Microbiology Commons^™

Microbial Nad Metabolism: Lessons From Comparative Genomics, Francesca Gazzaniga, Rebecca Stebbins, Sheila Z. Chang, Mark A. Mcpeek, Charles Brenner

Dartmouth Scholarship

NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life …

Exploring Diversity Of Nitrate Reducing Thermophiles In Nevada Hot Springs, Jenny Lam, Jeremy A. Dodsworth, Brian P. Hedlund

Undergraduate Research Opportunities Program (UROP)

High rates of denitrification have been measured in Nevada geothermal hot springs, but little is known about the thermophiles that contribute to this activity. We hypothesize that heterotrophic bacteria in the genus Thermus are the most important denitrifiers in the springs. Alternatively, other microorganisms including chemolithotrophs may also be important. To test these hypotheses, several different strategies will be used to try to enrich and isolate nitrate-reducing microorganisms. Isolates will be identified by 16S rRNA gene PCR and sequencing. Subsequently, representative isolates will be chosen for nitrate reductase gene (narG) sequencing and for studies on the kinetics of nitrate reduction …

The Role Of Rpoe In Stationary Phase Mutagenesis In Bacillus, Turquoise C. Alexander, Eduardo A. Robleto

Undergraduate Research Opportunities Program (UROP)

Stationary phase mutagenesis is a phenomenon whereby random mutations are generated in non-dividing cells. In order to understand how these mutations arise, we use Bacillus subtilis, a gram positive rod-shaped model organism. It is hypothesize that increased transcription promotes stationary phase mutagenesis in this organism. We therefore examined the role of rpoE, a gene that encodes RNA polymerase ! subunit and proposed to influence efficiency of transcription. To this end, we will first generate a strain bearing a deletion in the rpoE gene. In order to determine if this gene is important for mutagenesis, we will examine the accumulation of …

Attempts To Cultivate Bacteria From Deep Subsurface Aquifers And Mountaintop Plant Communities, Eric D. Hughes, J. C. Bruckner, Duane P. Moser

Undergraduate Research Opportunities Program (UROP)

In the late 1990s, the limits of life were pushed even further when microorganisms were discovered thriving 2.5 km below the surface of the Earth in deep South African gold mines. These very simple communities were dominated by a single species of bacteria from within the phylum, Firmicutes. Desulforudis audaxviator remains unique to a sizeable portion of the South African deep subsurface. At depths below 2.5km, it comprises well over 99% of all organisms present, which presents a unique circumstance in which the environment has provided a natural pure culture. From this naturally occurring pure culture, environmental genomics was applied …

The Role Of Recn In Stationary Phase Mutagenesis In Bacillus Subtilis, Lauren E. Johnson, Katherine R. Ona, Eduardo A. Robleto, Ronald E. Yasbin

Undergraduate Research Opportunities Program (UROP)

Here, we examine mutagenic programs that are independent of growth, such aspects of the evolutionary process are novel and have been implicated in the formation of cancers in animal cells and the acquisition of antibiotic resistance in animal pathogens. Adaptive or stationary phase mutagenesis is a genetic program to in increase diversity in cells under conditions of stress whereby cells escape non-dividing conditions. Previous research has shown that recombination functions are required to generate mutations that promote growth in Escherichia coli cells starved for carbon. This project tests the hypothesis that recombination functions are required for the generation of mutations …

Adding Upstream Sequence And A Downstream Reporter To The Bile Acid Inducible Promoter Of Clostridium Scindens Vpi 12708, Bryan Patrick Mason

Masters Theses & Specialist Projects

Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model …

Cloning And Expression Of A Putative Exported Protein Coding Gene Sas1089 From An Invasive Community Associated Staphylococcus Aureus Strain Mssa476, Nailah Alzaki

All-Inclusive List of Electronic Theses and Dissertations

Originally, methicillin resistant Staphylococcus aureus (MRSA) occurred primarily in the hospital setting and was designated hospital- associated MRSA (HA-MRSA). In recent years, isolates of Staphylococcus aureus from the community (CA-MRSA), found to be methicillin resistant, are not as resistant tooth~ antibiotics as HA-MRSA. However, community strains are noted for their invasiveness causing soft tissue infections with propensity for invasion to deeper sites.

