Performing Department
School of Plant and Environmental Sciences
Non Technical Summary
Problem Statement. There is growing circumstantial evidence that bacteria and fungi contribute to the formation of precipitation in clouds by catalyzing the freezing of very small cloud droplets that then grow into the size of much larger rain drops that are heavy enough to fall to the earth surface as precipitation. The process of catalyzing the freezing of water is called ice nucleation activity (INA) and any particle that has this activity is called an ice nucleus (IN). The biological basis of INA is only known for a single group of bacteria, called the Gammaproteobacteria. These bacteria all contain versions of the same gene, the ice nucleation activity (ina) gene. The protein encoded by this gene is inserted into the bacterial outer membrane and is the only identified and characterized biological INA molecule today. Searching for yet unknown bacteria with INA (also called Ice+ bacteria) in precipitation, the first Gram-positive Ice+ bacterium, Lysinibacillus parviboronicapiens (Lp), was identified. Similar to some fungal IN, INA of this bacterium is associated with secreted submicron sized IN. The secreted Lysinibacillus INA molecule (called LINA molecule) and secreted fungal INA molecules represent yet un-explored INA mechanisms.Relevance to Advancing Virginia, the Region, and the U.S. Virginia's agriculture, which totaled almost $3.5 billion in cash receipts in 2017, heavily depends on regular rainfall. Both droughts as well as excessive rainfall negatively affect Virginia farmers by reducing overall crop yield and quality. Also, the fruit industry in Virginia can be severely affected by spring frosts during bloom, a risk that increases after mild winters. Finally, Virginia ski resorts heavily depend on natural snow fall and on the ability to make artificial/technical snow with the use of snow cannons/fans, which is a costly endeavor. Therefore, the proposed study of INA molecules that affect freezing of water, and that possibly play a role in precipitation, is not only relevant to Virginia, but also the region, and the entire U.S. The long-term goal is to take advantage of our increased understanding of INA to develop interventions that can alleviate the listed problems.Approach. The short-term objectives of this project are to identify and characterize the genetic basis of the biosynthesis of the LINA molecule, to characterize the LINA molecule itself, to determine the distribution and frequency of LINA-producing bacteria, and to apply what is being learned to facilitate the identification and characterization of the INA molecules secreted by some fungi. First, the LINA molecule will be investigated using a combination of molecular biology, microbial genetic, bioinformatic, and analytical chemistry approaches. Secondly, LINA-producing bacteria will be isolated from the environment and putative LINA genes will be sequenced directly from environmental samples without culturing. Finally, fungi that secrete INA molecules will be compared to closely related fungi that do not secrete INA molecules using a combination of DNA sequencing and analytical chemistry approaches.Anticipated Outcomes and Impacts. Identifying the LINA molecule in a relatively simple bacterial system can be expected to facilitate the identification of similar molecules produced by fungi. Further, this project can be expected to provide the basis for possibly developing the LINA molecule and fungal INA molecules into powerful tools for weather modification to either mitigate droughts or prevent catastrophic precipitation events by cloud seeding for increased crop yield, or on a smaller scale, for snow making in ski resorts. Finally, identifying the secreted LINA molecule and secreted fungal INA molecules will provide new tools to advance our basic understanding of the physical process of ice nucleation. In fact, the purified molecules can be added to water in the absence of bacterial cells making it relatively easy to analyze how they catalyze freezing. This is very different compared to the INA protein produced by the Gammaproetobacteria, for which whole bacteria need to be added to water since the INA protein of the Gammaproetobacteria only displays its activity when part of the bacterial membrane.