No description available.

In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)

The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1. / Thesis / Master of Science (MS)

Histophilus somni is a commensal of the mucosal surfaces of respiratory and reproductive tracts of cattle and sheep. However, as an opportunistic pathogen, H. somni can cause diseases such as pneumonia, myocarditis, abortion, arthritis, and meningo-encephalitis. Previously, several virulence factors/mechanisms had been identified in H. somni of which the phase-variable lipooligosaccharide, induction of host cell apoptosis, intraphagocytic survival, and immunoglobulin Fc binding proteins were well characterized. To further understand the biological properties of H. somni, the genomes of pneumonia strain 2336 and preputial strain 129Pt have been sequenced. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, putative genes that encode proteases, restriction-modification enzymes, hemagglutinins, glycosyltransferases, kinases, helicases, and adhesins have been identified in H. somni. Most of the H. somni strain-specific genes were found to be associated with prophage-like sequences, plasmids, and/or transposons. Therefore, it is likely that these mobile genetic elements played a significant role in creating genomic diversity and phenotypic variability among strains of H. somni. Functional characterization of H. somni luxS in the genomic context revealed that the gene encodes S-ribosylhomocysteinase that can complement biosynthesis of AI-2 quorum sensing signal molecules in Escherichia coli DH5alpha. It was also found that several pathogenic isolates of H. somni form a prominent biofilm and that luxS as well as phosphorylcholine expression can influence biofilm formation by H. somni. In conclusion, comparative analyses of the genomes and functional characterization of putative genes have shed new light on the versatility and evolution of H. somni. / Ph. D.

The purpose of this research was to determine if bacteriophage (phage) could be used to treat and reduce the incidence of Listeria in food manufacturing facilities, and thereby reduce the risk of food products being cross-contaminated with Listeria monocytogenes. Listeria incidence in food manufacturing ready-to-eat environments was surveyed at 31 ready-to-eat (RTE) food plants. A total 4,829 samples were collected from all locations. Nine (29%) facilities had zero samples positive for Listeria spp., whereas 22 (71%) had one or more samples positive. The total incidence of Listeria spp. in all RTE food plants was 4.5%. The effectiveness of phage against Listeria was determined when applied to stainless steel, polyurethane thermoplastic, and epoxy. Each material was inoculated with a cocktail containing L. monocytogenes and L. innocua (4 to 5-log10 CFU/cm2) and treated with two different concentrations of phage (2x10^7 and 1x10^8 PFU/cm2). Treated samples were held at 4 or 20°C for 1 and 3h. After treatment with phage, Listeria reductions ranged from 1.27–3.33 log10 CFU/cm2 on stainless steel, 1.17–2.76 log10 CFU/cm2 on polyurethane thermoplastic, and 1.19–1.76 log10 CFU/cm2 on epoxy. Listeria reduction occurred on all materials tested, under all conditions. Higher phage concentration, longer time, and higher environmental temperatures led to significantly (P<0.05) greater reduction of Listeria on stainless-steel and polyurethane thermoplastic. The effectiveness of a phage against Listeria spp. was evaluated in two food manufacturing facilities, operating at either 4°C or 20°C. First, a moderate application of a 2x10^7 PFU/mL phage was applied once per day over three days and samples were collected and analyzed for Listeria at 0, 24, 48 and 72 h. This phage treatment led to a decrease in the incidence of Listeria by 67%. A second application method was studied with phage being applied in the food manufacturing environment in an intensified manner (3 times in 18 hours) at a higher concentration of phage (1x10^8 pfu/mL). This intensified application led to a 32% overall reduction in the incidence of Listeria in the production environment. Applications of Listeria specific phage can be an additional intervention strategy for controlling pathogenic Listeria organisms in food production facilities. / Doctor of Philosophy / Listeriosis is a serious illness caused by the bacterium Listeria monocytogenes. Annually in the United States it is estimated that 95.7% of all listeriosis illnesses are caused by the consumption of contaminated food, and it is generally recognized that L. monocytogenes is caused by cross contamination of ready-to-eat foods from an environmental source. The purpose of this research was to determine if food manufacturing facilities could use bacteriophage (phage) to treat and reduce the incidence of Listeria in food manufacturing plant, and thereby reduce the risk of food products being cross-contaminated with L. monocytogenes. The incident rate of Listeria was surveyed by collecting 4,829 samples from 31 ready-to-eat (RTE) food plants across the United States. Nine (29%) facilities had zero samples positive for Listeria spp., whereas 22 (71%) plants had one or more samples positive. The total incidence of Listeria spp. in all RTE food plant samples was 4.5%. Second, research was completed to determine if phage at different levels reduced Listeria on three common food manufacturing plant materials (stainless steel, polyurethane belting and epoxy flooring). After Listeria was attached to each material (4 to 5-log10 CFU/cm2), they were treated with two different levels of phage (2x10^7 and 1x10^8 PFU/cm2) at two temperatures (4 or 20°C) for 1 and 3 hours. After treatment with phage, Listeria reduction of 93.2 to 99.9% occurred on all materials tested, under all conditions. Finally, two different methods of applying phage (moderate and intensified) in a food manufacturing plant was studied. The moderate application was a single treatment with lower concentration of phage (2x10^7 PFU/mL) once per day for three days, while the intensified treatment was the application of high phage concentrations (1x10^8 pfu/mL) three different times, all in a single day. Both application methods reduced the incidence of Listeria in the food manufacturing plant. The total reduction across all trials was 67% using the moderate application method and 32% using the intensified application of phage.

