The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe. The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria. Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed. Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host. Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results. Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Experimental evolution has been used to investigate both general and specific evolutionary processes. More recently, it has also been used to resolve protein-protein interactions. Viruses assemble through a series of protein-protein interactions which must remain more favorable than any competing off-pathway reaction. By constructing chimeric viruses with genes or segments of genes from another species, foreign elements are introduced into this system of assembly. Characterization of the resulting chimeras provides information about which proteins interact, the protein-protein interacting interface, the role of particular domains, and the importance of specific residues. Chimeric viruses often exhibit a reduction in fitness, as the foreign element is unable to interact as efficiently in the system as the native element. Through experimental evolution, mutations accumulate that affect interacting partners in the system, leading to a more optimal assembly pathway. The microviruses are well-characterized single-stranded (ss) DNA bacteriophages. They are divided into three clades, represented by φX174, G4, and α3. Incidences of horizontal gene transfer between microvirus clades are unusually rare and may be due to a complex assembly pathway with multiple stages: a foreign element has the potential to disrupt a multitude of morphogenetic steps. In this study, we exchanged major spike genes between the two microvirus species φX174 and G4, then monitored the evolutionary recovery. Results can be interpreted within this thermodynamic paradigm. Although the G4-φXG chimera could only form plaques at low temperature and exhibited reduced fitness, its evolutionary recovery was relatively straightforward. The other chimera, φX-G4G, could only form plaques when complemented with two wild-type φX174 genes. Isolating a complementation-independent chimera required the passaging of mutants through a series of different environments. The first selection yielded mutations of the largest effects. First, the truncation of a protein involved in DNA synthesis was recovered, resulting in a global decrease in gene expression. Next, a recombination event at the 3' end of the foreign gene resulted in a modification of the protein’s C-terminus. These mutations were subjected to further analysis to determine why they were so critical at this early stage of experimental evolution. Subsequent passages of the φX-G4G chimera eventually yielded viable strains, with additional mutations affecting stages of late assembly. Overall, results indicate how gene exchange can drastically affect flux through the pathway. When the system is initially perturbed, the process of experimental evolution allows the pathway to return to a normalized state. The mutations isolated during this recovery stage indicates how the flux was initially altered, and how it can be restored.

Viruses infecting bacteria (phages) are the most abundant and ubiquitous entities on Earth and likely critical to any ecosystem, as they influence nutrient cycling, mortality and evolution. Ultimately, their impact depends on whether phage—host interactions lead to intracellular phage coexistence (temperate phage) or cell death (lytic phage). Temperate phages in the lysogenic cycle replicate their genome (either integrated into the host chromosome or extrachromosomally), until induced to become lytic, when they create and release progeny via cell lysis. While knowledge on lytic versus lysogenic outcomes is vast, it largely derives from few model systems that underrepresent natural diversity. Further, less is known about the efficiency of phage—host interactions and the regulation of optimal versus sub-optimal lytic infections, which are predicted as relevant under environmental (nutrients, temperature) and host (availability, density) conditions that are common in the ocean. In this dissertation I characterize the phage—host interactions in a new marine model system, phage ϕ38:1 and its Cellulophaga baltica bacterial host, member of the ubiquitous Bacteroidetes phylum. First, I show ϕ38:1’s ability to infect numerous, genetically similar strains of the C. baltica species, two of which display contrasting infection outcomes–lytic versus sub-optimally lytic or lysogenic on the original versus alternative hosts, respectively. Second, I collaboratively apply new gene marker-based approaches (phageFISH and geneELISA) to study ϕ38:1’s infection at the single-cell level and show that it is sub-optimal on the alternative host, rather than lysogenic. Third, I collaboratively develop whole-genome transcriptome datasets for ϕ38:1 infecting both, the optimal and sub-optimal hosts, to characterize the cellular response to infection and hypothesize potential transcriptional and post-transcriptional regulation of the sub-optimal infection. Together, these findings advance our knowledge of naturally-occurring phage—host interactions with a focus on nearly-unstudied sub-optimal infections.

