Life Sciences Commons^™

Bacteriophage Ecology In A Commercial Cucumber Fermentation, Jean Lu, Ilenys M. Perez-Diaz, Janet S. Hayes, Fred Bredit Jr.

Faculty and Research Publications

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent …

Natural And Lignocellulose-Enriched Microbial Communities In Great Boiling Spring, Nv, Jessica Cole

UNLV Theses, Dissertations, Professional Papers, and Capstones

The natural microbial communities present in Great Boiling Spring were investigated and contrasted against those present after in situ enrichment with lignocellulose. High-throughput cultivation-independent DNA sequencing of the V8 region of the small subunit (SSU) rRNA gene generated a total of 274,119 quality-filtered pyrosequencing fragments. Twelve natural spring samples were analyzed, including four high-temperature water samples and eight sediment samples ranging from 87 - 62 °C. Eight lignocellulosic enrichments incubated in the spring sediment and water at two high-temperature sites were analyzed. The natural water communities were found to be extremely uneven but relatively constant throughout time. The natural sediment …

Anti-Germinants As A New Strategy To Prevent Clostridium Difficile Infections, Amber Janece Howerton

UNLV Theses, Dissertations, Professional Papers, and Capstones

Clostridium difficileinfections (CDI) have emerged as a leading cause of hospital-associated complications. CDI is the major cause of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. The infective form of C. difficileis the spore, a dormant and hardy structure that forms under stress. Germination of C. difficile spores into toxin producing bacteria in the GI tract of susceptible patients is the first step in CDI establishment. Patient susceptibility occurs with a disruption of the natural gut microbiota by broad-spectrum antibiotics. Antibiotic treatments usually resolve CDI but refractory cases are on the rise. Of great concern is the …

The Evolution Of Host Specificity In The Vertebrate Gut Symbiont Lactobacillus Reuteri, Steven Frese

Department of Food Science and Technology: Dissertations, Theses, and Student Research

The vertebrate gut is home to one of the densest populations of life on Earth. This microbial community has a profound effect on host health, nutrition, development, behavior, and evolution. However, very little is known about how these microbes have evolved with their vertebrate hosts, how and whether they select hosts or how they remain associated with their hosts. Recent work identified Lactobacillus reuteri as an organism that is composed of host-specific sub-populations, each population associated with a different host animal. Representatives from each host-associated population were tested for their ability to colonize gnotobiotic mice, which only rodent strains could …

Fecal Indicator Bacteria In Knitting Mill Creek With A Comparison Of Methods Used To Enumerate E. Coli, Manju Nagarajan

OES Theses and Dissertations

This research seeks to evaluate the impact of an urban, multi-use neighborhood on the bacteriological quality of water in Knitting Mill Creek, a blind arm of the Lafayette River, a sub-estuary of the lower Chesapeake Bay. A principal objective was to determine the effects of rainfall, a surrogate for run-off, on water-column concentrations of fecal-indicator bacteria (FIB). I collected water samples weekly (September 2009-December 2010) and biweekly (January 2010-December 2011) at a storm-sewer outfall and a nearby marina and quantified their E. coli and enterococci concentrations using Colilert-18 and Enterolert, most-probable-number assays. In addition, I took monthly samples for these …

Microbial Sunscreens: Opportunities And Obstacles, Tanya Soule

Tanya Soule

No abstract provided.

Staphylococcus Aureus Response To Long Chain Antimicrobial Fatty Acids, Benjamin Arsic

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is a common pathogen that has the ability to infect virtually every tissue and organ system of the body. Despite its propensity to cause invasive infection, S. aureus is also a commensal organism, asymptomatically colonizing ~25% of the population. Much research has gone into resolving this paradox, focusing on both human and bacterial factors that may contribute to colonization. Antimicrobial fatty acids present on the skin and in the nasal mucosa are important components of the innate immune system, and thus we undertook to further understand how S. aureus responds to these fatty acids, and how this response …

Borrelia Burgdorferi Cp32 Bpab Modulates Expression Of The Prophage Nucp Nuclease And Ssbp Single-Stranded Dna-Binding Protein, Alicia M. Chenail, Brandon L. Jutras, Claire A. Adams, Logan H. Burns, Amy Bowman, Ashutosh Verma, Brian Stevenson

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The Borrelia burgdorferi BpaB proteins of the spirochete's ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5′-TTATA-3′. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5′ noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, …

Genomic Characteristics Of An Environmental Microbial Community Harboring A Novel Human Uncultured Tm7 Bacterium Associated With Oral Diseases, Michael Abrams, David Barton, Eamon Vandaei, Diana Romero, Adam Caldwell, Cleber C. Ouverney

