Release of Cell-Free Ice Nucleators from Three Recombinant Ice+Zymomonas mobilis Strains

2015 ◽  
Vol 25 (4) ◽  
pp. 277-283
Author(s):  
Athanasia Varsaki ◽  
Angelos Perisynakis ◽  
Constantin Drainas
Keyword(s):  
Growth Medium ◽  
Zymomonas Mobilis ◽  
Ice Nucleation ◽  
Temperature Threshold ◽  
Decarboxylase Activity ◽  
Ice Nucleation Protein ◽  
Produce Cell ◽  
Ice Nucleators ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

<b><i>Background/Aims:</i></b> This work is a study of the ability of three recombinant <i>Zymomonas mobilis </i>strains to release ice nucleators into their growth medium. <b><i>Methods:</i></b> The recombinant ice<sup>+</sup><i>Z. mobilis</i> cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. <b><i>Results:</i></b> Cell-free ice nucleators were obtained from all the recombinant ice<sup>+</sup><i>Z. mobilis</i> cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 μm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. <b><i>Conclusion:</i></b> These findings indicate that the recombinant ice<sup>+</sup><i>Z. mobilis</i> cells could be valuable for future use as a source of active cell-free ice nucleation protein.

scholarly journals Effects of atmospheric conditions on ice nucleation activity of <i>Pseudomonas</i>

2012 ◽  
Vol 12 (22) ◽  
pp. 10667-10677 ◽  
Author(s):  
E. Attard ◽  
H. Yang ◽  
A.-M. Delort ◽  
P. Amato ◽  
U. Pöschl ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Acidic Ph ◽  
Bacterial Cells ◽  
Atmospheric Conditions ◽  
Ice Nucleation Protein ◽  
Ice Melt ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

Abstract. Although ice nuclei from bacterial origin are known to be efficient at the highest temperatures known for ice catalysts, quantitative data are still needed to assess their role in cloud processes. Here we studied the effects of three typical cloud conditions (i) acidic pH (ii) NO2 and O3 exposure and (iii) UV-A exposure on the ice nucleation activity (INA) of four Pseudomonas strains. Three of the Pseudomonas syringae strains were isolated from cloud water and the phyllosphere and Pseudomonas fluorescens strain CGina-01 was isolated from Antarctic glacier ice melt. Among the three conditions tested, acidic pH caused the most significant effects on INA likely due to denaturation of the ice nucleation protein complex. Exposure to NO2 and O3 gases had no significant or only weak effects on the INA of two P. syringae strains whereas the INA of P. fluorescens CGina-01 was significantly affected. The INA of the third P. syringae strain showed variable responses to NO2 and O3 exposure. These differences in the INA of different Pseudomonas suggest that the response to atmospheric conditions could be strain-specific. After UV-A exposure, a substantial loss of viability of all four strains was observed whereas their INA decreased only slightly. This corroborates the notion that under certain conditions dead bacterial cells can maintain their INA. Overall, the negative effects of the three environmental factors on INA were more significant at the warmer temperatures. Our results suggest that in clouds where temperatures are near 0 °C, the importance of bacterial ice nucleation in precipitation processes could be reduced by some environmental factors.

Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates

2008 ◽  
Vol 5 (6) ◽  
pp. 373 ◽  
Author(s):  
Roya Mortazavi ◽  
Christopher T. Hayes ◽  
Parisa A. Ariya
Keyword(s):  
Bacterial Species ◽  
Ice Nucleation ◽  
Detailed Knowledge ◽  
Bacterial Populations ◽  
Inorganic Aerosols ◽  
Ice Nucleators ◽  
Species Population ◽  
Nucleation Activity ◽  
Ice Nucleation Activity ◽  
Snow Samples

