Ice nucleation active bacteria in precipitation are genetically diverse and nucleate ice by employing different mechanisms

A growing body of circumstantial evidence suggests that ice nucleation active (Ice+) bacteria contribute to the initiation of precipitation by heterologous freezing of super-cooled water in clouds. However, little is known about the concentration of Ice+ bacteria in precipitation, their genetic and phenotypic diversity, and their relationship to air mass trajectories and precipitation chemistry. In this study, 23 precipitation events were collected over 15 months in Virginia, USA. Air mass trajectories and water chemistry were determined and 33 134 isolates were screened for ice nucleation activity (INA) at −8 °C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at −8 °C in repeated tests. Concentrations of Ice+ strains in precipitation were found to range from 0 to 13 219 colony forming units per liter, with a mean of 384±147. Most Ice+ bacteria were identified as members of known and unknown Ice+ species in the Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae families, which nucleate ice employing the well-characterized membrane-bound INA protein. Two Ice+ strains, however, were identified as Lysinibacillus, a Gram-positive genus not previously known to include Ice+ bacteria. INA of the Lysinibacillus strains is due to a nanometer-sized molecule that is heat resistant, lysozyme and proteinase resistant, and secreted. Ice+ bacteria and the INA mechanisms they employ are thus more diverse than expected. We discuss to what extent the concentration of culturable Ice+ bacteria in precipitation and the identification of a new heat-resistant biological INA mechanism support a role for Ice+ bacteria in the initiation of precipitation.

[1]  Corinna Hoose,et al.  Heterogeneous ice nucleation on atmospheric aerosols: a review of results from laboratory experiments , 2012 .

[2]  T. Fujiwara,et al.  Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. , 2007, International journal of systematic and evolutionary microbiology.

[3]  G. Vali Sizes of Atmospheric Ice Nuclei , 1966, Nature.

[4]  B. Murray,et al.  Ice nucleation by particles immersed in supercooled cloud droplets. , 2012, Chemical Society reviews.

[5]  B. Murray,et al.  A technique for quantifying heterogeneous ice nucleation in microlitre supercooled water droplets , 2014 .

[6]  C. Morris,et al.  The Relationship of Host Range, Physiology, and Genotype to Virulence on Cantaloupe in Pseudomonas syringae from Cantaloupe Blight Epidemics in France. , 2000, Phytopathology.

[7]  P. Ariya,et al.  Ice nucleation activity of bacteria isolated from snow compared with organic and inorganic substrates , 2008 .

[8]  J Vandekerckhove,et al.  Identification and purification of a bacterial ice-nucleation protein. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Lindow,et al.  A model of the three-dimensional structure of ice nucleation proteins. , 1993, Journal of molecular biology.

[10]  C. Dearden,et al.  Ice formation and development in aged, wintertime cumulus over the UK: observations and modelling , 2011 .

[11]  H. Bauer,et al.  Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen , 2012 .

[12]  P. Fu,et al.  Evidence for a missing source of efficient ice nuclei , 2017, Scientific Reports.

[13]  S. Soubeyrand,et al.  Emigration of the plant pathogen Pseudomonas syringae from leaf litter contributes to its population dynamics in alpine snowpack. , 2012, Environmental microbiology.

[14]  Y. Travi,et al.  Soil water flow is a source of the plant pathogen Pseudomonas syringae in subalpine headwaters. , 2014, Environmental microbiology.

[15]  S. Lindow THE ROLE OF BACTERIAL ICE NUCLEATION IN FROST INJURY TO PLANTS , 1983 .

[16]  H. Antoun,et al.  Ice Nucleation Activity in Fusarium acuminatum and Fusarium avenaceum , 1992, Applied and environmental microbiology.

[17]  Markus D. Petters,et al.  Revisiting ice nucleation from precipitation samples , 2015 .

[18]  James R. Cole,et al.  Ribosomal Database Project: data and tools for high throughput rRNA analysis , 2013, Nucleic Acids Res..

[19]  David C. Sands,et al.  Ubiquity of Biological Ice Nucleators in Snowfall , 2008, Science.

[20]  P. Amato,et al.  Effects of atmospheric conditions on ice nucleation activity of Pseudomonas , 2012 .

[21]  M R Barer,et al.  Bacterial viability and culturability. , 1999, Advances in microbial physiology.

[22]  M. Bonn,et al.  Ice-nucleating bacteria control the order and dynamics of interfacial water , 2016, Science Advances.

[23]  P. Amato,et al.  Survival and ice nucleation activity of bacteria as aerosols in a cloud simulation chamber , 2015 .

[24]  R. Fall,et al.  Release of cell-free ice nuclei by Erwinia herbicola , 1986, Journal of bacteriology.

[25]  P. Connolly,et al.  Investigating the discrepancy between wet-suspension- and dry-dispersion-derived ice nucleation efficiency of mineral particles , 2015 .