Rapid Evolution Of Sex-Pheromone-Producing Enzyme In Drosophila, Troy R. Shirangi, Héloïse D. Dufour, Thomas M. Williams, Sean B. Carroll

Biology Faculty Publications

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We …

Contribution Of Ledgf/P75 To Prostate Cancer Chemoresistance, Melanie Mediavilla-Varela

Loma Linda University Electronic Theses, Dissertations & Projects

Prostate cancer (PCa) is the second leading cause of male cancer death in the United States, and is considered a health disparity because of its disproportionate incidence and mortality in African American (AA) males. A major challenge in PCa treatment is its recurrence into hormone refractory prostate cancer (HRPC). Docetaxel is the standard of care for first line chemotherapy to HRPC. Unfortunately, most patients ultimately manifest resistance to DTX and succumb to the disease. Circumventing DTX resistance in HRPC will require better understanding of the mechanisms by which this drug kills prostate tumor cells, and the genes that promote this …

Studies In Prolonged Oxidative Stress Resistance In Porphyromonas Gingivalis, Rachelle Mckenzie

Loma Linda University Electronic Theses, Dissertations & Projects

Porphyromonas gingivalis is a gram negative, anaerobic bacterium implicated as a major etiological agent in chronic adult periodontitis. In the inflammatory environment of the periodontal pocket, the organism must overcome oxidative stress as a result of exposure to air in the oral cavity as well as to reactive oxygen species (ROS) generated by host immune cells. A comprehensive mechanism for oxidative stress resistance in P. gingivalis is yet to be fully elucidated. In this study we examined the modulation of gene expression under conditions of oxidative stress by whole-genome DNA microarray analysis. Our findings indicated that numerous genes are involved …

Localization And Expression Of The Aer Receptor In Escherichia Coli, Daniel Salcedo

Loma Linda University Electronic Theses, Dissertations & Projects

In Escherichia coli, chemotactic movement towards an energetically-favorable environment is mediated by five transmembrane chemoreceptors. These sensory proteins respond to numerous environmental signals, including amino acids, sugars, and pH. The aerotaxis receptor, Aer, is an intracellular sensor that responds to oxygen concentration and other parameters affecting cellular energy levels. Bacterial chemotaxis receptors form dimeric signaling units that organize into tetramers and hexamers and form large clusters at the cell poles. A cluster arrangement could increase receptor sensitivity by lateral communication between dimers, thus amplifying the signal from one dimer. We have shown that Aer forms dimers, trimer of dimers, …

Altered Regulation Of Aquaporin Gene Expression In Allergen And Il-13-Induced Mouse Models Of Asthma, Carissa M. Krane, Bijia Deng, Venkateshwar Mutyam, Casey A. Mcdonald, Stephen Pazdziorko, Lawrence Mason, Samuel Goldman, Marion Kasaian, Divya Chaudhary, Cara Williams, Melisa W.Y. Ho

Biology Faculty Publications

IL-13 is known to affect many processes that contribute to an asthmatic phenotype, including inflammation, fibrosis, and mucus production. Members of the aquaporin (AQP) family of transmembrane water channels are targets of regulation in models of lung injury and inflammation. Therefore, we examined AQP mRNA and protein expression in allergen and IL-13-induced mouse models of asthma. Lungs from ovalbumin sensitized and ovalbumin challenged (OVA/OVA) and IL-13 treated mice showed airway thickening, increased mucus production, and pulmonary eosinophilia. Pulmonary function tests showed a significant increase in methacholine-induced airway hyperreactivity in OVA/OVA and IL-13-treated mice as compared with controls. Quantitative PCR analysis …

Comparison Of Replication-Competent, First Generation, And Helper-Dependent Adenoviral Vaccines, Eric A. Weaver, Pramod N. Nehete, Stephanie S. Buchl, Julien S. Senac, Donna Palmer, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and …

High Resolution Single Molecule Optical Localization Of Multiple Fluorophores On Dna Origami Constructs Fluorophores On Dna Origami Constructs, Anuradha Rajulapati

Theses, Dissertations and Capstones

An ongoing challenge in the development of nanoelectronics and nanophotonics is the nondestructive, high-resolution localization in space of single molecules and multi-molecular assemblies. The apparent barrier to the use of optical microscopy at the sub-100 nm scale is the well known Abbe Limit, the diffraction limit to resolution. This laboratory has adapted a technique called Single-molecule high resolution imaging with photobleaching (SHRIMP). We have developed methods for utilizing SHRIMP for the determination of the separation of two fluorophores in single DNA origami constructs.