Environmental context. Biological ice nucleators have been found to freeze water at very warm temperatures. The potential of bio-aerosols to greatly influence cloud chemistry and microphysics is becoming increasingly apparent, yet detailed knowledge of their actual role in atmospheric processes is lacking. The formation of ice in the atmosphere has significant local, regional and global influence, ranging from precipitation to cloud nucleation and thus climate. Ice nucleation tests on bacteria isolated from snow and laboratory-grown bacteria, in comparison with those of known organic and inorganic aerosols, shed light on this issue. Abstract. Ice nucleation experiments on bacteria isolated from snow as well as grown in the laboratory, in comparison with those of known organic and inorganic aerosols, examined the importance of bio-aerosols on cloud processes. Snow samples were collected from urban and suburban sites in the greater Montreal region in Canada (45°28′N, 73°45′W). Among many snow bacterial isolates, eight types of bacterial species, none belonging to known effective ice nucleators such as Pseudomonas or Erwinia genera, were identified to show an intermediate range of ice nucleation activity (–12.9 ± 1.3°C to –17.5 ± 2.8°C). Comparable results were also obtained for molten snow samples and inorganic suspensions (kaolin and montmorillonite) of buffered water solutions. The presence of organic molecules (oxalic, malonic and succinic acids) had minimal effect (<2°C) on ice nucleation. Considering experimental limitations, and drawing from observation in snow samples of a variety of bacterial populations with variable ice-nucleation ability, a shift in airborne-species population may significantly alter glaciation processes in clouds.

scholarly journals Urediospores of <i>Puccinia</i> spp. and other rusts are warm-temperature ice nucleators and harbor ice nucleation active bacteria

2012 ◽  
Vol 12 (10) ◽  
pp. 26143-26171 ◽  
Author(s):  
C. E. Morris ◽  
D. C. Sands ◽  
C. Glaux ◽  
J. Samsatly ◽  
S. Asaad ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Plant Diseases ◽  
Spatial And Temporal Variability ◽  
Warm Temperature ◽  
Ice Nuclei ◽  
Ice Nucleators ◽  
The Usa ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

Abstract. In light of various features of the biology of the rust fungi and of the epidemiology of the plant diseases they cause that illustrate the important role of rainfall in their life history, we have characterized the ice nucleation activity (INA) of the aerially disseminated spores (urediospores) of this group of fungi. Urediospores of this obligate plant parasite were collected from natural infections from 7 species of weeds in France, from coffee in Brazil and from field and greenhouse-grown wheat in France, the USA, Turkey and Syria. Immersion freezing was used to determine freezing onset temperatures and the abundance of ice nuclei in suspensions of washed spores. Microbiological analyses of spores and subsequent tests of the ice nucleation activity of the bacteria associated with spores were deployed to quantify the contribution of bacteria to the ice nucleation activity of the spores. All samples of spores were ice nucleation active having freezing onset temperatures as warm as −4 °C. Spores in most of the samples carried cells of ice nucleation-active strains of the bacterium Pseudomonas syringae (at rates of less than 1 bacterial cell per 100 urediospores), but bacterial INA accounted for only a small fraction of the INA observed in spore suspensions. Changes in the INA of spore suspensions after treatment with lysozyme suggest that the INA of urediospores involves a polysaccharide. Based on data from the literature, we have estimated the concentrations of urediospores in air at cloud height and in rainfall. These quantities are very similar to those reported for other biological ice nucleators in these same substrates. We suggest that air sampling techniques have ignored the spatial and temporal variability of atmospheric concentrations that occur under conditions propitious for precipitation that could increase their local abundance intermittently. Nevertheless, we propose that the relative low abundance of warm-temperature biological ice nucleators in the atmosphere corresponds to optimal conditions for the processes of evolution to positively select for INA.

scholarly journals Effects of atmospheric conditions on ice nucleation activity of <i>Pseudomonas</i>

2012 ◽  
Vol 12 (4) ◽  
pp. 9491-9516 ◽  
Author(s):  
E. Attard ◽  
H. Yang ◽  
A.-M. Delort ◽  
P. Amato ◽  
U. Pöschl ◽  
...  
Keyword(s):  
Environmental Factors ◽  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Acidic Ph ◽  
Bacterial Cells ◽  
Atmospheric Conditions ◽  
Ice Nucleation Protein ◽  
Ice Melt ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

