Microbiology Commons^™

Structure/Function Analysis Of Fega And Fhua In Bradyrhizobium Sp., Alexander James Herd

Senior Honors Projects, 2010-2019

Bradyrhizobium japonicum is a Gram-negative soil bacterium commonly known for its agriculturally significant mutualistic relationship with soybean. In this symbiosis, the bacteria and plant undergo complex molecular signaling characterized by sent and received signals resulting in the formation of infection threads and root nodules. This research aimed to compare two related bacterial outer membrane proteins, FegA and FhuA, associated with the molecular signaling between the bacteria and plant. Previous work has led to the hypothesis that the N-terminal domain (NTD) of FegA in B. japonicum is needed for a functional symbiosis to occur. Recombinant bacterial strains expressing altered FegA proteins …

Probiotics: Reiterating What They Are And What They Are Not, Azza A. Gadir, Raja Dhir, Gregor Reid

Microbiology & Immunology Publications

It has been over seventeen years since the scientific definition of probiotics was crafted, along with guidelines ensuring the appropriate use of the term. This definition is now used globally, yet on a consistent basis scientists, media and industry misrepresent probiotics or make generalized statements that illustrate a misunderstanding of their utility and limitations. The rate of discovery of novel organisms with potentially therapeutic benefit for both human and environmental health is progressing at an unprecedented rate. However, the term “probiotic” is often misapplied to describe any microbe with plausible therapeutic utility in the human host. It is argued that …

Enhanced Expression Of Receptor Tyrosine Kinase Mer (Mertk) On Socs3-Treated Polarized Raw 264.7 Anti-Inflammatory M2c Macrophages, Sankhadip Bhadra

Browse all Theses and Dissertations

Macrophages are phagocytic cells located in tissues, organs and even circulated within our body as white blood cells. They are critical in detecting tissue damage and infection. Resident tissue macrophages initiate the signals for inflammation recruiting neutrophils and blood monocytes which mature into macrophages at sites of infection and in the resolution of inflammation. Based on the local cytokine milieu in tissue sites, macrophages may be polarized into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. Receptor tyrosine kinase Mer (MERTK) helps in clearing dead neutrophils and other apoptotic cells from damaged tissue sites preventing chronic inflammation and autoimmune disorders. MERTK aids …

Bog Flora In The Grand Rapids, Michigan, Area: A Comparative Study Across Sites And Over Time O 1901 To 2017, Devani Antuma Jolman, Jenna L. Van Donselaar, David P. Warners, Garrett E. Crow

Faculty and Professional Research

Michigan’s natural landscape includes a diverse assemblage of ecosystems, among the most distinctive of which are peatlands, and more specifically, those known colloquially as “bogs.” When botanist Emma J. Cole published the Grand Rapids Flora in 1901, she included a large number of bog species documented in the greater Grand Rapids area (16 townships in two counties, an area of 585 square miles). Cole’s Flora is still the most comprehensive catalog of vascular plants found in West Michigan. This study is part of an ongoing update of Cole’s work undertaken by the Calvin University Herbarium to relocate and inventory Emma …

Four Draft Single-Cell Genome Sequences Of Novel, Nearly Identical Kiritimatiellaeota Strains Isolated From The Continental Deep Subsurface, Joshua D. Sackett, Brittany R. Kruger, Eric Becraft, Jessica K. Jarett, Ramunas Stepanauskas, Tanja Woyke, Duane P. Moser

Biology

The recently proposed bacterial phylum Kiritimatiellaeota represents a globally distributed monophyletic clade distinct from other members of the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum. Here, we present four phylogenetically distinct single-cell genome sequences from within the Kiritimatiellaeota lineage sampled from deep continental subsurface aquifer fluids of the Death Valley Regional Flow System in the United States.

Cornus Officinalis Significantly Improves Oxidative Capacity And Promotes The Calcium-Dependent Transcription Factor, Nfatc2, In Human 1.1b4 Pancreatic Cell Line, Arielle Sharp, Alexis Coiner, Brant Burkhardt

Molecular Biosciences Faculty Publications

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of pancreatic β cells (β-cells) and subsequent loss of insulin production. The only treatment for T1D is using exogenous insulin coupled with continual glucose monitoring following significant autoimmune destruction of β-cells. Novel interventional therapies are needed that can preserve and protect existing pancreatic β cells in individuals with early identified T1D autoimmunity. Our initial in-vitro evidence indicates Cornus officinalis (CO) may be able to serve in this function. What sets ethnopharmacology apart from conventional medicine is the simultaneous targeting of multiple mechanisms using a single herb due to …