Faculty Publications, Biological Sciences

The diversity of prokaryotes associated with humans has been dominated by uncultured species (not isolated in pure culture). For instance, nearly 80% of the human gut and 68% of the human oral microbes are thought to be uncultured; some of which have been associated with human oral, digestive, vaginal, and cardiovascular diseases. The revalence of uncultured pathogens is expected to continue to increase within the near future. In fact, public databases such as GenBank have nearly quadrupled the number of candidate phyla (those made entirely of uncultured organisms) since the 1980s and currently lists many new lineages of unclassified Bacteria …

The Mycoplasma Genitalium Mg_454 Gene Product Resists Killing By Organic Hydroperoxides, Sankaralingam Saikolappan, Smitha Sasindran, Hongwei Yu, Joel Basema, Subramanian Dhandayuthapani

Hongwei Yu

Mycoplasma genitalium is the smallest self-replicating organism and a successful human pathogen associated with a range of genitourinary maladies. As a consequence of its restricted genome size, genes that are highly conserved in other bacteria are absent in M. genitalium. Significantly, genes that encode antioxidants like superoxide dismutase and catalase-peroxidase are lacking. Nevertheless, comparative genomics has revealed that MG_454 of M. genitalium encodes a protein with putative function as an organic hydroperoxide reductase (Ohr). In this study, we found that an M. genitalium transposon mutant that lacks expression of MG_454 was sensitive to killing by t-butyl hydroperoxide and cumene hydroperoxide. …

The Crispr/Cas Adaptive Immune System Of Pseudomonas Aeruginosa Mediates Resistance To Naturally Occurring And Engineered Phages, Kyle C. Cady, Joe Bondy-Denomy, Gary E. Heussler, Alan R. Davidson, George A. O'Toole

Dartmouth Scholarship

Here we report the isolation of 6 temperate bacteriophages (phages) that are prevented from replicating within the laboratory strain Pseudomonas aeruginosa PA14 by the endogenous CRISPR/Cas system of this microbe. These phages are only the second identified group of naturally occurring phages demonstrated to be blocked for replication by a nonengineered CRISPR/Cas system, and our results provide the first evidence that the P. aeruginosa type I-F CRISPR/Cas system can function in phage resistance. Previous studies have highlighted the importance of the protospacer adjacent motif (PAM) and a proximal 8-nucleotide seed sequence in mediating CRISPR/Cas-based immunity. Through engineering of a protospacer …

Minor Pilins Of The Type Iv Pilus System Participate In The Negative Regulation Of Swarming Motility, S L. Kuchma, E. F. Griffin, G. A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming …

Sequesteration Of Lead, Cadmium And Arsenic By Lactobacillus Species And Detoxication Potential, Marc A. Monachese

Electronic Thesis and Dissertation Repository

Toxic metals are a class of elements with no biological role but with extreme toxicity. On average only 50% of ingested toxins are absorbed into the human body, for reasons still unknown. It was hypothesized that the gut microbiota plays a role in reducing toxin absorbance. The aim of this study was to determine if constituents of the gut, namely Lactobacillus species, are able to sequester arsenic, lead and cadmium from the environment. Lactobacilli were incubated with the metals, both in vitro and with a Caco-2 cell line. Analysis of metal concentrations was conducted to determine if these were reduced …

Regulation Of Toxin Synthesis By Clostridium Difficile, Charles Darkoh

Dissertations & Theses (Open Access)

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce …

Production And Degradation Of 4-Ethylphenol In Lactobacillus Sp. Pep8 Cultures And In Blended Swine Lagoon Enrichments, Clinton W. Copp

Masters Theses & Specialist Projects

4-Ethylphenol (4-EP) is a malodorant of swine waste and is derived from a component of lignin called p-coumaric acid (p-CA). The production of 4-EP from lignin in swine waste is untested. Additionally, the effect of Fe (III) on 4-EP levels is unknown. Four experiments were performed to determine if Lactobacillus sp. pep8 cultures, as well as enriched swine lagoon slurries, could liberate p-CA from lignin and convert p-CA to 4-EP. Furthermore, it was tested if the addition of Fe (III) influences the conversion of p- CA to 4-EP.

Experiment 1 tested Lactobacillus sp. pep8 cultures to determine if the addition …

Accumulation Of Extracellular Carbohydrates In A Scytonemin-Deficient Mutant Of Nostoc Punctiforme Exposed To Uva Stress, Tanya Soule

Tanya Soule

No abstract provided.

