Microbiology Commons^™

Antifungal Genome Mining And Genetics In Filamentous Actinomycete Bacteria Isolated From Local Soils, Jacob Alex, Kevin Lou, Bilal Makhdoom

Undergraduate Research Symposium Posters

Actinomycetes are gram positive, filamentous bacteria that produce useful antibiotics, antitumor agents, and agricultural products. A series of enrichments were undertaken to isolate actinomycetes from local soils, varying enrichment media, antibacterials, and soil treatments (including heat and CaCO3). Isolates were characterized by 16S rDNA sequencing, phenotypic and morphological observations, and antibiotic production. The genetic tractability of select isolates was analyzed using a panel of integrating vectors derived from ɸC31, ɸBT1, and OzzyJ phage using intergeneric conjugation. Further, a semi-degenerate multiplex PCR assay to detect ɸBT1 genomic integrants was designed and tested for the first time. Finally, PCR screens were used …

Evolution Of Equine Arteritis Virus During Persistent Infection In The Reproductive Tract Of The Stallion And The Male Donkey, Bora Nam

Theses and Dissertations--Veterinary Science

Equine arteritis virus (EAV) establishes persistent infection in the stallion reproductive tract, and the carrier stallion continues to shed virus in semen for weeks to years or lifelong. The objective of this study was to elucidate the intra-host evolution of EAV during persistent infection in stallions. Seven EAV seronegative stallions were experimentally infected with EAV KY84 strain and followed for 726 days post-infection, and sequential clinical samples including semen were collected for virus isolation and next-generation sequencing (NGS). In addition, archived sequential semen samples from two stallions that were naturally infected with EAV KY84 for a long-period (up to 10 …

An Undergraduate Cell Biology Lab: Western Blotting To Detect Proteins From Drosophila Eye, Neha Gogia, Ankita Sarkar, Amit Singh

Biology Faculty Publications

We have developed an undergraduate laboratory to allow detection and localization of proteins in the compound eye of Drosophila melanogaster, a.k.a fruit fly. This lab was a part of the undergraduate curriculum of the cell biology laboratory course aimed to demonstrate the use of Western Blotting technique to study protein localization in the adult eye of Drosophila. Western blotting, a two-day laboratory exercise, can be used to detect the presence of proteins of interests from total protein isolated from a tissue. The first day involves isolation of proteins from the tissue and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel …

Real-Time Quantitative Pcr To Demonstrate Gene Expression In An Undergraduate Lab, Abijeet Singh Mehta, Amit Singh

Biology Faculty Publications

The objective of this teaching note is to develop a laboratory exercise, which allows students to get a hands-on experience of a molecular biology technique to analyze gene expression. The short duration of the biology laboratory for an undergraduate curriculum is the biggest challenge with the development of new labs. An important part of cell biology or molecular biology undergraduate curriculum is to study gene expression. There are many labs to study gene expression in qualitative manner. The commonly used reporter gene expression studies are primarily qualitative. However, there is no hands-on experience exercise to quantitatively determine gene expression. Therefore, …

Contributions Of Gene Copy Number Variation To Genome Evolution And Local Adaptation Of The Cyanobacterium Acaryochloris, Amy L. Gallagher

Graduate Student Theses, Dissertations, & Professional Papers

Acaryochloris is a recently discovered genus of cyanobacteria, unique in its use of an uncommon chlorophyll as its major photosynthetic pigment, and in its peculiar genome dynamics. Members of this genus exhibit increased genic copy number variation (CNV), which is thought to be primarily derived from gene duplications and horizontal gene transfer (HGT). Acaryochloris provides an ideal system to explore mechanisms behind maintenance of gene duplicates and the influence of CNV in local adaptation. Here, I propose a mechanism for retention of gene duplicates of the bacterial recombinase, RecA, in Acaryochloris genomes and provide preliminary evidence that these paralogs are …