DNA Origami is extremely useful because it can address nanocomponents down to 2nm separation. It should be …

Compensatory Evolution Of Gene Regulation In Response To Stress By Escherichia Coli Lacking Rpos, Daniel M. Stoebel, Karsten Hokamp, Michael S. Last, Charles J. Dorman

All HMC Faculty Publications and Research

The RpoS sigma factor protein of Escherichia coli RNA polymerase is the master transcriptional regulator of physiological responses to a variety of stresses. This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition. This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they evolve in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than …

X4 Human Immunodeficiency Virus Type 1 Gp120 Down-Modulates Expression And Immunogenicity Of Codelivered Antigens, Avi-Hai Hovav, Michael Santosuosso, Maytal Bivas-Benita, Andre Plair, Alex Cheng, Mazal Elnekave, Elda Righi, Tao Chen, Satoshi Kashiwagi, Michael W. Panas, Shi-Hua Xiang, Karina Furmanov, Norman L. Letvin, Mark C. Poznansky

Nebraska Center for Virology: Faculty Publications

In order to increase the immune breadth of human immunodeficiency virus (HIV) vaccines, strategies such

as immunization with several HIV antigens or centralized immunogens have been examined. HIV-1 gp120

protein is a major immunogen of HIV and has been routinely considered for inclusion in both present and

future AIDS vaccines. However, recent studies proposed that gp120 interferes with the generation of immune

response to codelivered antigens. Here, we investigate whether coimmunization with plasmid-encoded gp120

alters the immune response to other coadministered plasmid encoded antigens such as luciferase or ovalbumin

in a mouse model. We found that the presence of gp120 …

Identification And Characterization Of A New Kaposi’S Sarcoma-Associated Herpesvirus Replication And Transcription Activator (Rta)- Responsive Element Involved In Rta-Mediated Transactivation, Hui-Ju Wen, Veenu Minhas, Charles Wood

Nebraska Center for Virology: Faculty Publications

Kaposi’s sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) is well established as a key transcriptional activator that regulates the KSHV life cycle from latency to lytic replication. It is expressed immediately after infection and activates a number of viral genes leading to virus replication. The RTA-responsive element (RRE) in the RTA target gene promoters is critical for RTA to mediate this transactivation. A number of non-conserved RREs have been identified in various RTA-responsive promoters, and AT-rich sequences have been proposed to serve as RTA targets, but no consensus RRE sequence has been identified so far. Two nonconserved RREs (RRE1 …

Use Of Average Mutual Information For Studying Changes In Hiv Populations, Khalid Sayood, Federico Hoffman, Charles Wood

Nebraska Center for Virology: Faculty Publications

Average mutual information (AMI) has been used in a number of applications in bioinformatics. In this paper we present its use to study genetic changes in populations; in particular populations of HIV viruses. Disease progression of HIV-1 infection in infants can be rapid resulting in death within the the first year, or slow, allowing the infant to survive beyond the first year. We study the development of rapid and slow progressing HIV population using AMI charts based on average mutual information among amino acids in the env gene from a population of 1142 clones derived from seven infants with slow …

Comparison Of Replication-Competent, First Generation, And Helper-Dependent Adenoviral Vaccines, Eric A. Weaver, Pramod N. Nehete, Stephanie S. Buchl, Julien S. Senac, Donna Palmer, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

All studies using human serotype 5 Adenovirus (Ad) vectors must address two major obstacles: safety and the presence of pre-existing neutralizing antibodies. Helper-Dependent (HD) Ads have been proposed as alternative vectors for gene therapy and vaccine development because they have an improved safety profile. To evaluate the potential of HD-Ad vaccines, we compared replication-competent (RC), first-generation (FG) and HD vectors for their ability to induce immune responses in mice. We show that RC-Ad5 and HD-Ad5 vectors generate stronger immune responses than FG-Ad5 vectors. HD-Ad5 vectors gave lower side effects than RC or FG-Ad, producing lower levels of tissue damage and …

Development Of Pcr Assays To Detect And Characterise Salmonella Isolates From Cheese And Meat Products By Utilizing An Integrated Genomics Approach, Noelle Geraldine Mccarthy

Theses

Salmonella is an extremely important foodborne pathogen which is responsible for millions of cases of gastroenteritis annually. Salmonella outbreaks have been associated with numerous common food stuffs such as meat, dairy and seafood. Traditional culture based methods for the detection Salmonella are labourious and costly. Rapid detection and serovar identification would dramatically decrease the time required to identify Salmonella outbreaks and potential health risks.