Epoxide-Mediated Cifr Repression Of Cif Gene Expression Utilizes Two Binding Sites In Pseudomonas Aeruginosa, Alicia E. Ballok, Christopher D. Bahl, Emily L. Dolben, Allia K. Lindsay, Jessica D. St. Laurent, Deborah Hogan, Dean Madden, George A. O'Toole

Dartmouth Scholarship

Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB, the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the -10 and +1 sites of both the cifR …

Serratia Marcescens: The Miracle Bacillus, Alan L. Gillen, Rebekah Gibbs

Faculty Publications and Presentations

The objectives of this article are to explain the mysterious appearance of crimson-colored bacteria on food and communion bread/wafers, over the centuries, as well as to describe the biological basis

Efficacy Comparison Of Various Pcr Master Mixes And Thermal Cycling Parameters For Detection Of Brucella Abortus In Multiplex Assays, Kevin J. Testo, Jason A. Olivas, Alda C. Carrillo, Pejman Naraghi-Arani

STAR Program Research Presentations

Diagnostic testing for bioterrorism agents in food is essential for the safety and welfare of the general populace. Multiplex assays provide the means to test for multiple pathogen signatures with a single polymerase chain reaction (PCR) test. PCR, Luminex xMAP ® infrared fluorophore bead technology, and flow cytometry, multiplex diagnostic assays are incredibly specific and sensitive to highly conserved regions at various loci in a microbe’s genome. In order to increase the effectiveness of the PCR and the resulting fluorescent response of the beads, different PCR master mix commercial kits were compared to the standard AgPath RT-PCR mix. This study …

Identification Of Persistent Long Range Interactions In GA95 And GB95 Through Thermal Unfolding Simulations, Milen Redai Tesfamariam

Chemistry & Biochemistry Theses & Dissertations

For over five decades, different experiments have been performed to research how proteins attain their native three dimensional structures. However, the folding problem continues to be a puzzle in modern science. The design of two proteins that have maximal sequence identity but different folds and functions is one method that is being used to study the relationship between protein structure and amino acid sequence. In particular, mutant proteins of Streptococcus protein G, G_A and G_B, have 95% sequence identity and a 3a helix fold and β4/a fold, respectively. Molecular dynamics simulations of G_A95 …

Cyclic Di-Gmp Phosphodiesterases Rmda And Rmdb Are Involved In Regulating Colony Morphology And Development In Streptomyces Coelicolor. Journal Of Bacteriology, Travis D. Hull, Min-Hyung Ryu, Matthew J. Sullivan, Ryan C. Johnson, Nikola T. Klena, Robert M. Gelger, Mark Gomelsky, Jennifer A. Bennett

Biology and Earth Science Faculty Scholarship

Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator ofmorphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. …

Lapg, Required For Modulating Biofilm Formation By Pseudomonas Fluorescens Pf0-1, Is A Calcium-Dependent Protease, Chelsea D. Boyd, Debashree Chatterjee, Holger Sondermann, George A. O'Toole

Dartmouth Scholarship

Biofilm formation by Pseudomonas fluorescens Pf0-1 requires the cell surface adhesin LapA. We previously reported that LapG, a periplasmic cysteine protease of P. fluorescens, cleaves the N terminus of LapA, thus releasing this adhesin from the cell surface and resulting in loss of the ability to make a biofilm. The activity of LapG is regulated by the inner membrane-localized cyclic-di-GMP receptor LapD via direct protein-protein interactions. Here we present chelation and metal add-back studies demonstrating that calcium availability regulates biofilm formation by P. fluorescens Pf0-1. The determination that LapG is a calcium-dependent protease, based on in vivo and in vitro …

Colonization Of Zea Mays By The Nitrogen Fixing Bacterium Gluconacetobacter Diazotrophicus, Nikita Eskin

Electronic Thesis and Dissertation Repository

To achieve high yields, corn fields are supplemented with nitrogen fertilizers. Nitrogen fertilizers account for a significant portion of production costs, and are harmful to the environment. Gluconacetobacter diazotrophicus, an endophytic nitrogen-fixing bacterium supplies its natural host sugarcane with a significant amount of nitrogen. This study investigated the colonization of G. diazotrophicus in seven different corn genotypes consisting of sweet and grain corn via three different methods of inoculation: soil drench, root dip, and aseptic inoculation. Sucrose content of the corn genotypes and nitrogenase activity were also analysed. Colonization was confirmed by PCR analysis. G. diazotrophicus colonization was detected using …

Host Pathogen Interactions: Is Arabidopsis Thaliana Remembered By Its Nemesis Pseudomonas Syringae?, Daniel Z. Kreiser

