Nitrate reducing commensal bacterium stimulates germination through nitric oxide production-a hypothesis [abstract]

Abstract only availableThe pink-pigmented (PPFM) commensal bacterium bacterium, Methylobacterium extorquens AM1, stimulates germination of soybean and other plants. I am testing the hypothesis that germination is stimulated by bacterial-produced nitric oxide (NO). A potential source of NO is nitrate reductase (NR) action on nitrite. Our goal was to identify NR gene(s), disrupt it(them) and examine germination stimulation of the mutants. Two nitrate reductase (NR) sequences were identified in the M. extorquens genome. Primers were designed and used to amplify both full-length and internal fragments of each NR gene. The internal fragments were cloned into a suicide vector (pAYC61) which encodes PPFM-expressed resistance gene to tetracycline (tetR). Tri-parental mating was then used to introduce pAYC61, carrying the internal NR sequences, into the PPFM strain, and tet-resistant PPFM colonies were selected. Methanol was used as the sole carbon source, thus selecting against the E. coli partners in the mating. A screen was devised to test among the tet-resistant progeny "exconjugates" for those with homologous integration of the internal NR sequence integrated into the respective host PPFM NR gene. Such integration will create two non-functional incomplete copies of the host NR gene. The screen will be based on PCR whereby primers will match vector sequence with NR sequence that is NOT part of the internal fragment. Once each NR gene is confirmed to be interrupted, the mutants will be tested for their ability to reduce nitrate to an utilizable nitrogen source. Germination stimulation and seedling root development will be examined by imbibing seed with progenitor and NR- PPFMs. The experiment will confirm or refute our observations of increased germination and lateral root formation in plants with PPFMs. Further, it will indicate whether such effects are due to nitric oxide produced by bacterial NR action on nitrate. Controls will be bacterium grown with without nitrate and seedlings grown with external NO sources and with an NO trap.MU Monsanto Undergraduate Research Fellowshi

What do pink pigs and soybeans have in common? Selecting for mutants in the ureide pathway

Abstract only availablePink Pigmented Facultative Methylotrophic bacteria (PPFMs) have been found to be the most abundant microorganisms among phylloplane microflora, and have been recovered from all plants examined. PPFMs are seed-transmitted and have been shown to enhance germination. PPFMs may contribute nitrogen, which is an essential nutrient, to the plant. In an attempt to determine this and gain a better understanding of the genes involved in ureide (allantoin, allantoate, etc,) utilization in PPFMs, we tried to ellicit mutants along the pathway. We performed a bi-parental mating between the PPFM soybean isolate (wild-type) and an E. coli strain containing a plasmid for a kanamycin resistance. This antibiotic resistance plasmid was randomly inserted into the PPFM's genome. The goal is to select mutants that lose the ability to break down ureides. Selection on different media is used to isolate the different mutants along the pathway. Seed surface sterilization does not remove PPFMs since they are found below the seed coat. We have devised a method to eliminate the bacteria by heating the seeds at 50°C for 48 hours. This treatment does not damage the seed. Heat-treated and un-heated soybean seeds were inoculated with a kanamycin resistant soybean isolate strain of PPFM (B140). These plants were grown to maturity in the greenhouse and seeds were collected. We germinated this second generation of seed and are looking to recover kanamycin resistant PPFM bacteria both from the seed as well as the first unifoliate leaves of the soybean. We were able to isolate several putative PPFM ureide utilization mutants. This research will give insight into the interactions between PPFMs and soybean that may be applied to many other plants. Experiments to recover kanamycin resistant bacteria from the seed and from the first unifoliate leaves of the plants are in progress.MU Monsanto Undergraduate Research Fellowshi

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19