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 degrees C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at -8 degrees 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 nanometersized 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.