Bacteriology Commons^™

Evaluation Of Virb Binding Site Contribution To The Regulation Of The Icsp Promoter In Shigella Flexneri, Juan C. Duhart, Maria I. Castellanos, Helen J. Wing

Undergraduate Research Opportunities Program (UROP)

Shigella species are gram-negative, rod-shaped bacteria that are closely related to Escherichia coli. Virulent Shigella spp. are intracellular pathogens that invade, replicate and spread through epithelial cells of the lower intestine and cause bacillary dysentery in humans. This disease is characterized by a robust inflammatory response that results in fever, abdominal pain, and bloody diarrhea (3). According to the CDC, approximately 14,000 cases are reported each year in the United States alone. This number however, does not reflect the actual incidence of this disease as many cases go unreported. The molecular pathogenesis of these bacteria lies in the large virulence …

Transcriptional Regulation Of The Shigella Flexneri Icsp Promoter: Silencing And Anti-Silencing By H-Ns And Virb, Dustin Harrison

UNLV Theses, Dissertations, Professional Papers, and Capstones

Shigella species are gram-negative intracellular pathogens that cause bacillary dysentery in humans. Many genes required for virulence of Shigella are carried on a large 230 kb plasmid and many of these are under the transcriptional control of the histone-like nucleoid structuring protein (H-NS) and by the major virulence regulator VirB. At the non-permissive temperature of 30⁰C, H-NS represses transcription, while at 37⁰C VirB alleviates this repression. This mechanism of gene regulation has been coined "silencing/anti-silencing" and is commonly found in many important bacterial pathogens including Salmonella spp. and Yersinia spp. The icsP gene, encoded by the Shigella virulence plasmid, is …

Constraint-Based Model Of Shewanella Oneidensis Mr-1 Metabolism: A Tool For Data Analysis And Hypothesis Generation, Grigoriy E. Pinchuk, Eric A. Hill, Oleg V. Geydebrekht, Jessica De Ingeniis, Xiaolin Zhang, Andrei Osterman, James H. Scott

Dartmouth Scholarship

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based …

Natural Competence In Thermoanaerobacter And Thermoanaerobacterium Species, A Joe Shaw, David A. Hogsett, Lee R. Lynd

Dartmouth Scholarship

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant.

In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence- mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to …

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 …

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.

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 …

Letter From The Dean, Lalit Verma

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

No abstract provided.

The Regulation Of The Icsp Promoter Of Shigella Flexneri By The Virulence Factor Virb, Maria Castellanos, Dustin Harrison, Helen Wing

Undergraduate Research Opportunities Program (UROP)

Shigella flexneri is a pathogenic bacterium that causes severe dysentery in humans commonly known as shigellosis. Shigella encodes an outer membrane protease called IcsP. The regulation of icsP expression is under direct control of a transcriptional factor called VirB, which controls the expression of many virulence genes in Shigella. Previous work has shown through deletion analysis of the icsP promoter region that sequences as far as 1368 base pairs upstream of the transcription starting site are important for the regulation of the icsP gene by VirB. However, it is still unclear whether VirB activation requires sequences within the icsP promoter …

Genomic Foundations Of Carbon Fixation In Bacteria Living In Hot Springs, Rachel K. Skinner, Brian P. Hedlund, Jeremy A. Dodsworth

Undergraduate Research Opportunities Program (UROP)

Photosynthesis does not occur above 73°C, so organisms living above this temperature must obtain useable carbon by some other mechanism. It is generally assumed that carbon is fixed by thermophiles through the process of chemolithoautotrophy; however, primary production has never been demonstrated to occur in hot springs >73°C. We have shown that two organisms, Thermocrinis and Pyrobaculum, make up more than 90% of the cells in an 80°C Great Basin hot spring, Great Boiling Spring. We hypothesize that these organisms fix carbon in the hot spring via the reverse tricarboxylic acid (rTCA) cycle. To test this hypothesis we will: i) …

Comparison Of Dna Pyrosequencing With Alternative Methods For Identification Of Mycobacteria, Loree C. Heller, Michael Jones, Ray H. Widen

Bioelectrics Publications

Identification of mycobacterial clinical isolates by pyrosequencing within the hypervariable A region of the 16S rRNA gene was compared to other identification methods. For >90% of isolates, these identifications correlated to the level of complex or species. For identification of many mycobacteria, pyrosequencing offers an inexpensive alternative to traditional sequencing.