Abstract. Although ice nuclei from bacterial origin are known to be efficient at the highest temperatures known for ice catalysts, quantitative data are still needed to assess their role in cloud processes. Here we studied the effects of three typical cloud conditions (i) acidic pH (ii) NO2 and O3 exposure and (iii) UV-A exposure on the ice nucleation activity (INA) of four Pseudomonas strains. Three of the Pseudomonas syringae strains were isolated from cloud water and the phyllosphere and Pseudomonas fluorescens strain CGina-01 was isolated from Antarctic glacier ice melt. Among the three conditions tested, acidic pH caused the most significant effects on INA likely due to denaturation of the ice nucleation protein complex. Exposure to NO2 and O3 gases had no significant or only weak effects on the INA of two P. syringae strains whereas the INA of P. fluorescens CGina-01 was significantly affected. The INA of the third P. syringae strain showed variable responses to NO2 and O3 exposure. These differences in the INA of different Pseudomonas suggest that the response to atmospheric conditions could be strain-specific. After UV-A exposure, a substantial loss of viability of all four strains was observed whereas their INA decreased only slightly. This corroborates the notion that under certain conditions dead bacterial cells can maintain their INA. Overall, the negative effects of the three environmental factors on INA were more significant at the warmer temperatures. Our results suggest that in clouds where temperatures are near 0 °C, the importance of bacterial ice nucleation in precipitation processes could be reduced by some environmental factors.

scholarly journals Freezing nucleation apparatus puts new slant on study of biological ice nucleators in precipitation

2014 ◽  
Vol 7 (1) ◽  
pp. 129-134 ◽  
Author(s):  
E. Stopelli ◽  
F. Conen ◽  
L. Zimmermann ◽  
C. Alewell ◽  
C. E. Morris
Keyword(s):  
Light Transmission ◽  
Freezing Temperature ◽  
Partial Solution ◽  
Ice Nucleation ◽  
Precipitation Sample ◽  
Ice Nucleators ◽  
New Apparatus ◽  
Nucleation Activity ◽  
Ice Nucleation Activity ◽  
Scatter Light

Abstract. For decades, drop-freezing instruments have contributed to a better understanding of biological ice nucleation and its likely implications for cloud and precipitation development. Yet, current instruments have limitations. Drops analysed on a cold stage are subject to evaporation and potential contamination. The use of closed tubes provides a partial solution to these problems, but freezing events are still difficult to be clearly detected. Here, we present a new apparatus where freezing in closed tubes is detected automatically by a change in light transmission upon ice development, caused by the formation of air bubbles and crystal facets that scatter light. Risks of contamination and introduction of biases linked to detecting the freezing temperature of a sample are then minimized. To illustrate the performance of the new apparatus we show initial results of two assays with snow samples. In one, we repeatedly analysed the sample (208 tubes) over the course of a month with storage at +4 °C, during which evidence for biological ice nucleation activity emerged through an increase in the number of ice nucleators active around −4 °C. In the second assay, we indicate the possibility of increasingly isolating a single ice nucleator from a precipitation sample, potentially determining the nature of a particle responsible for a nucleation activity measured directly in the sample. These two seminal approaches highlight the relevance of this handy apparatus for providing new points of view in biological ice nucleation research.

scholarly journals Urediospores of rust fungi are ice nucleation active at > −10 °C and harbor ice nucleation active bacteria

2013 ◽  
Vol 13 (8) ◽  
pp. 4223-4233 ◽  
Author(s):  
C. E. Morris ◽  
D. C. Sands ◽  
C. Glaux ◽  
J. Samsatly ◽  
S. Asaad ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Ice Nucleation ◽  
Rust Fungi ◽  
Plant Diseases ◽  
Ice Nuclei ◽  
Ice Nucleators ◽  
The Usa ◽  
Immersion Freezing ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

