Graduation date: 1999

Graduation date: 1997

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 129-143).

The timing of host lysis is the only decision made in the bacteriophage lytic cycle. To optimize timing, double-stranded DNA phages use a 2-component lysis system consisting of a muralytic enzyme, the endolysin, and a small membrane protein, the holin, which controls the timing of lysis. The best characterized holin gene to date is the S gene of bacteriophage λ. One unusual feature of the S gene is that it produces two proteins of opposing function: the holin, S105, and the antiholin, S107. Raab et al isolated and characterized a number of S mutants, but all of them expressed both the holin and the antiholin; it is possible, then, that the true extent of the holin-holin interactions were masked by interactions with the antiholin. Thus, a large number of S105 mutants were created, and their phenotypes characterized in the absence of the antiholin. The interaction between those mutants and the wild-type were examined in an attempt to better understand what determines the timing of hole formation by S105. S105 and S107 differ only by two amino acids at the N-terminus; S107 has an additional Met-Lys sequence. Previous studies have shown that S107 may have a different topology to S105, where the N-terminus of S107 is located in the cytoplasm and is cannot flip through the membrane because of the extra cationic side chain. This study investigates the role of the N-terminal transmembrane domain of the S proteins in terms of hole formation and its role in the antiholin character of S107. Previous results suggest that S105 forms hole via a large oligomeric structure termed the “death raft”. The death raft model states that after S105 is inserted into the membrane, it forms “rafts”, which grow in size until a spontaneous channel forms leading to depolarization of the membrane and hole formation. This study investigates the pathway of hole formation at the single-cell level, using a C-terminal fusion of S105 and green fluorescent protein, and attempts to address several of the predictions posed by the death raft model.

Structural biology lays the molecular foundation for the modern field of life sciences. In this thesis, X-ray crystallography is the primary resource for atomic detail structural information and is the major technology employed in our research. Three examples show how structural biology addresses the basic processes of life. Firstly, two crystal structures of R21, corresponding to two biological states, reveal a new activation mechanism of SAR-endolysin, which not only complements the previous model, but is also more generally applicable to the endolysin family. The structural information was further corroborated by NMR data in solution. The second example is the crystal structure of mycobacteriophage lysin B, which identified the function of the protein, and tackles the unique problem of how mycobacteriophage circumvent the mycolic acid-rich outer membrane of mycobacterium. The last example is the homology modeling of the Plasmodium ribosomal L4 protein. The action mode for the drug in Plasmodium was proposed based on that, which accounts for the anti-malaria effect of azithromycin.

The need for clean potable water and sustainable energy are two current and pressing issues with implications affecting the global population. Renewed interests in alternative energy have prompted researchers to investigate the full capacity of biofuels. These interests have led to not only the examination of current method limitations, but also to the investigation of new conversion methods. One promising method for bioenergy production is pyrolysis of lignocellulosic feedstocks. Through pyrolysis, a single crop may produce ethanol, bio-oil, and/or gaseous energy (syngas). The remaining solid phase product is a black carbon dubbed 'biochar'. In the current study, biochar was used as a both an unamended sorbent and a precursor to form powdered activated carbons (PACs) capable of removing waterborne viruses. Biochar was activated with KOH, ZnCl2, and H3PO4 and analyzed using the Brunauer, Emmett and Teller (BET) method, a combination of Kjeldahl digest and ICP-MS, and scanning electron microscopy (SEM). Sorbents were tested in batch studies using phosphate buffered saline (PBS), surface water, and groundwater. Bacteriophages MS2 and thetaX174 served as viral surrogates. All activation treatments significantly increased surface area, up to 1495.5 m2/g (KOH-activated). While the non-activated biochar was not effective in virus removal, the KOH-activated PAC had tremendous removal in the PBS/MS2 batch (mean 98.7% removal, up to 6.2 x 109 particles/mL, as compared to the Darco S-51: 82.3%). As evidenced by this study, sorption efficiency will be governed by viral species, carbon type and concentration, and water quality. The results of this study indicate that biochar can serve as a precursor for a highly porous and effective PAC, capable of removing waterborne viruses from environmental waters.

The 4.6 Mb circular E. coli chromosome is compacted by segregation into 400-500 supercoiled domains, created by both active and passive mechanisms like transcription and DNA-binding proteins. We find that transposable prophage Mu, transcriptionally silent by definition, is organized into an independent domain as determined by the close proximity of Mu termini L and R separated by a 37 kb Mu genome. Cre-loxP recombination is used in this study in vivo and in vitro. Critical to formation/maintenance of the Mu 'domain' configuration are a strong gyrase site SGS at the center of Mu, the Mu L end, the MuB protein, and the E. coli nucleoid-associated proteins IHF, Fis and HU. The Mu domain was observed at two structurally different chromosomal locations, and was specific to the Mu prophage, i.e. was not observed for the [mathematical symbol] prophage. A model is proposed that by employing its cis-elements to create a domain barrier for segregation and compaction of its genome, the large selfish DNA element Mu profits from the transposition-ready arrangement of its ends, while simultaneously providing a fitness advantage to the host. / text

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M13 filamentous bacteriophage has been used as a biotemplate for the nucle- ation of materials. Phage is an ideal and diverse scaffold with its large aspect ratio and ability to display biomolecules to bind a range of targets. To form more complex patterned materials, interactions between the phage must be specific and reliable. We develop a phage labeling method using sortase enzymes to create multi-phage nanostructures. We exploit two sortases and functionalize the N-termini of the pIII, pIX, and pVIII proteins with small and large moieties. For the pVIII, we show a 100 fold improvement in display of GFP molecules on the phage surface. Taking advantage of orthogonal sortases, we simultaneously label two capsid proteins on a single phage particle. Using these N-terminal labeling techniques, we demonstrate fluorescent staining of cells and construct a lampbrush phage structure linking the pIII of one phage to the pVIII of another using a biotin-streptavidin linkage. To further expand our labeling repertoire, C-terminal sortase labeling of phage was pursued. To achieve this goal, we transfer a loop structure from cholera toxin to pIII and label it with a fluorophore and a multi-domain protein. With this archi- tecture, we form end-to-end dimers using sortase to conjugate the loop structure to phage containing the nucleophile motif. Lastly, we investigate DNA hybridization as a method for crosslinking phage. Using sortase, we label the pVIII on two sets of phage: one with ssDNA and the other with a complementary DNA oligonucleotide. We anneal these phages together and observe phage networks that are dispersed by heat and reform upon cooling.