The nucleotide sequence of the PRD1 terminal protein gene has been determined. The coding region for PRD1 terminal protein is 777 base pairs long and encodes 259 amino acid residues (29,326 daltons). The deduced amino acid sequence of PRD1 terminal protein reveals no overall homology with other known terminal proteins or related proteins. A closer examination revealed a highly conserved amino acid sequence, YSRLRT, exist among all identified DNA terminal proteins including PRD1, PZA, Nf, φ29 and adenovirus. This is the first conserved amino acid sequence that has been found in all identified DNA terminal proteins. Not only is the YSRLRT sequence conserved, but its spatial location is similar as well. Therefore, the significance of the YSRLRT conserved sequence is suggested by both its conservative spatial location and high degree of homology across species. To study the structure-function relationship of the YSRLRT sequence of PRD1 terminal protein, in vitro site-directed mutagenesis was performed to determine the role of each amino acid in this conserved region. The PRD1 terminal protein and DNA polymerase genes were cloned into phagemid pEMBLex3, and the recombinant plasmid used for constructing mutants. Eleven PRD1 terminal protein mutant clones were examined for their priming complex formation activities. Our results have strongly demonstrated that the positive charge residue of arginine-174 plays an important role for PRD1 terminal protein function. There are 13 tyrosine residues in the predicted PRD1 terminal protein. It was of interest to known which tyrosine is actually linked to terminal nucleotide of the PRD1 DNA. We used a new approach involving replacing the tyrosine residues with phenylalanine residues in the carboxyl terminal portion of the protein. From analyses, the tyrosine-190 has been determined to be the most likely linkage site between terminal protein and PRD1 DNA.

Filamentous bacteriophages were engineered to express foreign genes with the ultimate purpose of displaying transmission control anti-malarial peptides as in phage display. It was hypothesized that expression of foreign genes would be possible using the phage’s promoters. This hypothesis was tested by assuming that promoters for the phage major coat protein (MCP) gene would also promote the expression of any foreign gene inserted downstream of the MCP gene. As proof of principle, the bacteriophages Pf3, Pf1, and M13 were engineered in this way to successfully synthesize Enhanced Green Fluorescent Protein (EGFP). Type 88 phage display on the EGFP recombinant Pf3 was attempted by fusing a second copy of its MCP gene to the existing EGFP gene. This resulted in a phage display Pf3 replacement vector which was then used to construct a phage for displaying an anti-malarial peptide.

A multi-scale approach was used to investigate the occurrence and physical characteristics of viral surrogates in water systems. This approach resulted in a methodology to quantify the dynamics and physical parameters of viral surrogates, including bacteriophages and nanoparticles. Physical parameters impacting the occurrence and survival of viruses can be incorporated into models that predict the levels of viral contamination in specific types of water. Multiple full-scale water systems (U.S., Italy and Australia) were tested including surface water, drinking water, stormwater and wastewater systems. Water quality parameters assessed included viral markers (TTV, polyomavirus, microviridae and adenovirus), bacteriophages (MS2 and ΦX-174), and coliforms (total coliforms and E. coli). In this study, the lack of correlations between adenovirus and that of bacterial indicators suggests that these bacterial indicators are not suitable as indicators of viral contamination. In the wastewater samples, microviridae were correlated to the adenovirus, polyomavirus, and TTV. While TTV may have some qualities which are consistent with an indicator such as physical similarity to enteric viruses and occurrence in populations worldwide, the use of TTV as an indicator may be limited as a result of the detection occurrence. The limitations of TTV may impede further analysis and other makers such as coliphages, and microviridae may be easier to study in the near future. Batch scale adsorption tests were conducted. Protein-coated latex nanospheres were used to model bacteriophages (MS2 and ΦX-174) and includes a comparison of the zeta potentials in lab water, and two artificial groundwaters with monovalent and divalent electrolytes. This research shows that protein-coated particles have higher average log10 removals than uncoated particles. Although, the method of fluorescently labeling nanoparticles may not provide consistent data at the nanoscale. The results show both that research on viruses at any scale can be difficult and that new methodologies are needed to analyze virus characteristics in water systems. A new dynamic light scattering methodology, area recorded generalized optical scattering (ARGOS) method, was developed for observing the dynamics of nanoparticles, including bacteriophages MS2 and ΦX-174. This method should be further utilized to predict virus fate and transport in environmental systems and through treatment processes. While the concentration of MS2 is higher than ΦX-174 as demonstrated by relative total intensity, the RMSD shows that the dynamics are greater and have more variation in ΦX-174 than MS2 and this may be a result of the hydrophobic nature of ΦX-174. Relationships such as these should be further explored, and may reflect relationships such as particle bonds or hydrophobicity.