In the US 99% of all Salmonella infections are caused by members of Salmonella enterica subspecies 1. Serovars within subspecies 1 can vary due to host specificity and pathogenicity. DNA sequencing projects have provided valuable genomic …

Electroporation For The Delivery Of Dna-Based Vaccines And Immunotherapeutics: Current Clinical Developments, Angela M. Bodles-Brakhop, Richard Heller, Ruxandra Draghia-Akli

Bioelectrics Publications

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. the recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. current research has revealed that EP can be successfully used in many species, including humans. clinical trials are …

Development Of A Gene Silencing Dna Vector Derived From A Broad Host Range Geminivirus, Edward M. Golenberg, D Noah Sather, Leandria C. Hancock, Kenneth J. Buckley, Natalie M. Villafranco, David M. Bisaro

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.

Results

The construction of a gene silencing vector derived from the …

Letter From The Dean, Lalit Verma

Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences

No abstract provided.

Patterning Defects In Silkworm Embryos Analysed Through Cuticle Preparations, Amit Singh, Madhuri Kango-Singh, K. P. Gopinathan

Biology Faculty Publications

The mulberry silkworm, Bombyx mori, a holometabolous lepidopteran insect, has a metameric body plan. Due to its functional adaptation, B. mori presents some unique deviations in its pal/ern from the evolutionarily advanced dipteran insect, Drosophila melanogaster. Previous studies on mutant phenotype analysis in B. mori have been carried out in late stages of larval development. Here we employ, the cuticle preparation approach during embryonic development to study morphological landmarks associated with B. mori, Eri, another race a/silkworm, and pattern defects associated with Ekp mutant of B.mori. The homeotic mutant Ekp, generates ectopic abdominallegs, a feature …

Intradermal Delivery Of Plasmid Vegf(165) By Electroporation Promotes Wound Healing, Bernadette Ferraro, Yolmari Cruz, Domenico Coppola, Richard Heller

Bioelectrics Publications

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF₁₆₅ (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal …

Human Ubc9 Contributes To Production Of Fully Infectious Human Immunodeficiency Virus Type 1 Virions, Tareq Jaber, Christopher R. Bohl, Gentry L. Lewis, Charles Wood, John T. West Jr., Robert A. Weldon Jr.

Nebraska Center for Virology: Faculty Publications

Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, …

Expression Of Hpv16 E5 Produces Enlarged Nuclei And Polyploidy Through Endoreplication, Lulin Hu, Tamara A. Potapova, Shibo Li, Susannah Rankin, Gary J. Gorbsky, Peter C. Angeletti, Brian P. Ceresa

Nebraska Center for Virology: Faculty Publications

Anogenital cancers and head and neck cancers are causally-associated with infection by high-risk

human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to

oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can

transform cells when expressed independently. HPV16 E6 and E7 have well-described roles

causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell

transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid

nuclei that are dependent on the level and duration of HPV16 E5 expression. Live-cell imaging

data indicate these changes do not …

Human Papillomavirus 16 E5 Induces Bi-Nucleated Cell Formation By Cell-Cell Fusion, Lulin Ha, Kendra Plafker, Valeriya Vorozhoko, Rosemary E. Zuna, Marie H. Hanigan, Gary J. Gorbsky, Scott M. Plafker, Peter C. Angeletti, Brian P. Ceresa

Nebraska Center for Virology: Faculty Publications

Human Papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes – E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, …

A Comparative Study Of Hiv-1 Clade C Env Evolution In A Zambian Infant With An Infected Rhesus Macaque During Disease Progression, For Yue Tso, Federico G. Hoffmann, Damien C. Tully, Philippe Lemey, Robert A. Rasmussen, Hong Zhang, Ruth M. Ruprecht, Charles Wood

Nebraska Center for Virology: Faculty Publications

Objective—To evaluate whether HIV-1 clade C (HIV-C) envelope variations that arise during disease progression in rhesus macaque model reflect changes that occur naturally in human infection.

Design—An infant macaque was infected with SHIV-1157i, an R5 tropic clade C SHIV (SHIV-C) which expresses a primary HIV-C envelope derived from an infected human infant, and monitored over a five-year period. Genetic variation of the V1-V5 envelope region, which is the main target for humoral immune responses, derived from the infected macaque and infant was examined.

Methods—V1-V5 envelope region were cloned and sequenced from longitudinal PBMC samples collected from the infected macaque and …

Protection Against Mucosal Shiv Challenge By Peptide And Helper-Dependent Adenovirus Vaccines, Eric A. Weaver, Pramod N. Nehete, Bharti P. Nehete, Stephanie J. Buchl, Donna Palmer, David C. Montefiori, Philip Ng, K. Jagannadha Sastry, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helper- dependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5- immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than …