Structural Basis Of Phosphatidylcholine Recognition By The C2–Domain Of Cytosolic Phospholipase A,2Α, Yoshinori Hirano, Yong-Guang Gao, Daniel J. Stephenson, Ngoc T. Vu, Lucy Malinina, Dhirendra K. Simanshu, Charles E. Chalfant, Dinshaw J. Patel, Rhoderick E. Brown

Molecular Biosciences Faculty Publications

Ca²⁺-stimulated translocation of cytosolic phospholipase A2α (cPLA2α) to the Golgi induces arachidonic acid production, the rate-limiting step in pro-inflammatory eicosanoid synthesis. Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid …

Conserved Glycines Control Disorder And Function In The Cold-Regulated Protein, Cor15a, Oluwakemi T. Sowemimo, Patrick Knox-Brown, Wade M. Borcherds, Tobias Rindfleisch, Anja Thalhammer, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

Cold-regulated (COR) 15A is an intrinsically disordered protein (IDP) from Arabidopsis thaliana important for freezing tolerance. During freezing-induced cellular dehydration, COR15A transitions from a disordered to mostly α-helical structure. We tested whether mutations that increase the helicity of COR15A also increase its protective function. Conserved glycine residues were identified and mutated to alanine. Nuclear magnetic resonance (NMR) spectroscopy was used to identify residue-specific changes in helicity for wildtype (WT) COR15A and the mutants. Circular dichroism (CD) spectroscopy was used to monitor the coil–helix transition in response to increasing concentrations of trifluoroethanol (TFE) and ethylene glycol. The impact of the COR15A …

Host Restriction Factors In The Replication Of Tombusviruses: From Rna Helicases To Nucleocytoplasmic Shuttling, Cheng-Yu Wu

Theses and Dissertations--Plant Pathology

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication.

Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment.

In addition, the features rendering the abundant plant DEAD-box helicases either …

Staphylococcus Aureus Evasion Of The Innate Immune System, James Paul Grayczyk

Dissertations

Upon entry into the host, pathogens must overcome innate immunity in order to cause disease. The innate immune system represents a fast-acting initial line of defense to prevent infection. In order to withstand innate defenses, bacterial pathogens like the Gram-positive bacterium Staphylococcus aureus, produce a wide array of virulence factors that can inhibit innate immune cell recruitment and antimicrobial activity, or directly target and kill phagocytic leukocytes thereby facilitating pathogenesis. Infection with S. aureus can cause disease in virtually any tissue site and is a significant burden to human health. In this dissertation, we sought to understand how S. aureus …

Generating Biologic And Genetic Research Tools To Investigate Serotype I Feline Coronaviruses, Robert Christian Mettelman

Dissertations

Serotype I feline infectious peritonitis virus (FIPV) is an alphacoronavirus of high veterinary importance due to the 99% mortality rate observed in cats. Since the 1980s, robust experimentation on these viruses has been limited by availability of culturable pathogenic strains, few permissive cell lines, and a lack of standardized methods to study their basic virology. These complications have resulted in variable and conflicting literature reports, have slowed study of clinical strains and hindered effective vaccine design. The goal of this dissertation was to develop a laboratory "toolbox" containing standardized methods, permissive cell lines and genetic techniques to alleviate some of …

Strain-Specific Protein Interaction And Localization Of Two Strains Of Potato Yellow Dwarf Virus And Functional Domains Of Their Matrix Protein, Chanyong Jang

Theses and Dissertations--Plant Pathology

Potato yellow dwarf virus (PYDV) is the type species of the genus nucleorhabdovirus which is typified by its nucleotropic characters of the members. The virus accomplishes its replication and morphogenesis in the nuclei of infected cells. Two strains, Constricta strain (CYDV) and Sanguinolenta strain (SYDV) have been described at the level of vector-specificity. CYDV is vectored by Agallia constricta and SYDV is transmitted by Aceratagllia sanguinolenta. The full-length genome of CYDV was sequenced. The 12,792 nt antisense genome encodes seven open reading frames in the order of, nucleocapsid protein (N), unknown protein (X), phosphoprotein (P), movement protein (Y), matrix …

