Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographies.

Antimicrobial efficacy of recently approved lytic bacteriophage preparation Salmofresh™ against Salmonella was evaluated on chicken breast fillets as dip and surface application, which reduced Salmonella by 0.7-0.9 log CFU/g and 0.8-1 log CFU/g, respectively. Surface application of Salmofresh™ on Salmonella inoculated chicken breast followed by storage under modified atmosphere packaging (95% CO2/ 5% O2) reduced Salmonella by 1.2 log CFU/g. The combined application of Salmofresh™ with cetylpyridinium chloride (CPC) and lauric arginate (LAE) reduced Salmonella on chicken breast fillets by 1.2-1.4 log CFU/g and 0.9-1 log CFU/g, respectively. The sequential application of chemical antimicrobial (CPC, LAE, chlorine and peracetic acid) and Salmofresh™ in reducing Salmonella was tested in a chicken skin model. Dip treatment in peracetic acid (400ppm) followed by surface application of phage revealed the highest reduction of Salmonella up to 2.5 log CFU/cm2 on chicken skin.

Lysozyme from the bacteriophage lambda, 1aL, complexed with a chitohexasaccharide has been solved to 2.6 Å by molecular replacement using a mutant form of 1aL as a model. The protein packs as a dimer in the crystal with the backbones of both monomers being nearly identical. The inhibitor molecule resides in the deep cleft in the middle of the bilobal enzyme. Subsites A to Dare occupied by one (GlcNAc)₆ molecule, while the remaining sites interact with two rings from the adjacent (GlcNAc)₆ molecule. The binding mode of 1aL is compared to other lysozymes (HEWL, HuL, GEWL, T4) and Slt70. Interesting differences are noted in the stacking interactions in ring D and the extensive interactions in ring E. It is hypothesized in the thesis that one possible role of the peptide moiety is to interact with Tyr-132, preventing it from forming stacking interactions with ring D, allowing the sugar to penetrate deeper into the active site. Ring E is buried deeply in the enzyme and has a low thermal factor. In addition, the active site is much narrower in 1aL than in other lysozyme structures. A possible explanation has been suggested that rings E and F stay in the active site longer than those in lysozyme to prevent water molecules instead of the O6 atom of ring D to participate in the nucleophilic attack at the end of the reaction. / Thesis / Master of Science (MS)

McMaster University MASTER OF SCIENCE (2020) Hamilton, Ontario (Department of Biochemistry and Biomedical Sciences) TITLE: Identifying new compounds capable of inducing model phages AUTHOR: Anisha Nandy SUPERVISOR: Dr. Alexander P. Hynes NUMBER OF PAGES: xi, 80 / Prophages are the genomes of bacteriophages (phages, bacterial viruses) that integrate into the chromosome of their host upon infection, lying dormant until conditions favour their reactivation. A cell harbouring a prophage is called a lysogen, as, upon exposure to certain signals, the prophage will initiate a replicative cycle ending in lysis of the host bacterium and release of phages. This process is known as induction. Canonically, induction occurs through activation of the bacterial SOS-response, a DNA repair cascade initiated by detection of DNA damage. Studies of prophage induction have almost exclusively relied on challenges with compounds that result in the initiation of the host SOS response. Recent studies have identified some signals that affect prophage induction independently of the SOS response, but these approaches have not been systematic. To identify non-canonical triggers of prophage induction, I screened 3,936 compounds against two model lysogens. The first, carrying phage HK97, is a model for induction. The second, carrying phage Mu—a prophage thought to be uninducible—serves as a control. Any compound which inhibited bacterial growth in only our HK97 lysogen was considered to have resulted in a phage-mediated response. The 171 compounds identified in this screen were then used to re-challenge the lysogen at a range of concentrations and monitor the resulting release of free phages associated with induction. Increases in phage counts were seen for 86 compounds. While 38 of these were known SOS activators, 49 were novel, ‘non-canonical’ inducers. Unexpectedly, the screening also revealed seven unique chemical inducers for the supposedly un-inducible model prophage, Mu. The 56 new phage-inducers identified by this work include compounds likely to be driving phage induction through non-canonical pathways. As prophages are thought to respond to bacterial stress, these may reflect stressors acting through new mechanisms. Using these compounds as tools opens up an avenue to probe other stress pathways in bacteria, and, as evidenced by induction of Mu, potentially help discover new phages that don’t respond to canonical inducers. / Thesis / Master of Science (MSc) / Bacterial viruses (phages) can lie dormant as prophages in their host bacterium until a signal triggers their activation, production of viruses, and rapid killing of the host. This switch from dormant prophage to active phage called induction. Almost all molecules that result in prophage inductions belong to a limited set of compounds which elicit a specific stress response in bacteria. Screening 3936 compounds for their ability to inhibit the growth of bacteria carrying known prophages resulted in the identification of a small subset associated with increased phage production. For one Escherichia coli prophage—HK97, a model of induction—we found 49 compounds not previously known as inducers. For another model prophage—Mu, a prophage thought to be chemically uninducible—we identified seven such compounds. These compounds will serve as tools to determine what signals prophages can respond to, and potentially identify new stress pathways of interest in bacteria.

