Identification and characterization of ice nucleation active bacteria isolated from precipitation
Failor, Kevin Christopher
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Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera.
- Doctoral Dissertations