Functional Inhibition Of Acid Sphingomyelinase Disrupts Infection By Intracellular Bacterial Pathogens, Chelsea L. Cockburn, Ryan S. Green, Sheela R. Damle, Rebecca K. Martin, Naomi N. Ghahrai, Punsiri M. Colonne, Marissa S. Fullerton, Daniel H. Conrad, Charles E. Chalfant, Daniel E. Voth, Elizabeth A. Rucks, Stacey D. Gilk, Jason A. Carlyon

Molecular Biosciences Faculty Publications

Intracellular bacteria that live in host cell–derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking—Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, …

Functional Analysis Of The Replication Fork Proteome Identifies Bet Proteins As Pcna Regulators, Sarah R. Wessel, Kareem N. Mohni, Jessica W. Luzwick, Huzefa Dungrawala, David Cortez

Molecular Biosciences Faculty Publications

Identifying proteins that function at replication forks is essential to understanding DNA replication, chromatin assembly, and replication-coupled DNA repair mechanisms. Combining quantitative mass spectrometry in multiple cell types with stringent statistical cutoffs, we generated a high-confidence catalog of 593 proteins that are enriched at replication forks and nascent chromatin. Loss-of-function genetic analyses indicate that 85% yield phenotypes that are consistent with activities in DNA and chromatin replication or already have described functions in these processes. We illustrate the value of this resource by identifying activities of the BET family proteins BRD2, BRD3, and BRD4 in controlling DNA replication. These proteins …

P53 Phosphomimetics Preserve Transient Secondary Structure But Reduce Binding To Mdm2 And Mdmx, Robin Levy, Emily Gregory, Wade Borcherds, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient …

The Otud5–Ubr5 Complex Regulates Fact-Mediated Transcription At Damaged Chromatin, Angelo De Vivo, University Of South Florida, Jose Yegres, Jeonghyeon Kim, Sylvia Emly, Younghoon Kee

Molecular Biosciences Faculty Publications

Timely stalling and resumption of RNA polymerases at damaged chromatin are actively regulated processes. Prior work showed an importance of FACT histone chaperone in such process. Here we provide a new role of OTUD5 deubiquitinase in the FACT-dependent process. Through a DUB RNAi screen, we found OTUD5 as a specific stabilizer of the UBR5 E3 ligase. OTUD5 localizes to DNA double strand breaks (DSBs), interacts with UBR5 and represses the RNA Pol II elongation and RNA synthesis. OTUD5 co-localizes and interacts with the FACT component SPT16 and antagonizes the histone H2A deposition at DSB lesions. OTUD5 interacts with UBR5 and …

Six-Nine Months Long Term Culture Of Mouse Bone Marrow Cells Differentiated To Macrophages And Eosinophils, Olena B. Svitlova

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Mouse models of eosinophil-associated diseases have been used to study the mechanisms of disease pathogenesis. In this study, mouse-derived bone marrow cells were used in long-term (6 and 9 months) cell cultures of differentiated eosinophils and macrophages. IL-5 was used to differentiate the stem cells to eosinophils and GM-CSF was used to propagate macrophages from the bone marrow stem cells. The maximum time period for observing the eosinophil cultures was 252 days which is censurably longer than the 18 days culture period observed by others. The results were assessed by describing the microscopic cell morphology by Wright staining, modified Giemsa …

Crystallographic Studies On Host-Interacting Proteins Of Phage K Virion, Antonio Pichel Beleiro

Theses

Phage K is a lytic bacteriophage that infects a broad range of staphylococci, including some methicillin-resistant Staphylococcus aureus strains. Phage K attaches to its host via a baseplate. The contracted conformation of the baseplate contains a fiber and two receptorbinding proteins responsible for the interaction with the host. The native conformation of the baseplate also includes a tail central spike that shears off after contraction. The aim of the thesis is the structural characterization of phage K receptor-binding proteins and tail central spike. Experimentally, the thesis relies on crystallographic methods, which encompass: production of suitable sample, crystallization, diffraction, and data …

The Occurrence Of Listeria Monocytogenes In The Mushroom Production Chain And The Use Of Bacteriophage For Its Control, Vincenzo Pennone

Theses

In Agaricus bisporus production, the occurrence of L. monocytogenes is still poorly understood. A number of studies have shown the presence of L. monocytogenes in the mushroom processing environment, suggesting its source from raw materials storage areas (Viswanath et al, 2013; Murugesan et al, 2015). In this study, 10 mushroom producers and three raw material (compost and casing) producers participated in two surveys on the occurrence of L. monocytogenes in the most high-risk areas of the production environment. In the first survey, floors, drains, platforms, crates, raw materials and mushrooms were tested for L. monocytogenes presence and the results were …