No description available.

The seed germination process during sprout production provides suitable environmental conditions for the growth of pathogenic bacteria, such as Listeria monocytogenes. A potential way to control this bacterial growth is through the use of bacteriophages, which are naturally occurring viruses that specifically attack bacterial targets and have been shown to be effective antimicrobials in some foods. Therefore, the objective of this study was to evaluate the antimicrobial susceptibility of L. monocytogenes to bacteriophage on alfalfa sprouts during seed germination and subsequent refrigerated storage at 4 °C. Alfalfa sprout seeds were dip-inoculated with 5.5 x 105 CFU/ml L. monocytogenes serogroups 1 and 4. This was followed by treatment with the commercial bacteriophage LISTEX™ P100 at a concentration of 5.3 x 107 PFU/ml. The seeds were then soaked and germinated for 80 h using the glass jar method. The concentration of L. monocytogenes was determined every 24 h using PALCAM agar plated in triplicate. When compared to the spiked, untreated control, treatment of sprout seeds with LISTEX™ P100 resulted in a statistically significant (p < 0.05) reduction of 1.6 log10 CFU/g L. monocytogenes after the initial 24 h of germination. However, the bacteriophage did not show a lasting inhibitory effect, with no statistically significant reductions in L. monocytogenes growth as compared to the control at subsequent time points. The bacteriophage remained stable over the entire germination and storage period. Although biocontrol of Listeria with bacteriophages has high potential to serve as an alternative strategy to control foodborne illnesses, factors such as phage delivery and dose optimization in sprouts need to be further investigated.

By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these "hairy toroids", part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

Intracellular symbioses between bacteria and insects are numerous, and alter the ecology and evolution of host and symbiont alike. Long-term persistence results from either exploitation (e.g., reproductive manipulations) or mutually beneficial interactions (e.g., nutritional mutualisms). The endosymbiont Hamiltonella defensa, while not essential for growth or survival of healthy aphids, protects aphids from attack by parasitoid wasps. In this thesis, I have used a variety of sequenced-based techniques to illuminate the population and genome dynamics of H. defensa and to disentangle how these factors contribute to its ability to persist and protect its hosts.I characterized the phylogenetic relationships among H. defensa strains from aphids and a whitefly using a multilocus approach. Most loci evolve in a clonal manner, and one cluster of strains may have given rise to an obligate symbiosis. Some H. defensa strains were infected with the bacteriophage APSE, which encodes putative eukaryotic specific toxins and has been suggested to be involved in protecting aphids. I sequenced the toxin locus and the flanking regions from the APSE strains and found that although the phage genome backbone was highly conserved, strains contained non-orthologous toxin-cassettes. Sequenced cassettes contained one of three putative toxin families: Shiga toxin, cytolethal distending toxin, and YD-repeat toxins. A correlation was noted that of several genetically identical H. defensa strains, the one without phage APSE encoding the YD-repeat toxin failed to protect its aphid host. This APSE strain carrying the YD-repeat toxin has since been demonstrated to be essential for protection in several related H. defensastrains.To examine additional bacterial encoded loci that might facilitate the persistence in and protection of aphids by H. defensa, I sequenced the genome of one strain and obtained partial genomes of two additional strains. These genomes exhibit a streamlined metabolism, but are littered with mobile DNA and putative virulence factors. Horizontal gene transfer, recombination and rearrangements are common, and phage and plasmids have played an important role in resorting genes. Thus, although H. defensa benefits its host, its facultative lifestyle has resulted in a pattern of genome evolution associated with reproductive parasites rather than long-term mutualists.

Staphylococcal phage 80α can serve as a helper bacteriophage for a family of mobile genetic elements called Staphylococcus aureus pathogenicity islands (SaPIs). The prototype island, SaPI1, is able to hijack the 80α capsid assembly process and redirect capsid formation to yield smaller, phage-like transducing particles carrying SaPI DNA. Capsid size redirection is accomplished through two SaPI1-encoded gene products, CpmA and an alternate scaffold protein, CpmB. The normal 80α scaffold and the SaPI1 CpmB scaffold share a small block of conserved residues at their C-termini, several of which had been shown to be essential for CpmB function. This led to the hypothesis that the C-termini of both the phage and SaPI scaffolds interact in similar ways with the major capsid protein. The goal of this study was to test this hypothesis and to identify the amino acid residues at the capsid-scaffold interface, using a genetic approach.