Bacteriophage Felix O1, also known as enterobacteria phage O1, has been used to type Salmonella Typhi and is an excellent candidate for use in bioremedial and therapeutic applications. It has extremely high intra-species specificity and is strictly virulent in nature, unable to undergo lysogeny. To facilitate the development of the bacteriophage for use in these areas, the full sequence of the genome had been elucidated previously. In this work, identification and classification of functional coding sequences via reverse transcriptase-polymerase chain reaction was performed. All of the 115 putative open reading frames (ORFs) studied were found to be functional. 53.0%, 9.6%, and 18.3% of the ORFs investigated were found to initiate expression early, middle or late in the lytic cycle, respectively. Expression of the remaining 19.1% ORFs was evident when the amount of total RNA was increased and when samples were taken at a later time point. Comparisons between bacteriophage Felix O1 and the phage with the most shared homologs, phage T4, revealed many similarities in times of gene expression. / Master of Science

An assay was designed to measure the sensitivity of Escherichia coli mutants to bacteriophage infection via growth curves, using a Tecan temperature controlled plate reader. I screened 3985 single-gene deletion strains in Escherichia coli K12 from the Keio collection and identified 43 strains displaying varying degrees of resistance to four different phages, three non-contractile tailed phages (λcI857, HK97, and HK243) and the myoviridae T6, including 20 genes not previously implicated in phage infection. Additional assays, such as adsorption and tests of DNA-injection, were designed to further characterize resistant strains. The use of these assays helped identify varying sensitivities to LPS structure and LamB receptor concentration in the three non-contractile tailed phages, showing HK97 is the most sensitive to changes and HK243 the least. I also found that the periplasmic chaperone, FkpA, is required for HK97 DNA-injection.

An assay was designed to measure the sensitivity of Escherichia coli mutants to bacteriophage infection via growth curves, using a Tecan temperature controlled plate reader. I screened 3985 single-gene deletion strains in Escherichia coli K12 from the Keio collection and identified 43 strains displaying varying degrees of resistance to four different phages, three non-contractile tailed phages (λcI857, HK97, and HK243) and the myoviridae T6, including 20 genes not previously implicated in phage infection. Additional assays, such as adsorption and tests of DNA-injection, were designed to further characterize resistant strains. The use of these assays helped identify varying sensitivities to LPS structure and LamB receptor concentration in the three non-contractile tailed phages, showing HK97 is the most sensitive to changes and HK243 the least. I also found that the periplasmic chaperone, FkpA, is required for HK97 DNA-injection.

<p>The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.</p><p>Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.</p><p>In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.</p><p>&#8232;For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of <italic>Escherichia coli</italic> K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 <underline>+</underline> 13.2 and 38.2 <underline>+</underline> 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.</p><p>Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of <italic>E. coli</italic> K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h<super>-1</super>, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 10<super>8</super> PFU/mL and 10<super>8</super> CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.</p><p>In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.</p><p>Finally, for an industrial application, the use of phages to inhibit invasive <italic>Lactobacilli</italic> in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of <italic>Lactobacillus plantarum</italic> during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.</p><p>In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.</p> / Dissertation

Phytopathogenic Dickeya species inflict large economic losses on a variety of crops. A lack of effective chemical control methods has generated interest in the use of bacteriophages (phages) as a novel tool for biocontrol. In the last decade, six phages have been isolated in Belgium and Poland using Dickeya solani as the host. Previous work in this laboratory has isolated ninety phages capable of infecting D. solani. The majority have been morphologically classified as members of the Ackermannviridae family. In agreement with findings in Salmonella and Klebsiella species, the capsule of D. solani is a likely receptor of Ackermannviridae family phages. Analysis of D. solani strains carrying reporter fusions suggested that the capsule genes are expressed in response to nutritional stress, however disruption of the capsular polysaccharide cluster did not significantly impact virulence. Experiments assessing capsular polysaccharide as a putative receptor for Ackermannviridae family phages in nosocomial pathogen Serratia produced inconclusive results. Phageresistance due to random transposon mutagenesis identified genes encoding transcription factors and regulators, but none directly linked to capsular polysaccharide production. Thirteen phages were capable of infecting a wider host range of Dickeya species. Morphological and genomic analysis showed that six were Podoviridae family members, whilst the other seven were Myoviridae family members. These are part of the recently defined 'hairy Myoviridae', characterised by a distinct morphology. Another member of this grouping was isolated during this study, but is more closely related to phages of Erwinia amylovora. A subset of the Ackermannviridae family phages were shown to be capable of facilitating transduction. This makes them unsuitable for use in the environment due to the risk of deleterious horizontal gene transfer. This is also true for the Myoviridae family members, but not for one of the Podoviridae family members. This phage could therefore be a promising candidate for therapeutic use.