biogeography, gene flow, atmospheric processes, human health and transmission of pathogens that affect humans, plants and animals. The extent of their impact depends essentially on cell-survival rates during the process of aerosolization. A central factor for cell-survival is water availability prior to and upon aerosolization. Also, the ability of cells to successfully cope with stress induced by drying determines their chances of survival. In this study, we used the ice-nucleation active, plant pathogenic Pseudomonas syringae strain R10.79 as a model organism to investigate the effect of drying on cell survival. Two forms of drying were simulated: drying of cells in small droplets aerosolized from a wet environment by bubble bursting and drying of cells in large droplets deposited on a surface. For drying of cells both in aerosol and surface droplets, the relative humidity (RH) was varied in the range between 10 and 90%. The fraction of surviving cells was determined by live/dead staining followed by flow cytometry. We also evaluated the effect of salt concentration in the water droplets on the survival of drying cells by varying the ionic strength between 0 and 700 mM using NaCl and sea salt. For both aerosol and surface drying, cell survival increased with decreasing RH (p < 0.01), and for surface drying, survival was correlated with increasing salt concentration (p < 0.001). Imaging cells with TEM showed shrunk cytoplasm and cell wall damage for a large fraction of aerosolized cells. Ultimately, we observed a 10-fold higher fraction of surviving cells when dried as aerosol compared to when dried on a surface. We conclude that the conditions, under which cells dry, significantly affect their survival and thus their success to spread through the atmosphere and colonize new environments as well as their ability to affect atmospheric processes.

The relationship between age of smoking initiation and current smoking: an analysis of school surveys in three European countries.

INTRODUCTION: It is held that younger smoking initiates are more likely to become regular smokers. The definitions of smoking initiation (a puff, part of a cigarette, a whole cigarette) are inconsistent and raise questions about the robustness of the view. We sought to re-examine the relationship using adolescent smoking data from 3 European countries. METHODS: A stratified secondary, logistic regression analysis of Global Youth Tobacco Survey data was conducted using a design-based analysis. Subgroup analyses were conducted of 13- to 15-year olds from Latvia (high smoking prevalence), Slovenia (moderate prevalence), and Montenegro (low prevalence) who had initiated smoking. The outcome was current smoking--smoking everyday for the past 30 days, or smoking 10 or more days in the past 30 days. Smoking initiation was operationalized as a single puff of a cigarette, and age of smoking initiation was a derived continuous measure. RESULTS: In Latvia, there was a significant association between age of smoking initiation and current smoking for males (p < .05) and females (p < .001) when smoking was operationalized as smoking every day. It was only significant in female adolescents (p < .001) for smoking 10 or more days. In Slovenia and Montenegro, there was no significant relationship between age of smoking initiation and current smoking for either males or females. CONCLUSIONS: The evidence about the relationship between age of smoking initiation and current smoking is not clear. Explanations for the findings may relate to a lack of power, the specificity of the measure, or problems with the theory.