Crystallographic Structure Determination Of Bacteriophage-Encoded Enzymes That Specifically Target Pathogenic Bacteria, Marta Sanz Gaitero

Theses

Antibiotic resistance is becoming a serious public health concern. Infections that some decades ago could be treated with antibiotics now sometimes do not respond to traditional treatment, causing higher mortality and economic losses. An alternative to the use of antibiotics are bacteria's natural predators, bacteriophages (or phages), and specifically their lytic enzymes. These proteins are produced by phages to degrade bacterial peptidoglycan to inject their genetic material into the bacteria (virion-associated peptidoglycan hydrolases) or to release their progeny once the infection is finished (endolysins). They can be applied exogenously to lyse Gram-positive bacteria or be genetically engineered to lyse Gram-negative …

Development And Evaluation Of Vaccine Candidates For Senecavirus A, Bishwas Sharma

Electronic Theses and Dissertations

Senecavirus A (SVA) has been associated with several outbreaks of vesicular disease (VD) since 2014 in major swine-producing countries around the world. The virus causes VD that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no commercial vaccines available for SVA. Here, we developed a live attenuated SVA strain by mutating a wild type SVA strain SD15-26 (wt SVA). The live attenuated virus, rSVA mSacII, replicated less efficiently in cell cultures in vitro and expressed lower amounts of VP1 and VP2 capsid proteins than wt SVA. Most importantly, the rSVA mSacII virus failed to induce clinical …

Shrimp Production Environment And The Gut Microbiome: Effects Of Aquaculture Practices And Selective Breeding On The Gut Microbiome Of Pacific Whiteleg Shrimp, Litopenaeus Vannamei, Angela Landsman

Electronic Theses and Dissertations

Knowledge of the functional role of the gut microbiome in animal health and nutrition may provide solutions to shrimp aquaculture challenges, such as improving disease resistance and optimizing growth particularly with low cost feeds. Successful manipulation of bacteria found in the gut requires a deeper understanding of shrimp microbial communities and how their compositional structure is influenced by environmental conditions, and inherent host factors such as genetics. The initial research investigated the intestinal bacterial communities of the Pacific whiteleg shrimp (Litopenaeus vannamei) reared in pond systems compared to indoor aquaculture facilities as an exploration of the effects of aquaculture practices …

Isolation And Identification Of Potential Bioinoculants Based On Phosphate Solubilizing And Plant Growth Promoting Benefits, Rachel Raths

Electronic Theses and Dissertations

Conservative models have shown that as populations rise, food production needs to double by 2050. Population increase and the green revolution have caused fertilizer inputs to increase since the 1960’s, increasing environmental issues and production costs. These intensive practices have led to degraded arable land and there has been an increase in urbanization meaning we need to make best use of the farming land that is available and ensure it is sustainable for future food production. Due to this, there has been a higher demand for research on more environmentally and economically friendly approaches to food production. Plant growth promoting …

Hydrodynamic Cavitation As In-Line Process To Control Common Dairy Sporeformers, Pratibha Chaudhary

Electronic Theses and Dissertations

Sporeforming bacteria and their endospores have the ability to survive thermal treatments and proliferate in the milk and dairy products to cause spoilages. We applied the effect of hydrodynamic cavitation in controlling these bacteria and their spores at pilot scale level. Our aim was to investigate a technology that would improve the microbial quality of milk and dairy products, and is capable of being scaled up to be adapted at industrial level. Cavitation damages the cell walls of the bacteria and their endospores (releasing dipicolinic acid, responsible for endospore resistance to extreme conditions), thereby making them susceptible to thermal treatments …

Comparing Gut Microbiome And Virome In The Breast Milk- And Formula-Fed Late Preterm Infants, Zyi Wang

Electronic Theses and Dissertations

The neonatal intestinal microbiome consists of all microorganisms in the gut. Although the microbiome is critical to human health and disease, its colonization remains incompletely understood, particularly in the preterm infant. We aimed to characterize the intestinal bacteria microbiome and virome in moderate-to-late preterm infants. We hypothesized that the bacteria microbiome and virome differs between breast milk and formula-fed infants. We collected stool samples from twenty infants born between 32 0/7 and 36 6/7 weeks gestation. Samples were collected after infants reached full volume enteral feedings. Ten infants were breast milk fed and ten received infant formula. DNA and RNA …

Assessing Listeria Risk During Different Stages Of Ice Cream Manufacturing And Storage, Neha Singh