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Goal 1: Determine the genetic basis of how the LINA molecule is synthesized and use analytical chemistry to characterize the LINA molecule itself.Based on the preliminary data, our current hypothesis is that the LINA molecule is produced by enzymes encoded by an operon consisting of a PKS, a NRPS, and a thioesterase. We will test this hypothesis by: 1. genetically complementing the already obtained UV mutant strains that have mutations in the PKS/NRPS/thioesterase gene cluster and test the genetically complemented mutants for having regained INA, 2. cloning the PKS/NRPS/thioesterase operon and ectopically expressing it in Escherichia coli and Bacillus subtilis and testing the obtained strains for INA. Also, comparison of the cell-free culture supernatant of the wild-type Ice+ Lp strain with our mutant Ice- strains will facilitate the use of analytical chemistry to characterize the LINA molecule itself.Goal 2: Determine the environmental distribution of Lysinibacillus bacteria with INA and the presence of the putative LINA genes in environmental samples.Most Lysinibacillus species described to date were isolated by chance without any comprehensive sampling campaign. Therefore, the exclusive occurrence of INA in the L. parviboronicapiens species may be an artefact of insufficient sampling. To gain a more comprehensive view of the distribution of Lysinibacillus bacteria with INA, we will use the enrichment medium we developed to systematically search for Lysinibacillus strains in precipitation, soil, and plants. Isolated Lysinibacillus strains will be tested for INA and their genomes will be sequenced for precise species-level identification. In parallel, culture-independent sequencing approaches will be used to determine the distribution of putative LINA genes in environmental samples.Goal 3: Leverage expertise gained from the study of the LINA mechanism to identify the genetic basis and chemistry of INA molecules secreted by fungi.To start identifying the genetic basis and chemistry of secreted fungal INA molecules, we will first obtain a collection of fungi that have been found to produce secreted INA molecules, in particular, Fusarium species and Mortierella species. After confirming INA, the genomes of these fungi will be sequenced and compared to the genomes of closely related fungi that do not have INA. Genes only present in the fungi with INA will then be characterized using bioinformatic tools. In parallel, the cell-free culture supernatants of fungi with INA will be compared to cell-free culture supernatants of fungi without INA and analytical chemistry will be used to start investigating the chemical composition of the secreted INA molecules.
Project Methods
Goal 1Experimental designComplementation of existing UV mutants: We will attempt to complement our existing Ice- UV mutant strains to determine if the identified PKS and NRPS genes are necessary for LINA. We already cloned the PKS gene into an E. coli - Bacillus/Lysinibacillus shuttle vector. Next, the NRPS gene will be cloned. In parallel, we will optimize the transformation protocol of Lp to facilitate the introduction of DNA constructs into the UV mutants. Putatively complemented UV mutants will be confirmed by DNA sequencing and by testing for INA.Cloning and ectopic expression of the putative LINA gene cluster: To determine if the PKS/NRPS/thioesterase gene cluster is sufficient for biosynthesis of the LINA molecule, we will clone the gene cluster and express it in E. coli and in B. subtilis. The gene cluster will be cloned by assembling smaller DNA fragments since it is too long to be cloned in a single step. Also, promoters known to be functional in E. coli and in B. subtilis will be added. The advantage is that E. coli and B. subtilis are established genetic systems for which efficient protocols for transformation already exist.Analytical chemistry of the LINA molecule: Our biggest limitation so far in identifying the LINA molecule is the small quantity we can produce. We will thus compare different growth media and conditions under which Lp not only grows better but also produces larger amounts of the LINA molecule. Fortunately, purification is not problematic since the molecule is in the supernatant and can be resuspended from the 100kDa filter after washing with 50% methanol. As described in the preliminary data section, we will also start concentrating the 100 kDa retentate using lyophilization. The use of this washed and concentrated filtrate can be expected to be pure enough for gel filtration and liquid chromatography - mass spectrometry (LC-MS). Comparing wild-type and mutant preparations should allow us to identify the molecular weight of a peak only present in the wild-type but not in the mutant and that should correspond to the LINA molecule.Statistical analyses: The only aspect of this goal that is quantitative and needs a statistical analysis is the comparison of the strength of INA between wild-type Ice+ Lp, the Ice- mutants, and the complemented mutants. We will apply the analysis described in our previous work, which is based on the work by Vali.Potential difficulties and alternative plansWe expect to be able to genetically complement the Ice- mutants to confirm the putative LINA biosynthetic genes. However, if transformation were to fail, we could continue with the UV mutant screen to saturation to confirm that the PKS and NRPS genes are necessary for LINA. If we ran into problems with the cloning of the PKS/NRPS/thioesterase gene cluster, we could construct a genomic library and then transform a clone containing the gene cluster