[26]  Ice nucleation by water-soluble macromolecules , 2014 .

[27]  Paul J. DeMott,et al.  Measurement of Ice Nucleation-Active Bacteria on Plants and in Precipitation by Quantitative PCR , 2013, Applied and Environmental Microbiology.

[28]  S. J. Gilmour,et al.  Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria. , 2005, FEMS microbiology ecology.

[29]  C. Upper,et al.  Plants as Sources of Airborne Bacteria, Including Ice Nucleation-Active Bacteria , 1982, Applied and environmental microbiology.

[30]  L R Maki,et al.  Ice Nucleation Induced by Pseudomonas syringae , 1974, Applied microbiology.

[31]  S. S. Hirano,et al.  Atmospheric dispersal of ice nucleation-active Bacteria : the role of rain , 1990 .

[32]  R. Campbell,et al.  Novel dimeric β-helical model of an ice nucleation protein with bridged active sites , 2011, BMC Structural Biology.

[33]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[34]  C. Morris,et al.  A User's Guide to a Data Base of the Diversity of Pseudomonas syringae and Its Application to Classifying Strains in This Phylogenetic Complex , 2014, PloS one.

[35]  S. Borrmann,et al.  The ice nucleating ability of pollen: Part III: New laboratory studies in immersion and contact freezing modes including more pollen types , 2005 .

[36]  S. Lindow,et al.  Frost sensitivity of Zea mays increased by application of Pseudomonas syringae , 1976, Nature.

[37]  Ulrich Pöschl,et al.  Ice nucleation activity in the widespread soil fungus Mortierella alpina , 2015 .

[38]  C. Morris,et al.  Features of air masses associated with the deposition of Pseudomonas syringae and Botrytis cinerea by rain and snowfall , 2014, The ISME Journal.

[39]  S. Kreidenweis,et al.  Biogenic ice nuclei in boundary layer air over two U.S. High Plains agricultural regions , 2012 .

[40]  S. Lindow,et al.  Xanthomonas campestris pv. translucens strains active in ice nucleation , 1987 .

[41]  S. S. Hirano,et al.  Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury. , 1985, Plant physiology.

[42]  Johannes Quaas,et al.  Frequency of occurrence of rain from liquid‐, mixed‐, and ice‐phase clouds derived from A‐Train satellite retrievals , 2015 .

[43]  C. Morris,et al.  Ice nucleation active bacteria and their potential role in precipitation , 2004 .

[44]  R. Fall,et al.  High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature , 1993, Journal of bacteriology.

[45]  T. Clauss,et al.  Characterization of airborne ice-nucleation-active bacteria and bacterial fragments , 2015 .

[46]  G. Vali Quantitative Evaluation of Experimental Results an the Heterogeneous Freezing Nucleation of Supercooled Liquids , 1971 .

[47]  B. Murray,et al.  The relevance of nanoscale biological fragments for ice nucleation in clouds , 2015, Scientific Reports.

[48]  Jan P. Meier-Kolthoff,et al.  Genomic and Genetic Diversity within the Pseudomonas fluorescens Complex , 2016, PloS one.

[49]  P. Amato,et al.  Ice nucleation activity of bacteria isolated from cloud water , 2013 .

[50]  C. Morris,et al.  The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle , 2008, The ISME Journal.

[51]  T. Fujiwara,et al.  Lysinibacillus parviboronicapiens sp. nov., a low-boron-containing bacterium isolated from soil. , 2009, International journal of systematic and evolutionary microbiology.

[52]  K. Lewis,et al.  Isolating "Uncultivable" Microorganisms in Pure Culture in a Simulated Natural Environment , 2002, Science.

[53]  S. Lindow Erwinia herbicola: A Bacterial Ice Nucleus Active in Increasing Frost Injury to Corn , 1978 .

[54]  R. E. Lee,et al.  Isolation of ice-nucleating active bacteria from the freeze-tolerant frog, Rana sylvatica. , 1995, Cryobiology.

[55]  U. Pöschl,et al.  Bioprecipitation: a feedback cycle linking Earth history, ecosystem dynamics and land use through biological ice nucleators in the atmosphere , 2014, Global change biology.

[56]  David S. Guttman,et al.  Evolution of the Core Genome of Pseudomonas syringae, a Highly Clonal, Endemic Plant Pathogen , 2004, Applied and Environmental Microbiology.

[57]  Ruprecht Jaenicke,et al.  The ice nucleating ability of pollen:: Part II. Laboratory studies in immersion and contact freezing modes , 2002 .

[58]  C. Morris,et al.  Ice nucleation active particles are efficiently removed by precipitating clouds , 2015, Scientific Reports.

[59]  E. Bigg,et al.  Persistent after-effects of heavy rain on concentrations of ice nuclei and rainfall suggest a biological cause , 2015 .