Analysis Of Microbial Diversity In Disturbed Soil, Tyler G. Sanda

Williams Honors College, Honors Research Projects

This paper uses the composition and abundance of microbial species to analyze soil recovery in disturbed land. Surface mining disturbs ecological communities throughout the world. As organizations seek to reclaim these disturbed lands, a proper analysis of recovery is needed. In previous studies, recovery of disturbed land was limited to surface examinations, which do not characterize the possible unseen devastating effects of the subsoil. Soil microorganisms are extremely sensitive to environmental changes such as strip mining. It is proposed that these microorganisms may serve as better indicators of recovery post disturbance. Our analysis indicates microbial recovery, however it may not …

Reactivation Of Hiv-1 Proviruses In Immune-Compromised Mice Engrafted With Human Voa-Negative Cd4+ T Cells, Zhe Yuan, Guobin Kang, Wuxun Lu, Qingsheng Li

Nebraska Center for Virology: Faculty Publications

Background: HIV-1 infection remains incurable on antiretroviral therapy (ART) due to virus latency. To date, enhanced co-culture assays, including viral outgrowth assays (VOA), are commonly used to measure HIV-1 latent reservoirs and evaluate latency-reversing agents (LRAs). However, VOA can only reactivate a small fraction of intact proviruses.

Methods: To explore the utility of NOD scid gamma (NSG) mice as an in vivo model to reactivate HIV-1 proviruses from VOA-negative CD4+ T cells, resting CD4+ T cells from an HIV-1 latently infected individual were isolated and the human CD4+ T cells corresponding to VOA-positive and VOA-negative CD4+ T cells were engrafted …

The Role Of The Dosage Compensation Complex As A Pathway For Spiroplasma To Induce Male Lethality In Drosophila Melanogaster, Becky Cheng

CMC Senior Theses

Drosophila melanogaster and many other insects harbor intracellular bacterial symbionts that are transmitted vertically from infected host mothers to their offspring. Many of these bacteria alter host reproductive developmental processes in order to increase their transmission success. For example, Spiroplasma, a spirochete that naturally infects D. melanogaster, selectively kills males during mid-embryogenesis while sparing females. Previous studies suggested that Spiroplasma interacts genetically with the male-specific dosage compensation pathway, which causes ~2-fold up-regulation of most genes located on the male’s single X chromosome so that their expression matches the levels found in females who have two Xs. To further …

Detection Of Antibiotic Resistance Loci In Wastewater And Fresh Water Samples, Mohammed Hamed Almalki

Masters Theses

Proper treatment of municipal wastewater is important not only for prevention of eutrophication but also for public health concerns. One consideration for wastewater treatment is the presence of organisms carrying antibiotic resistance genes. If these organisms survive the wastewater treatment process and are released into the streams, they can pose significant risks to public health. In addition, their presence also raises concerns for lateral gene transfer events at the wastewater plant. The spread of antibiotic loci has a major impact in public health and clinical therapy because resistant organisms render conventional treatments of important illnesses difficult. Beta-lactam drugs are known …

A Rare Case Of Erythema Elevatum Diutinum Presenting As Diffuse Neuropathy, G H. Nguyen, E L. Guo, D Norris

Faculty, Staff and Students Publications

No abstract provided.

Comparing An Atomic Model Or Structure To A Corresponding Cryo-Electron Microscopy Image At The Central Axis Of A Helix, Stephanie Zeil, Julio Kovacs, Willy Wriggers, Jing He

Computer Science Faculty Publications

Three-dimensional density maps of biological specimens from cryo-electron microscopy (cryo-EM) can be interpreted in the form of atomic models that are modeled into the density, or they can be compared to known atomic structures. When the central axis of a helix is detectable in a cryo-EM density map, it is possible to quantify the agreement between this central axis and a central axis calculated from the atomic model or structure. We propose a novel arc-length association method to compare the two axes reliably. This method was applied to 79 helices in simulated density maps and six case studies using cryo-EM …

Cullin-4 Regulates Wingless And Jnk Signaling-Mediated Cell Death In The Drosophila Eye., Meghana Tare, Ankita Sarkar, Shimpi Bedi, Madhuri Kango-Singh, Amit Singh