A bioactive paper is a paper that can detect, capture and deactivate water and airborne pathogens. In this project, we presented a model "bioactive paper" made by attaching T4 bacteriophages to a cellulose substrate. T4 bacteriophages can be genetically engineered to possess copies of cellulose-binding modules (CBM) on their capsids. This allows them to bind specifically onto cellulose surfaces. Our model surface is a thin film of regenerated cellulose made by spin coating a glass or quartz substrate with a cellulose triacetate and subsequently hydrolyzing the surface back to cellulose. We successfully demonstrated the attachment of the CBM-T4 bacteriophages onto cellulose substrates by the phage viability test. The deposition kinetics were measured using an impinging jet apparatus combined with an evanescent wave light scattering (EWLS) system. We first tested the apparatus by using amidine latex particles deposited on the cellulose at different flow rates and found them to be in a good agreement with the constant potential double-layer model. The adhesion experiments were also performed in an impinging jet apparatus in which the CBM-T4 bacteriophages and the unassembled protein complexes from a suspension of 4.08 x 10 8 PFU/mL were allowed to diffuse to the cellulose surface, The competitive diffusion kinetics were again studied by the EWLS technique. For CBM-T4, the blocking time was found to be around 58 minutes and the maximum surface number density of phages was 5.9 x 1010 per m 2. / Key phrases: bioactive paper, cellulose film, cellulose binding module, bacteriophage T4, evanescent wave light scattering, unassembled protein complex, diffusion kinetics

The long, contractile tails of myophages are the conduit for phage DNA transfer into the bacterial host cell and the most important part of the myophage tail is the baseplate; a complex structure, distal to the phage head. To better understand the structure and function of myophage baseplates, a component of the phage P2 baseplate, gpW was studied. This protein is widely conserved among myophages and is essential for the formation of infectious phage particles. Bioinformatic work confirmed that gpW homologues are found in almost all myophages and in many prophages. Moreover, gpW was shown to be a structural component of the virion; and, using electron microscopy, it was found to be at the top of the P2 baseplate. It was also found that some single residue substitutions can completely disrupt gpW function. Finally, evidence is presented that at least eight different proteins may be required to form intermediate P2 baseplate structures while other proteins may be necessary for the formation of stable baseplate complexes.

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The long, contractile tails of myophages are the conduit for phage DNA transfer into the bacterial host cell and the most important part of the myophage tail is the baseplate; a complex structure, distal to the phage head. To better understand the structure and function of myophage baseplates, a component of the phage P2 baseplate, gpW was studied. This protein is widely conserved among myophages and is essential for the formation of infectious phage particles. Bioinformatic work confirmed that gpW homologues are found in almost all myophages and in many prophages. Moreover, gpW was shown to be a structural component of the virion; and, using electron microscopy, it was found to be at the top of the P2 baseplate. It was also found that some single residue substitutions can completely disrupt gpW function. Finally, evidence is presented that at least eight different proteins may be required to form intermediate P2 baseplate structures while other proteins may be necessary for the formation of stable baseplate complexes.

