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. / PHD / It is a common misconception that water freezes at 0°C (32°F). In clouds, water may remain liquid until -37 to -40°C (-35 to -40°F). At temperatures warmer than this, water molecules must collect around small particles that can help form ice, called ice nuclei. Numerous ice nuclei have been identified, ranging from dirt and dust, to volcanic ash, and even to pollen, fungi, and bacteria. One of these bacteria, Pseudomonas syringae, was identified as an ice nucleus in the 1970’s when it was discovered that it was increasing susceptibility of corn to frost damage. Since then, other Pseudomonas species as well as other bacteria within the same family of bacteria have been shown to have the ability to freeze water at relatively warm temperatures utilizing a specialized protein. Despite numerous studies on how these bacteria can exist in the atmosphere and how they can freeze water, the extent of this freezing ability, the concentration of bacteria in precipitation, and how cloud chemistry affects these bacteria has not been widely studied. In this study, precipitation was collected over the course of 15 months and the bacteria found within the collected precipitation were checked to see if they could act as ice nuclei. We found many of the previously described bacterial ice nuclei in the precipitation samples, but also identified a previously unidentified bacterium capable of freezing water. This bacterium, Lysinibacillus parviboronicapiens, does not use the same method of freezing as the other described bacterial ice nuclei. As such, we set out to determine the method it uses. We have determined that this bacterium utilizes a heat-stable, nanometer-sized particle that is not a protein. To better understand this molecule, representative strains of each species of this genus of bacteria were tested for their ability to freeze water, however, only this species has the ability. To further identify the molecule, UV radiation was used to disrupt the bacteria’s ability to freeze water, and the genes responsible were identified. Based on these results, we have tentatively identified the responsible genes as part of a polyketide synthase gene cluster. This gene cluster is responsible for producing small molecules that provide some survival advantage for the bacteria, in our case, possibly the ability to freeze water. As a final step, and to help serve other researchers, a comprehensive analysis of the entire Lysinibacillus genus has been performed and a reference guide has been generated to help describe and distinguish individual species.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/92196 |
| Date | 05 February 2018 |
| Creators | Failor, Kevin Christopher |
| Contributors | Plant Pathology, Physiology and Weed Science, Vinatzer, Boris A., Williams, Mark A., Schmale, David G. III, Barrett, John E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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