Biology Faculty Publications

In all multicellular organisms, the fundamental processes of cell proliferation and cell death are crucial for growth regulation during organogenesis. Strict regulation of cell death is important to maintain tissue homeostasis by affecting processes like regulation of cell number, and elimination of unwanted/unfit cells. The developing Drosophila eye is a versatile model to study patterning and growth, where complex signaling pathways regulate growth and cell survival. However, the molecular mechanisms underlying regulation of these processes is not fully understood. In a gain-of-function screen, we found that misexpression of cullin-4 (cul-4), an ubiquitin ligase, can rescue reduced eye mutant phenotypes. Previously, …

Comparative Genomic Analysis Of Two Serotype 1/2b Listeria Monocytogenes Isolates From Analogous Environmental Niches Demonstrates The Influence Of Hypervariable Hotspots In Defining Pathogenesis, Aidan Casey, Kieran Jordan, Aidan Coffey, Edward M. Fox, Olivia Mcauliffe

Department of Biological Sciences Publications

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but …

A Microbiomic Approach To The Characterization Of The Impacts And Influences Of Viral, Bacterial, And Harmful Algal Bloom Toxins On The Bottlenose Dolphin, Corey David Russo

Dissertations

As apex predators that display high site fidelity Tursiops truncatus (bottlenose dolphin) are indicators of marine ecosystem health. Bottlenose dolphins, additionally, display pathogenesis and immune response similar to that of humans. Humans and coastal bottlenose dolphins, in particular, are constantly exposed to the same industrial, agricultural and domestic toxins and pathogens, contaminants and pollutants. Thus, studies on the bottlenose dolphin are also valuable in bridging the gap between ocean and human health. Bottlenose dolphins are susceptible to viral bacterial and toxin infection. Infection in the bottlenose dolphin manifests itself in the form of mass stranding events, unusual mortality events, chronic …

A Novel Codon-Optimized Siv Gag-Pol Immunogen For Gene-Based Vaccination, Catherine M. Crosby, Eric A. Weaver, Reeti Khare, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Simian immunodeficiency virus (SIV) is a robust pathogen used in non-human primates to model HIV vaccines. SIV encodes a number of potential vaccine targets. By far the largest and most conserved protein target in SIV is its gag-pol protein that bears many epitopes to drive multivalent immune T cell responses. While gag-pol is an attractive antigen, it is only translated after a frame shift between gag and pol with the effect that gag and pol are expressed at an approximate 10/1 ratio. The codon bias of native lentiviral genes are also mismatched with the abundance of tRNAs in mammalian cells …

Peering Below The Diffraction Limit: Robust And Specific Sorting Of Viruses With Flow Cytometry, Shea T. Lance, David J. Sukovich, Kenneth M. Stedman, Adam R. Abate

Biology Faculty Publications and Presentations

Background: Viruses are incredibly diverse organisms and impact all forms of life on Earth; however, individual virions are challenging to study due to their small size and mass, precluding almost all direct imaging or molecular analysis. Moreover, like microbes, the overwhelming majority of viruses cannot be cultured, impeding isolation, replication, and study of interesting new species. Here, we introduce PCR-activated virus sorting, a method to isolate specific viruses from a heterogeneous population. Specific sorting opens new avenues in the study of uncultivable viruses, including recovering the full genomes of viruses based on genetic fragments in metagenomes, or identifying the …

Consensus Paper: Cerebellar Development, K Leto, M Arancillo, Ebe Becker, A Chiang, Et Al.

Faculty, Staff and Students Publications

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

Genetic Determinants Of Salmonella And Campylobacter Required For In Vitro Fitness, Rabindra Kumar Mandal

Graduate Theses and Dissertations

Non-typhoidal Salmonella (NTS) and Campylobacter play a major role in foodborne illness caused by the consumption of food contaminated by pathogens worldwide. A comprehensive understanding of the genetic factors that increase the survival fitness of these foodborne pathogens will effectively help us formulate mitigation strategies without affecting the nutrition ecology. The objective of this study was to identify the genetic determinants of Salmonella and Campylobacter that are required for fitness under various in vitro conditions. For the purpose, we used a high throughput Transposon sequencing (Tn-seq) that utilizes next generation sequencing (NGS) to screen hundreds of thousands of mutants simultaneously. …