Sara Of Staphylococcus Aureus Binds To The Sara Promoter To Regulate Gene Expression, Ambrose L. Cheung, Koren Nishina, Adhar C. Manna

Dartmouth Scholarship

The 375-bp sarA open reading frame is driven by three promoters, P1, P3, and P2. Using gel shift and DNase I footprinting assays, we found that SarA binds to two 26-bp sequences and one 31-bp sequence within the P1 and P3 promoters, respectively. Together with the results of transcription analyses, our data indicate that SarA binds to its own promoter to down-regulate sarA expression.

Assessment Of Key Parameters To Achieve Bacteria Transformability In E. Coli And A. Tumefaciens, Zach Regelin

Honors Program Theses

ln this project, growth and transformability of E. coli and A. tumefaciens were investigated. The growth of the bacterial species was monitored under different key parameters including: the strain of the bacteria, the inoculum source (a fresh plate or frozen glycerol stock), and the media type. Growth was monitored by measuring the optical density (OD). Using E. coli strains Dh5a and CdB\ it was determined that the strain, the media type, and the amount of inoculum had a great effect on the growth but the source of the inoculum had little effect. For A. tumefaciens, the strain and the media …

Cloning And Functional Analysis Of The Putative Tetracycline Resistance Gene Hp1165 In Helicobacter Pylori, Yuhuan Li

All-Inclusive List of Electronic Theses and Dissertations

Tetracycline has been used in combination with other antibiotics in treatment of Helicobacter pylori infection. However, tetracycline resistance has developed in clinical isolates of H. pylori, rendering treatment failure. Mutations in the 16S rRNA gene have been reported to mediate tetracycline resistance in some patient isolates. Nevertheless, the diversity of tetracycline resistance cases suggests multiple genes are involved. HPl 165, a putative tetracycline resistance gene in the genome of H. pylori 26695, displays 49.8% identity to the tetracycline efflux gene tetA (P) from Clostridium perfringens. To determine the function of the HPl 165 gene and gain a better understanding of …

Pcr And 16s Rdna Sequencing For Identifying And Comparing Gram-Negative Bacteria, Molly Truax

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Caenorhabditis Elegans: A Look At Koch's Postulates And Host-Pathogen Interactions, Nicole Helmrichs

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Identification Of The Polar Flagellum Genes In Azospirillum Brasilense, Enid Thanna Mckinley

Loma Linda University Electronic Theses, Dissertations & Projects

Azospirillum brasilenser, a nitrogen fixing rhizobacterium, can colonize plant roots and increase the yield of economically important cereals, such as rice and grasses. This organism has a mixed pattern of flagellation: a single polar flagellum for motility in liquid medium and lateral flagella which might be involved in motility across the root surface and or attachment to the root hairs of plants. Flagella dictate A. brasilense chemotactic ability and motility, which plays an important role in colonization of plant roots. However, nothing is known about the genes that code for the polar flagellum. To study the polar flagellum, different mutants …

Molecular Evolution Of Insecticidal Spore-Forming Bacteria, John Pool

Honors Theses

Molecular methods are increasingly being used to determine the phylogeny of microorganisms. This research was intended to determine phylogenetic relationships for bacteria of the species Bacillus thuringiensis and other members of the Bacillus cereus group. Each strain was analyzed by its sasp-B gene sequence to determine its species classification and relation to other strains studied. Results of this study indicated that according to the sasp-B gene tree, the species Bacillus thuringiensis is a paraphyletic with respect to both Bacillus cereus and Bacillus anthracis. Some unexpected results and implications for species designations are also discussed.

Construction Of Escherichia Coli Arginine Repressor Fusion Protein And Analysis Of Its Function In Xer Site-Specific Recombination, Kholis Abdulrachim

Student Works (2000-2009)

In addition to its role in L-arginine biosynthesis in Escherichia coli, arginine repressor (ArgR), the product of the argR gene, also plays an essential role as an obligate accessory protein in Xer site-specific recombination system. A structure-function relationship study of ArgR was performed to understand more about its role in Xcr site-specific recombination. Fusion proteins between ArgRWT (wild-type ArgR) and a biotinylated peptide as well as between ArgRNV (a mutant ArgR) and a biotinylated peptide were constructed. The biotinylated peptide was fused in frame to the amino-tenninus of ArgR WT and ArgRNV, respectively. Xer recombination assays showed that the ArgRWT-biotinylated …

