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

Dynamical response of the "GGG" rotor to test the Equivalence Principle: theory, simulation and experiment. Part I: the normal modes

Recent theoretical work suggests that violation of the Equivalence Principle might be revealed in a measurement of the fractional differential acceleration $\eta$ between two test bodies -of different composition, falling in the gravitational field of a source mass- if the measurement is made to the level of $\eta\simeq 10^{-13}$ or better. This being within the reach of ground based experiments, gives them a new impetus. However, while slowly rotating torsion balances in ground laboratories are close to reaching this level, only an experiment performed in low orbit around the Earth is likely to provide a much better accuracy. We report on the progress made with the "Galileo Galilei on the Ground" (GGG) experiment, which aims to compete with torsion balances using an instrument design also capable of being converted into a much higher sensitivity space test. In the present and following paper (Part I and Part II), we demonstrate that the dynamical response of the GGG differential accelerometer set into supercritical rotation -in particular its normal modes (Part I) and rejection of common mode effects (Part II)- can be predicted by means of a simple but effective model that embodies all the relevant physics. Analytical solutions are obtained under special limits, which provide the theoretical understanding. A simulation environment is set up, obtaining quantitative agreement with the available experimental data on the frequencies of the normal modes, and on the whirling behavior. This is a needed and reliable tool for controlling and separating perturbative effects from the expected signal, as well as for planning the optimization of the apparatus.Comment: Accepted for publication by "Review of Scientific Instruments" on Jan 16, 2006. 16 2-column pages, 9 figure

FLORA: a novel method to predict protein function from structure in diverse superfamilies

Predicting protein function from structure remains an active area of interest, particularly for the structural genomics initiatives where a substantial number of structures are initially solved with little or no functional characterisation. Although global structure comparison methods can be used to transfer functional annotations, the relationship between fold and function is complex, particularly in functionally diverse superfamilies that have evolved through different secondary structure embellishments to a common structural core. The majority of prediction algorithms employ local templates built on known or predicted functional residues. Here, we present a novel method (FLORA) that automatically generates structural motifs associated with different functional sub-families (FSGs) within functionally diverse domain superfamilies. Templates are created purely on the basis of their specificity for a given FSG, and the method makes no prior prediction of functional sites, nor assumes specific physico-chemical properties of residues. FLORA is able to accurately discriminate between homologous domains with different functions and substantially outperforms (a 2–3 fold increase in coverage at low error rates) popular structure comparison methods and a leading function prediction method. We benchmark FLORA on a large data set of enzyme superfamilies from all three major protein classes (α, β, αβ) and demonstrate the functional relevance of the motifs it identifies. We also provide novel predictions of enzymatic activity for a large number of structures solved by the Protein Structure Initiative. Overall, we show that FLORA is able to effectively detect functionally similar protein domain structures by purely using patterns of structural conservation of all residues

Comparative Map and Trait Viewer (CMTV): an integrated bioinformatic tool to construct consensus maps and compare QTL and functional genomics data across genomes and experiments

In the past few decades, a wealth of genomic data has been produced in a wide variety of species using a diverse array of functional and molecular marker approaches. In order to unlock the full potential of the information contained in these independent experiments, researchers need efficient and intuitive means to identify common genomic regions and genes involved in the expression of target phenotypic traits across diverse conditions. To address this need, we have developed a Comparative Map and Trait Viewer (CMTV) tool that can be used to construct dynamic aggregations of a variety of types of genomic datasets. By algorithmically determining correspondences between sets of objects on multiple genomic maps, the CMTV can display syntenic regions across taxa, combine maps from separate experiments into a consensus map, or project data from different maps into a common coordinate framework using dynamic coordinate translations between source and target maps. We present a case study that illustrates the utility of the tool for managing large and varied datasets by integrating data collected by CIMMYT in maize drought tolerance research with data from public sources. This example will focus on one of the visualization features for Quantitative Trait Locus (QTL) data, using likelihood ratio (LR) files produced by generic QTL analysis software and displaying the data in a unique visual manner across different combinations of traits, environments and crosses. Once a genomic region of interest has been identified, the CMTV can search and display additional QTLs meeting a particular threshold for that region, or other functional data such as sets of differentially expressed genes located in the region; it thus provides an easily used means for organizing and manipulating data sets that have been dynamically integrated under the focus of the researcher’s specific hypothesis

Evolution of enhanced innate immune evasion by SARS-CoV-2

Emergence of SARS-CoV-2 variants of concern (VOCs) suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on characterisation of spike changes in VOCs, mutations outside spike likely contribute to adaptation. Here we used unbiased abundance proteomics, phosphoproteomics, RNAseq and viral replication assays to show that isolates of the Alpha (B.1.1.7) variant3 more effectively suppress innate immune responses in airway epithelial cells, compared to first wave isolates. We found that Alpha has dramatically increased subgenomic RNA and protein levels of N, Orf9b and Orf6, all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful Alpha transmission, and may increase in vivo replication and duration of infection4. The importance of mutations outside Spike in adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the Delta and Omicron N/Orf9b regulatory regions

Combining specificity determining and conserved residues improves functional site prediction

<p>Abstract</p> <p>Background</p> <p>Predicting the location of functionally important sites from protein sequence and/or structure is a long-standing problem in computational biology. Most current approaches make use of sequence conservation, assuming that amino acid residues conserved within a protein family are most likely to be functionally important. Most often these approaches do not consider many residues that act to define specific sub-functions within a family, or they make no distinction between residues important for function and those more relevant for maintaining structure (e.g. in the hydrophobic core). Many protein families bind and/or act on a variety of ligands, meaning that conserved residues often only bind a common ligand sub-structure or perform general catalytic activities.</p> <p>Results</p> <p>Here we present a novel method for functional site prediction based on identification of conserved positions, as well as those responsible for determining ligand specificity. We define Specificity-Determining Positions (SDPs), as those occupied by conserved residues within sub-groups of proteins in a family having a common specificity, but differ between groups, and are thus likely to account for specific recognition events. We benchmark the approach on enzyme families of known 3D structure with bound substrates, and find that in nearly all families residues predicted by SDPsite are in contact with the bound substrate, and that the addition of SDPs significantly improves functional site prediction accuracy. We apply SDPsite to various families of proteins containing known three-dimensional structures, but lacking clear functional annotations, and discusse several illustrative examples.</p> <p>Conclusion</p> <p>The results suggest a better means to predict functional details for the thousands of protein structures determined prior to a clear understanding of molecular function.</p

Mu2e calorimeter readout system

The Mu2e electromagnetic calorimeter is made of two disks of un-doped parallelepiped CsI crystals readout by SiPM. There are 674 crystals in one disk and each crystal is readout by an array of two SiPM. The readout electronics is composed of two types of modules: 1) the front-end module hosts the shaping amplifier and the high voltage linear regulator; since one front-end module is interfaced to one SiPM, a total of 2696 modules are needed for the entire calorimeter; 2) a waveform digitizer provides a further level of amplification and digitizes the SiPM signal at the sampling frequency of $200\ \text{M}\text{Hz}$with 12-bits ADC resolution; since one board digitizes the data received from 20 SiPMs, a total of 136 boards are needed. The readout system operational conditions are hostile: ionization dose of$20\ \text{krads}$, neutron flux of$10^{12}\ \mathrm{n}(1\ \text{MeVeq})/\text{cm}^2$, magnetic field of$1\ \text{T}$and in vacuum level of$10^{-4}\ \text{Torr}$. A description of the readout system and qualification tests is reported