Abstract. Various features of the biology of the rust fungi and of the epidemiology of the plant diseases they cause illustrate the important role of rainfall in their life history. Based on this insight we have characterized the ice nucleation activity (INA) of the aerially disseminated spores (urediospores) of this group of fungi. Urediospores of this obligate plant parasite were collected from natural infections of 7 species of weeds in France, from coffee in Brazil and from field and greenhouse-grown wheat in France, the USA, Turkey and Syria. Immersion freezing was used to determine freezing onset temperatures and the abundance of ice nuclei in suspensions of washed spores. Microbiological analyses of spores from France, the USA and Brazil, and subsequent tests of the ice nucleation activity of the bacteria associated with spores were deployed to quantify the contribution of bacteria to the ice nucleation activity of the spores. All samples of spores were ice nucleation active, having freezing onset temperatures as high as −4 °C. Spores in most of the samples carried cells of ice nucleation-active strains of the bacterium Pseudomonas syringae (at rates of less than 1 bacterial cell per 100 urediospores), but bacterial INA accounted for only a small fraction of the INA observed in spore suspensions. Changes in the INA of spore suspensions after treatment with lysozyme suggest that the INA of urediospores involves a polysaccharide. Based on data from the literature, we have estimated the concentrations of urediospores in air at cloud height and in rainfall. These quantities are very similar to those reported for other biological ice nucleators in these same substrates. However, at cloud level convective activity leads to widely varying concentrations of particles of surface origin, so that mean concentrations can underestimate their possible effects on clouds. We propose that spatial and temporal concentrations of biological ice nucleators active at temperatures > −10 °C and the specific conditions under which they can influence cloud glaciation need to be further evaluated so as to understand how evolutionary processes could have positively selected for INA.

Clustering of ice nucleation protein correlates with ice nucleation activity

Cryobiology ◽  
1990 ◽  
Vol 27 (4) ◽  
pp. 416-422 ◽  
Author(s):  
Gunhild M. Mueller ◽  
Paul K. Wolber ◽  
Gareth J. Warren
Keyword(s):  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Nucleation Activity ◽  
Ice Nucleation Activity

scholarly journals Characterization and recombinant expression of a divergent ice nucleation protein from ‘Pseudomonas borealis’

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1164-1169 ◽  
Author(s):  
Zhongqin Wu ◽  
Lei Qin ◽  
Virginia K. Walker
Keyword(s):  
Recombinant Expression ◽  
Ice Nucleation ◽  
The Other ◽  
Chromosome Walking ◽  
Ice Nucleation Protein ◽  
Flat Surfaces ◽  
Nucleation Activity ◽  
Sequence Encoding ◽  
Ice Nucleation Activity ◽  
Selection Of

Isolates of ‘Pseudomonas borealis’ were recovered after ice-affinity selection of summer-collected soils. ‘P. borealis’ DL7 was further characterized and shown to have ice nucleation activity (INA), a property that allows the crystallization of ice at temperatures close to the melting point, effectively preventing the supercooling of water. INA was optimally detected after culturing at temperatures consistent with psychrophilic growth. The sequence encoding the ‘P. borealis’ ice nucleation protein (INP) was obtained using both PCR and chromosome walking. When expressed in Escherichia coli, the resulting inaPb recombinants had INA. The ‘P. borealis’ sequence, dubbed inaPb, is clearly related to previously cloned INP genes, but it shows greater divergence. Sequence analysis suggests that there are two opposite flat surfaces, one relatively hydrophobic that likely serves as an ice template, and the other that could function as a complementary face to facilitate interprotein interaction for ice-step formation.

Effects of Ice Nucleation Protein Repeat Number and Oligomerization Level on Ice Nucleation Activity

2018 ◽  
Vol 123 (3) ◽  
pp. 1802-1810 ◽  
Author(s):  
M. L. Ling ◽  
H. Wex ◽  
S. Grawe ◽  
J. Jakobsson ◽  
J. Löndahl ◽  
...  
Keyword(s):  
Ice Nucleation ◽  
Repeat Number ◽  
Ice Nucleation Protein ◽  
Nucleation Activity ◽  
Ice Nucleation Activity
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