Genomes Of Gardnerella Strains Reveal An Abundance Of Prophages Within The Bladder Microbiome, Kema Malki, Jason W. Shapiro, Travis Kyle Price, Evann Elizabeth Hilt, Krystal Thomas-White, Trina Sircar, Amy B. Rosenfeld, Michael J. Zilliox, Alan J. Wolfe, Catherine Putonti

Bioinformatics Faculty Publications

Bacterial surveys of the vaginal and bladder human microbiota have revealed an abundance of many similar bacterial taxa. As the bladder was once thought to be sterile, the complex interactions between microbes within the bladder have yet to be characterized. To initiate this process, we have begun sequencing isolates, including the clinically relevant genus Gardnerella. Herein, we present the genomic sequences of four Gardnerella strains isolated from the bladders of women with symptoms of urgency urinary incontinence; these are the first Gardnerella genomes produced from this niche. Congruent to genomic characterization of Gardnerella isolates from the reproductive tract, isolates …

Current Antibiotics And Future Herbal Extract Methods To Treat Methicillin-Resistant Staphylococcus Aureus (Mrsa): Focusing On Inhibition Of Penicillin-Binding Protein 2a (Pbp2a), Mgayya R. Makullah

Senior Honors Theses

Methicillin-Resistant Staphylococcus aureus (MRSA) has developed resistance towards a number of antibiotics. This resistance creates a challenge when trying to treat MRSA with a number of antibiotics. This is mainly caused by the penicillin-binding proteins 2a (PBP2a). PBP2a have significantly less affinity for beta-lactam antibiotics compared to the other penicillin-binding proteins (PBPs) expressed by non-resistant strains. New treatments involving a combination of antibiotics and herbal extracts are being developed and used to inactivate PBP2a, allowing the previous ineffective antibiotics to be more effective.

Draft Genome Sequence For A Urinary Isolate Of Nosocomiicoccus Ampullae, Evann Elizabeth Hilt, Travis Kyle Price, Katherine Diebel, Catherine Putonti, Alan J. Wolfe

Bioinformatics Faculty Publications

A draft genome sequence for a urinary isolate of Nosocomiicoccus ampullae (UMB0853) was investigated. The size of the genome was 1,578,043 bp, with an observed G+C content of 36.1%. Annotation revealed 10 rRNA sequences, 40 tRNA genes, and 1,532 protein-coding sequences. Genome coverage was 727× and consisted of 32 contigs, with an N₅₀ of 109,831 bp.

Genome Sequences And Annotation Of Two Urinary Isolates Of E.Coli, Travis Kyle Price, Arya Mehtash, Laurynas Kalesinskas, Kema Malki, Evann Elizabeth Hilt, Catherine Putonti, Alan J. Wolfe

Bioinformatics Faculty Publications

The genus Escherichia includes pathogens and commensals. Bladder infections (cystitis) result most often from colonization of the bladder by uropathogenic E. coli strains. In contrast, a poorly defined condition called asymptomatic bacteriuria results from colonization of the bladder with E. coli strains without symptoms. As part of an on-going attempt to identify and characterize the newly discovered female urinary microbiota, we report the genome sequences and annotation of two urinary isolates of E. coli: one (E78) was isolated from a female patient who self-reported cystitis; the other (E75) was isolated from a female patient who reported that she did …

Replication-Transciption Switch In Human Mitochondria, Karen Agaronyan

Graduate School of Biomedical Sciences Theses and Dissertations

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. Human mitochondrial RNA polymerase (mtRNAP) is a central enzyme involved in gene expression in mitochondria. It generates genome-size polycistronic transcripts and also makes replication primers at two origins of replication. MtRNAP is distantly related to phage T7 RNAP. While T7 RNAP is optimized to produce large amounts of transcripts to overcompete the bacterial …

Molecular Analysis Of Ftsz-Ring Assembly In E. Coli Cytokinesis, Kuo-Hsiang Huang