Electronic Theses and Dissertations

Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). Evidence about the presence of injured cells in ice …

A Full-Ocean-Depth Rated Modular Lander And Pressure-Retaining Sampler Capable Of Collecting Hadal-Endemic Microbes Under In Situ Conditions, Logan M. Peoples, Matthew Norenberg, David Price, Madeline Mcgoldrick, Mark Novotny, Alexander Bochdansky, Douglas H. Bartlett

OES Faculty Publications

The hadal zone remains one of the least studied environments because of its inaccessibility, in part because of hydrostatic pressures extending to 110 MPa. Few instruments are capable of sampling from such great depths. We have developed a full-ocean-depth-capable lander that can be fit with sampling packages for the collection of still images, video, motile megafauna, and hadal seawater. One payload includes a pressure-retaining sampler (PRS) able to maintain seawater samples under in situ pressure during recovery. We describe the technical specifications of the lander and the PRS and preliminary results from three deployments at depths in excess of 10,700 …

Metaproteomics Reveal That Rapid Perturbations In Organic Matter Prioritize Functional Restructuring Over Taxonomy In Western Arctic Ocean Microbiomes, Molly P. Mikan, H. Rodger Harvey, Emma Timmins-Schiffman, Michael Riffle, Damon H. May, Ian Salter, William S. Noble, Brook L. Nunn

OES Faculty Publications

We examined metaproteome profiles from two Arctic microbiomes during 10-day shipboard incubations to directly track early functional and taxonomic responses to a simulated algal bloom and an oligotrophic control. Using a novel peptide-based enrichment analysis, significant changes (p-value < 0.01) in biological and molecular functions associated with carbon and nitrogen recycling were observed. Within the first day under both organic matter conditions, Bering Strait surface microbiomes increased protein synthesis, carbohydrate degradation, and cellular redox processes while decreasing C1 metabolism. Taxonomic assignments revealed that the core microbiome collectively responded to algal substrates by assimilating carbon before select taxa utilize and metabolize nitrogen intracellularly. Incubations of Chukchi Sea bottom water microbiomes showed similar, but delayed functional responses to identical treatments. Although 24 functional terms were shared between experimental treatments, the timing, and degree of the remaining responses were highly variable, showing that organic matter perturbation directs community functionality prior to alterations to the taxonomic distribution at the microbiome class level. The dynamic responses of these two oceanic microbial communities have important implications for timing and magnitude of responses to organic perturbations within the Arctic Ocean and how community-level functions may forecast biogeochemical gradients in oceans.

Nitrogen Assimilation In Picocyanobacteria Inhabiting The Oxygen-Deficient Waters Of The Eastern Tropical North And South Pacific, Montserrat Aldunate, Carlos Henriquez-Castillo, Qixing Ji, Jessica Lueders-Dumont, Margaret R. Mulholland, Bess B. Ward, Peter Von Dassow, Osvaldo Ulloa

OES Faculty Publications

Prochlorococcus and Synechococcus are the most abundant free-living photosynthetic microorganisms in the ocean. Uncultivated lineages of these picocyanobacteria also thrive in the dimly illuminated upper part of oxygen-deficient zones (ODZs), where an important portion of ocean nitrogen (N) loss takes place via denitrification and anaerobic ammonium oxidation. Recent metagenomic studies revealed that ODZ Prochlorococcus have the genetic potential for using different N forms, including nitrate and nitrite, uncommon N sources for Prochlorococcus, but common for Synechococcus. To determine which N sources ODZ picocyanobacteria are actually using in nature, the cellular 15N natural abundance (δ15N) and assimilation rates of …

Stability Of Temperate Coral Astrangia Poculata Microbiome Is Reflected Across Different Sequencing Methodologies, Dawn B. Goldsmith, Zoe A. Pratte, Koty H. Sharp

Arts & Sciences Faculty Publications

The microbiome of the temperate coral Astrangia poculata was first described in 2017 using next-generation Illumina sequencing to examine the coral’s bacterial and archaeal associates across seasons and among hosts of differing symbiotic status. To assess the impact of methodology on the detectable diversity of the coral’s microbiome, we obtained near full-length Sanger sequences from clone libraries constructed from a subset of the same A. poculata samples. Eight samples were analyzed: two sets of paired symbiotic (brown) and aposymbiotic (white) colonies collected in the fall (September) and two sets collected in the spring (April). Analysis of the Sanger sequences revealed …