into B. subtilis.Goal 2Experimental designIsolation of Lysinibacillus strains from the environment: While our Ice+ Lp strains were isolated from precipitation, the type strain of Lp and many other Lysinibacillus species were isolated from soil and type strains of other Lysinibacillus species were isolated from plants. Therefore, to identify additional close relatives of the Ice+ Lp strains, we will use the enrichment medium described in the preliminary data section to isolate Lysinibacillus strains from newly collected precipitation, soil, and plants. Colonies will be analyzed using PCR primers specific to Lp (already designed and tested) and tested for INA.Genome sequencing: all newly isolated colonies identified as Lysinibacillus and all Lysinibacillus strains available in culture collections for which no genome sequence has yet been published will be genome-sequenced using Illumina MiSeq technology. Eighteen Lysinibacillus type strains (of a total of 28 Lysinibacillus species type strains) are already in our collection. We successfully sequenced the genomes of nine of these type strains. Genome sequences of another 11 type strains are publicly available.Comparative genomics: Lysinibacillus genomes will be compared using various bioinformatic pipelines. Using these programs, we will try to find answers to questions such as these: 1. What is the phylogeny of Lysinibacillus? 2. What is the distribution of the putative LINA biosynthetic gene cluster in the Lysinibacillus genus? 3. How many species exist within the Lysinibacillus genus and which of them include strains with INA?Culture-independent analysis of the distribution of the LINA gene cluster in the environment: We will query publicly available metagenomic data sets with the sequence of the putative LINA biosynthetic gene cluster to determine the distribution of this gene cluster in different environments. We will also complement searches of public data with our own metagenomic analysis of precipitation.Statistical analyses: same as for goal 1.Potential difficulties and alternative plansExperiments in this goal can be expected to give us detailed insight into the environmental distribution of the genus Lysinibacillus, and the distribution of the LINA gene cluster. We have over 15 years of experience in the analysis of genome sequences and in comparative evolutionary genomics. We recently started to use metagenomics as well. Since we already identified a putative LINA gene cluster and we already have Lysinibacillus and Lp genome sequences available, we can start with goal 2 experiments in parallel to goal 1 experiments even before we isolate new strains.Goal 3Experimental designTesting of fungal strains for INA: Fungal species with INA are common in the genera Fusarium and Mortierella. We will obtain strains of these species from collaborators and test them for INA. Testing for INA of fungi follows the same protocol as testing INA of bacteria. We thus have the necessary equipment and expertise.Genome sequencing and comparative genomics of fungal strains with INA: DNA of the fungal strains that test positive for INA will be extracted using standard protocols. Genome sequences will be assembled and genes will be predicted. The Gene content of fungal genomes will then be compared using comparative genomics pipelines, such as OrthoMCL and Get-Homologues. Genes only present in the genomes of fungal strains with INA will be considered as putative INA genes. Because INA molecules of Fusarium and Mortierella have been found to be secreted and partially heat resistant similar to the LINA molecule, we will also test the specific hypothesis that fungal INA molecules are polyketides. We will do this by searching for polyketides synthases among the genes that are unique to the fungal strains with INA.Analytical chemistry of fungal INA molecules: Since fungal INA molecules have been found to be secreted and to be similar in size to the LINA molecule, we will use the same differential filtration to isolate fungal INA molecules as we did for the LINA molecule. The purified INA molecules with then be analyzed as described for the LINA molecule in goal 1, including gel filtration and liquid chromatography - mass spectrometry (LC-MS). Moreover, we will compare preparations obtained from fungal strains with INA with those obtained from fungal strains without INA.Statistical analysis: the same as for goal 1.Potential difficulties and alternative plansGoal 3 is an exploratory goal to start investigating the genetic and chemical basis of fungal INA. With our expertise in testing INA and in genome sequence analysis and comparison, we do not anticipate major problems with this goal. Therefore, we expect to identify candidate INA genes and to get some initial insight into the fungal INA molecules. However, we do not expect to bring this goal to completion and to confirm the identity of all genes necessary for fungal INA or to determine the precise structure of any fungal INA molecule.