Genetic And Structural Analyses Of Cytoplasmic Filaments Of Wild-Type Treponema Phagedenis And A Flagellar Filament-Deficient Mutant, Jacques Izard, William A. Samsonoff, Mary Beth Kinoshita, Ronald J. Limberger

Department of Food Science and Technology: Faculty Publications

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments, cfpA, was identified in various treponemal species. Treponema phagedenis cfpA was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from T. denticola, T. vincentii, and T. pallidum subsp. pertenue. Wild-type T. phagedenis and a …

Insertional Inactivation Of Treponema Denticola Tap1 Results In A Nonmotile Mutant With Elongated Flagellar Hooks, Ronald J. Limberger, Linda L. Slivienski, Jacques Izard, William A. Samsonoff

Department of Food Science and Technology: Faculty Publications

The treponemal fla operon is comprised of numerous motility-related genes; however, the initial gene of this operon, tap1, has no known function. A recently developed system to generate specific mutants in Treponema denticola was utilized to determine if Tap1 was essential for motility. T. denticola tap1 and flanking DNA were identified, cloned, and sequenced, and a suicide plasmid that contained tap1 interrupted with an erythromycin resistance cassette (ermF and ermAM) was constructed. Because of potential polar effects from this cassette, a second plasmid that contained tap1 interrupted with a modified erythromycin resistance cassette that lacked the putative ermF transcription …

Characterization Of Metal-Regulated Genes In Pseudomonas Fluorescens, Melanie Pearson

Honors Theses

One side effect of today's industrialized world is increased levels of heavy metals in the environment. Many of these metals are necessary for biological function as trace elements, but at higher concentrations are toxic. Other metals, such as cadmium, are not beneficial at any level, and have only deleterious effects on living organisms. Cadmium is primarily thought to interfere with normal biological function of proteins. Human exposure to cadmium appears to primarily damage the kidneys, but may also affect the liver, lungs, immune system, and central nervous system. Bacteria, however, have developed several methods for handling toxic heavy metals: cation …

Genetics And Applications Of Nisin Production In Lactococcus Lactis Subsp. Lactis And Conjugal Exchange Of This Trait, Jeffery R. Broadbent

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative.

Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip⁺Suc⁺) to plasmid DNA in two well characterized L. lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip⁺Suc⁺ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. …

The Expression And Regulation Of The Hsdk Genes Of Escherichia Coli K-12, Ainu Parkash

Loma Linda University Electronic Theses, Dissertations & Projects

The DNA restriction and modification (R-M) genes in bacteria play a defensive role in the organism, offering protection to the host from invaders such as bacteriophages. The expression of these genes in E. coli K-12 had not previously been studied due to the complexity of the EcoK R-M enzymes. The use of lacZ gene fusion techniques allowed studies of the expression of the hsdK genes for the first time. Operon fusions of Mu(/ac) to the hsdK genes show that the two hsdK promoters, p _res and p _mod, are weak (50 Miller units) and that …

Characterization Of Hsd::Mudx128 Operon Fusion Mutant Of Escherichia Coli K-12, Marjorie Ann T. Reyno

Loma Linda University Electronic Theses, Dissertations & Projects

The study of the regulation of gene expression in complex genes can be facilitated by the use of operon fusions which place a well characterized lacZ gene under the control of a promoter of interest. An hsd::MudX128 operon fusion mutant of E. coli K-12 isolated by Prakash (1986) was observed to have a high β-galactosidase activity (1000 units), ten to twenty times higher than other hsd::MudX mutants. This β-galactosidase activity was suppressed to a very low level (25 units) by the introduction of an F’ plasmid, F'101, which carries the 98 min to 2 min region of the …

Genetic Transformation In Salmonella Typhimurium, Siao Ping Tasi

Loma Linda University Electronic Theses, Dissertations & Projects

Plasmid transformation is an essential step in many gene cloning experiments. In Salmonella tynhimurium, mutants of rough strain have shown improved transformation frequency previously. In this work a stable restriction-minus (r^-) and modification-plus (m⁺) strain and its derivatives were constructed. Effect of restriction barrier on transformation of plasmid pBR322 was tested. Several factors which may affect transformation frequency have been systematically examined to determine the optimum conditions for transformation in S. tvphimurium. Efforts were made to simplify the procedure.

Using pBR322 plasmid as an experimental model, the r^- strain demonstrated 10⁵ to …