Lawrence University Honors Projects

Plants contain innate immune systems that deter pathogen infection. Pattern recognition receptors bind microbe-associated molecular patterns (MAMPs), triggering immunity. MAMPs are proteins exclusive to pathogens that are typically indispensable for their survival. For this reason, MAMPs cannot be mutated or removed without causing pathogen death. However, this does not necessitate constitutive expression of MAMPs. In this study, the MAMP response of Arabidopsis thaliana was utilized to determine differential detection of MAMPs expressed by Pseudomonas syringe pv. tomato DC3000 when pretreated with A. thaliana. Results demonstrated that more MAMPs are detected when P. syringae had previously encountered A. thaliana, …

Discovery Of A Novel Antibiotic From A Bacillus Bacterium Cultivated From Its Natural Environment, Patrick J. Mcmonagle

Lawrence University Honors Projects

The current primary method of treating bacterial infections is using antibiotics. However, this continued treatment of these illnesses caused by pathogenic bacteria is causing the rate of evolution of these disease-inducing organisms to increase. Antibiotic resistance is forcing scientists to search for new forms of antibiotics to compete with these new ‘super bugs.’ I pursued the search for novel antibiotics through their natural source – antibiotic-producing microorganisms. As microbes have a direct advantage when producing antibiotics, it is my thought that they will be the best resource to discovering new and effective antibiotics. I conducted an exploratory search for antibiotic-producing …

Enumeration Of Sandy Sediment Bacteria: Search For Optimal Protocol, Slava Epstein, Jeffrey Rossel

Slava Epstein

We examined and compared a variety of existing protocols for enumeration of bacteria from marine sandy sediments. The focus was on how to dislodge bacteria from sediment particles; a commercial blender, an ultrasonic cleaner, and an ultrasonic cell disrupter were tested. The ultrasonic cell disrupter was found to be the most efficient device for bacterial dislodgment. With a 5 mm microtip vibrating at 109 μm amplitude and 20 kHz, the optimal sonication time of small (≤ 0.5 cm³) samples was 180 s. Having identified the optimal dislodgment treatment, we went through other steps of sediment bacteria enumeration (use of surfactants, …

Enumeration Of Sandy Sediment Bacteria: Are The Counts Quantitative Or Relative?, Slava S. Epstein, D. Alexander, K. Cosman, A. Dompé, S. Gallagher, J. Jarsobski, E. Laning, R. Martinez, G. Panasik, C. Peluso, R. Runde, E. Timmer

Slava Epstein

Several tests were carried out to enable evaluation of the precision with which sandy sediment bacteria could be enumerated. This represented the first attempt to place direct epifluorescence counts of benthic bacteria on a quantitative, rather than relative, ground. The tests combined in situ radioisotope ([³H], [¹⁴C]) labeling of sediment bacteria, bacterial dislodgment by ultrasonic treatment, and bacterial enumeration via fluorescent staining. The results provided direct and indirect evidence that the employed protocol for bacterial enumeration accounted for 88 to 98% of all bacteria present in sediments. The identified approach thus allowed for a rather complete quantification of sediment bacteria. …

Methodology Of In Situ Grazing Experiments: Evaluation Of A New Vital Dye For Preparation Of Fluorescently Labeled Bacteria, Slava S. Epstein, Jeffrey Rossel

Slava Epstein

A new fluorescent dye, cyanoditolyl tetrazolium chloride (CTC), is suggested for use in grazing studies to prepare fluorescently labeled bacteria (FLB). As food tracers, CTC-stained bacteria appeared to have advantages over FLBs prepared in accordance with previously published protocols. The CTC is a vital dye, non-toxic to the examined bacteria; in our study, the CTC-stained bacteria grew at the same rate as non-stained bacteria. This is an advantage over the commonly used DTAF-employing staining protocol, which results in heat-killed or immobilized prey particles. The CTC-stained bacteria were not toxic to the studied bacterial grazers; a test ciliate grew on FLBs …

Synthesis Of Fluorogenic Substrates For The Enzymatic Characterization Of Rv0045c From M. Tuberculosis, Kelly Jo Mckenna

Undergraduate Honors Thesis Collection

Mycobacterium tuberculosis is the pathogenic bacterial agent commonly responsible for tuberculosis, or TB. Although treatment exists for the active form of tuberculosis, no method has been developed for eliminating M tuberculosis in its dormant state. One hypothesized method for the elimination of dormant TB is to develop an inhibitor specific for M tuberculosis esterases and lipases, as these esterases and lipases are essential to the survival of dormant TB infection. In this research, the substrate specificity of the Rv0045c esterase from M tuberculosis was studied due to the essential role of Rv0045c in TB metabolism and its dissimilarity to other …

An Investigation Of Bacterial Ribonucleases As An Antibiotic Target, Ashley Denise Frazier

Electronic Theses and Dissertations

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance.

Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of …