Currently, most of the RNAs used in lab research are prepared by in vitro transcription or chemical synthesis, which can be costly. In vivo expression in bacterial cells is another approach to RNA preparation that allows large scale production at a lower cost. However, there are some obstacles in bacterial expression, including RNA degradation in host cell, as well as RNA extraction and purification. tRNA and 5S RNA have been reported as scaffolds to circumvent the degradation problem. These scaffolds can not only make the RNA product survive in the cell but also increase the stability after extraction. The packaging RNA (pRNA) of bacteriophage phi29 is a small non-coding RNA with a compact structure. The three-way junction (3WJ) region from pRNA is a thermodynamically stable RNA motif good for constructing therapeutic RNA nanoparticles. The 3WJ can not only integrate multiple RNA modules, but also stabilize them. Here I report a series of approaches made to express recombinant RNAs based on pRNA or 3WJ in bacteria, including 1) Investigating the mechanism of RNA folding in vitro and in vivo using 3WJ. 3WJ-based RNAs were expressed in E. coli using pET system. The results show that the folding of RNA is affected by both overall and regional energy landscape. 2) Expression of an RNA nanoparticle harboring multiple functional modules, a model of therapeutic RNA, in E. coli using a combination of tRNA scaffold and pRNA-3WJ. The expression was successful and all of the RNA modules were functional. 3) Expression of pRNA-based recombinant RNAs in B. subtilis. This is a novel system of expressing recombinant RNAs in Gram-positive bacteria.

The successful fabrication and characterization of two chemiresistive platforms for biomolecule detection was demonstrated by this work. The Si/Silica based single walled nanotube thin film (SWNTTF) platform was developed to understand the effect of device geometry on pH and M13 bacteriophage sensing capabilities as well as the underlying mechanisms governing SWNTTF chemiresistive biosensors. The dominant mechanism of sensing switched from direct chemical doping to electrostatic gating when the target analyte changed from H+/OH- ions in pH testing to whole viruses. The experimental limit of detection for M13 for this platform was 0.5pM and an increased sensitivity as well as variability was observed in devices with smaller channel widths. Preliminary device calibration was completed in order to correlate a resistance response to a bulk M13 concentration. The polyethylene terephthalate (PET) based SWNTTF platform was developed to demonstrate the commercial potential of SWNTTF chemiresistive biosensors by detecting relevant concentrations of brain natriuretic peptide (BNP) on economically viable substrates. The pH response of these chemiresistors confirmed that chemical doping was the cause for resistance change in the SWNTTFs. The preliminary results demonstrated successful BNP detection at 50pg/mL using both aptamers and antibodies as recognition elements. Using SWNTTFs as the transducing element of chemiresistors allowed for further understanding of electrical mechanisms of sensing as well as achieving sensitive, real-time and reproducible electrical virus and biomolecule detection. Although these platforms do not achieve ultrasensitive limits of detection, they demonstrate the commercial potential of platforms using SWNTTFs as the transducing element of electrical biomolecule sensors.

Bacteriophages (hereafter referred as phages) can translate their mRNAs efficiently by maximizing the use of codons decoded by the most abundant tRNAs of their bacterial hosts. Translation efficiency directly influences phage fitness and evolution. Reengineered phages find application in controlling their host population in both health and industry. The objective of this thesis work is to examine the factors shaping codon choices of single stranded DNA (ssDNA) and double stranded DNA (dsDNA) Escherichia coli phages. In chapter two, we employed two indices, rRSCU (correlation in relative synonymous codon usage between phages and their hosts) and CAI (codon adaptation index) to measure codon adaptation in phages. None of the analyzed ssDNA phages encode tRNAs while some dsDNA phages encode their own tRNAs. Both rRSCU and CAI are negatively correlated with number of tRNA genes encoded by these dsDNA phages. We observed significantly greater rRSCU for dsDNA phages (without tRNAs) than ssDNA phages. In addition, we propose that ssDNA phages have evolved a novel codon adaptation strategy to overcome the disruptive effect of their high C→T mutation rates in codon adaptation with host. In chapter three, we formulated an index phi to measure selection by host translation machinery and to present explicit linear and nonlinear models to characterize the effect of C→T mutation and host-tRNA-mediated selection on phage codon usage. The effect of selection (phi) on codon usage is detectable in most dsDNA and ssDNA phage species. C→T mutations also interfere with nonsynonymous substitutions at second codon positions, especially in ssDNA phages. Strand asymmetry along with the accompanying local variation in mutation bias can significantly affect codon adaptation in both dsDNA and ssDNA phages.