Dissertations, Theses, and Capstone Projects

An essential first step in bacterial division is the assembly of a cytokinetic ring (Z-ring) formed by the tubulin-like FtsZ at midcell. The highly conserved core domain of FtsZ has been reported to mediate assembly of FtsZ polymers in vivo and in vitro. Species-specific differences in the FtsZ C-terminal domain such as the FtsZ CTV region and interactions with several modulatory proteins such as ZapC and ZapD, restricted to certain bacterial classes, also serve as key determinants of FtsZ protofilament bundling. Here, we characterize (i) the roles of the FtsZ CTV region in mediating both longitudinal and lateral interactions …

Vascular Endothelial Growth Factor-A Gene Electrotransfer Promotes Angiogenesis In A Porcine Model Of Cardiac Ischemia, Anna A. Bulysheva, Barbara Hargrave, Nina Burcus, Cathryn G. Lundberg, Len Murray, Richard Heller

Bioelectrics Publications

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. …

Mucosal Vaccination By Adenoviruses Displaying Reovirus Sigma 1, Eric A. Weaver, Zenaido T. Camacho, Matthew L. Hillestad, Catherine M. Crosby, Mallory A. Turner, Adam J. Guenzel, Hind J. Fadel, George T. Mercier, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

We previously developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but had 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generated stronger T cell responses than Ad5 when used for mucosal immunization. New Ad5-fiber-sigma vectors were generated here by varying the number of fiber β-spiral shaft repeats (R) fused between fiber tail and the sigma. Ad5 virions encoding R3, R14, and R20 chimeras were rescued. Increasing chimera length led to their decreasing encapsidation of these proteins in the virions. Ad5-R3 …

The Role Of Mfd In Stationary-Phase Oxidative Damage Repair In Bacillus Subtilis, Katelyn E. Porter

UNLV Theses, Dissertations, Professional Papers, and Capstones

Since the 1950’s it has been shown that bacterial cells accumulate mutations even in non- dividing conditions, but how this type of mutation occurs is still highly debated. In Bacillus subtilis, Mfd, a precursor of the nucleotide excision repair (NER) system, mediates the formation of mutations in stationary-phase or non-replicating cells. In growing cells, Mfd recruits repair when RNA polymerase is stalled during transcription; it then recruits proteins from NER to repair damage. Here, we examine the hypothesis that Mfd mediates the formation of mutations by interacting with cellular components that repair reactive oxygen species (ROS), a natural byproduct of …

Relationship Between Morphogenesis And Secretion In The Filamentous Fungus Aspergillus Nidulans, Lakshmi Preethi Yerra

School of Biological Sciences: Dissertations, Theses, and Student Research

Filamentous fungi have a long history in biotechnology for the production of food ingredients, pharmaceuticals and enzymes. The advancements made in recent years have earned filamentous fungi such as Aspergillus species a dominant place among microbial cell factories. Although the model fungus A. nidulans has been extensively studied, the genetic and regulatory networks that underlie morphogenesis and development have yet to be fully characterized. The Rho GTPases (Cdc42 and RacA) are one of the most important regulators of the morphogenetic processes among diverse eukaryotic organisms. Although the functions of these GTPases are relatively well-characterized, little is known about their …

Separate F-Type Plasmids Have Shaped The Evolution Of The H30 Subclone Of Escherichia Coli Sequence Type 131., Timothy J Johnson, Jessica L Danzeisen, Bonnie Youmans, Kyle Case, Katharine Llop, Jeannette Munoz-Aguayo, Cristian Flores-Figueroa, Maliha Aziz, Nicole Stoesser, Evgeni Sokurenko, Lance B. Price, James R Johnson

Environmental and Occupational Health Faculty Publications

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H …

Genome Engineering In Mammalian Cells By Flp And Cre Dna Recombinase Variants, Riddhi Shah

Doctoral Dissertations

Genome engineering relies on DNA modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ self-sufficient, versatile site-specific DNA recombinase Flp and Cre can be developed into promising alternatives. We demonstrate that a Flp variant evolved to recombine an FRT-like sequence FL-IL10A, which is located upstream of the human interleukin-10 gene, can target this sequence in the model setting and native …