Thesis. 1977. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Microfiche copy available in Archives and Science. / Bibliography : leaves 272-276. / Ph.D.

Shiga toxin (Stx) is a bacteriophage (phage)-encoded virulence factor of the Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 implicated in the pathogenesis of renal tissue damage and bloody diarrhoea in human. Cattle are the main asymptomatic reservoir for EHEC O157:H7 with the lymphoid-follicle rich areas of the terminal rectum identified as the primary colonisation site. However, the significance of Stx during bovine intestinal colonisation by EHEC O157:H7 remains unclear with mixed findings described in published studies. The objective of this study was to investigate if Stx and the Stx-encoding phage significantly contribute to EHEC O157:H7 colonisation particularly at the bovine terminal rectum. The expression of Stx receptor, Globotrioasylceramide (Gb3) at the bovine terminal rectum was analysed by fluorescence microscopy, revealing a similar pattern of Gb3 detection in the bovine colon with scattered positive detections limited to sub-epithelial, mesenchymal-associated cells. Purified Stx2 treatment of Gb3+ and Gb3- epithelial cell lines for 6 to 18 hours produced no effect on the cell cycle and proliferation. CD3+/CD8+, CD3+/γδ+ and CD21+ cells were significantly different between calves infected with EHEC O157:H7 Strain 9000 (Stx2a+/Stx2c+) and the uninfected calves, but not in calves with Strain 10671 (Stx2c+). Stx did not interfere with IFN-gamma (IFN-γ)-activation of the JAK/STAT1 pathway in epithelial cells. Bovine EHEC O157:H7 strains isolated from Scottish cattle farms in the IPRAVE study (Phage type 21/28 and 32) were used for a series of bacterial phenotypic characterisation assays. Total Stx production, Verocytotoxicity, growth in a competitive environment, epithelial cell adherence and Galleria mellonella virulence assay were performed to compare the IPRAVE EHEC O157:H7 strains (PT21/28 and PT32) and the isogenic Stx-phage mutants. Stx levels produced by the bovine-originated EHEC O157:H7 strains were significantly lower than that of the human isolated strains. The absence of Gb3 on the bovine terminal rectal epithelium, the non-significant changes in the cell cycle along with the uninterrupted IFN-γ activation of the JAK/STAT1 pathway in intestinal epithelial cells and the minute quantities of Stx generated by EHEC O157:H7 bovine strains suggest that the toxin is not involved in colonisation directly, at least at the intestinal epithelial level. Although future work is required to explain the mechanisms underlying the observed EHEC O157:H7 phenotypic changes particularly in the Stx-phage mutant strains, the work done has proven that the Stx-encoding phage indeed has the ability to exert changes in the bacterial cell leading to changes in bacterial phenotypes, which in turn, might affect the colonisation of the bovine intestine.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

Wild-type and minute mutants of the T4 strain of bacteriophages were treated with 1.0-12.0 ug per ml concentrations of aminoacridine. Escherichia coli was the host organism for the bacteriophages.Concentrations of acridine less than 2.0 ug per ml had no noticeable effect on wild-type ormmutant plaque formation. Two and one half to seven ug per ml concentrations of acridine reduced the size (diameter) and number of plaques formed from both. Concentrations greater than 7.0 ug per ml completely inhibited the growth of both.The host organism (Escherichia coli) was not affected by the acridine.Ball State UniversityMuncie, IN 47306

Brachyspira species are bacteria that colonize the intestines of some mammalian and avian species with different degrees of pathogenicity. Brachyspira intermedia is a mild pig and bird pathogen with an unknown genomic sequence. In this project, we completed the genome of Brachyspira intermedia PWS/AT and did a comparative genomic analysis between B. intermedia PWS/AT and the already completed genomes of B. hyodysenteriae WA1, B. murdochii 56-150T and B. pilosicoli 95/1000. A table containing 15 classes of unique and shared genes was developed and analyzed in order to gain a better understanding of species-specific traits and clues behind the different degree of pathogenicity. Our result shows that genes are overall poorly annotated and further studies are of great importance for understanding different and shared properties. The largest number of unique features was found in B. intermedia and B. murdochii. B. hyodysenteriae and B. pilosicoli has most likely developed independently towards different biological niches and B. pilosicoli has undergone a major reductive evolution. One plasmid and six prophages were found in B. intermedia, where two of the phages appear to be capable of horizontal gene transfer. Further genome sequencing of more strains will probably increase the understanding of species-specific traits even more.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Jan. 26, 2010). Additional advisors: Asim K. Bej, Gail E. Christie, Peter E. Prevelige, Jr., R. Douglas Watson. Includes bibliographical references.