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1
Cramer, Todd James Lucas. "Genetic mosaicism between the bacteriophage [phi]80 and bacteriophage [lambda]." Bowling Green, Ohio : Bowling Green State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=bgsu1223514067.
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2
Cramer, Todd James. "Genetic Mosaicism Between The Bacteriophage φ80 And Bacteriophage λ". Bowling Green State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1223514067.
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3
Ryan, Elizabeth Michelle. "Polymeric bacteriophage delivery systems." Thesis, Queen's University Belfast, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558175.
Повний текст джерелаАнотація:
Phage therapy is the use of bacteriophages to treat bacterial infections. Once a prominent method of antibacterial therapy, phage therapy became almost forgotten following the discovery of antibiotics in 1928. The rising instance of antibiotic resistant bacteria in the last number of years has resulted in resurgence in interest in phage therapy. Research into delivery methods for bacteriophages has been very limited. Up to very recently, phages had only been delivered parentrally using crude and purified phage stocks and orally for gastrointestinal infections. In order for phage therapy to become suitable for use in mainstream medicine, suitable dosage forms and phage delivery platforms must be developed. This research project endeavoured to successfully formulate a model bacteriophage, T4 phage, into useful polymeric delivery systems, beginning with an alginate based microparticulate system. Bacteriophages were successfully stabilized within a Ca-alginate-trehalose system and m icroparticles were fully characterised. The release profi le of these m icroparticles suggest that they could be used to successfully treat gastrointestinal infections. The present study also devised a phage stable soluble microneedle system. It was found that increasing the trehalose concentration within the microneedles, improved rnicroneedle strength, as did the addition of a Poly (methyl vinayl ether-eo-maleic acid) backing layer. A poly(carbonate) hollow rnicroneedle system was used to successfully deliver phages both in vitro and in vivo using a rat model. This study was the first time that bacteriophages have been delivered transdermally in vivo. Aside from the development of bacteriophage delivery systems, novel work in the application of Phage-Antibiotic synergy (PAS) to E.co/i biofilrns was completed with extremely successful results. It was found that administering T4 bacteriophage in combination with the antibiotic Cefotaxime improved biofilrn eradication, compared to treatment with bacteriophage or antibiotic alone. This is the first time that PAS has been applied to a biofilm model.
4
Thanki, Anisha M. "Development of a phage-based diagnostic test for the identification of Clostridium difficile." Thesis, Loughborough University, 2016. https://dspace.lboro.ac.uk/2134/20340.
Повний текст джерелаАнотація:
Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes.
5
Terry, Tamsin Deborah. "Peptide display on filamentous bacteriophage." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627599.
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6
Nemavhulani, Shonisani. "Bacteriophage diversity in haloalkaline environments." University of the Western Cape, 2013. http://hdl.handle.net/11394/4313.
Повний текст джерелаАнотація:
>Magister Scientiae - MScThere are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities.
7
Abedon, Stephen Tobias. "The ecology of bacteriophage T4." Diss., The University of Arizona, 1990. http://hdl.handle.net/10150/185040.
Повний текст джерелаАнотація:
In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.
8
Forghani, Farnaz. "Protein engineering of bacteriophage Mu transposase." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60444.
Повний текст джерелаАнотація:
Bacteriophage Mu is an ideal system to study DNA transposition. The 70-KDa protein product of the phage early gene A, termed transposase, is absolutely required for transposition. Transposase binds specifically at sites located at both ends of the phage genome, termed attL and attR, and at an enhancer-like element at the left end of the genome, called IAS (internal activation sequence). It then nicks at these ends, and nicks a random target DNA sequence in a 5 base pair staggered fashion with 5$ sp prime$ extensions and promotes strand transfer between the Mu ends and the target DNA. The transposase protein can be roughly divided into three domains. The other activities of the protein have not been mapped even at the domain level. To further define the different functional domains of this complex enzyme, a series of insertion mutants at 8 different sites along the transposase protein were constructed using TAB linker mutagenesis. In this study, 1 and 2 TAB linkers were inserted into 8 HpaII sites in the Mu A gene, generating a set of 2 and 4 amino acid insertion mutants. Examination of these mutants for specific DNA-binding activity of transposase to the ends of the phage genome in vitro revealed temperature sensitive proteins. Transpositional activity of the mutant proteins revealed that the mutant proteins, which are temperature sensitive in specific DNA-binding activity, are deficient in transpositional activity.
9
Long, Graham Stanley. "Molecular cloning of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339539.
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10
Sorrell, Julian Anthony. "An investigation into strong bacteriophage promoters." Thesis, Bangor University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361171.
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11
Toropova, Katerina. "Cryo-electron microscopy of bacteriophage MS2." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503345.
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12
Alvarez, Ana Gonzalez. "Structural studies of filamentous bacteriophage proteins." Thesis, Keele University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.332387.
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13
Huang, Haomin. "Studies on transduction by bacteriophage P1." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/14124.
Повний текст джерелаАнотація:
The original aim of this thesis was to address the origin of the abortive transduction protein (TPA), which appears to join the ends of transduced DNA and render it refractory to degradation by host nuclease and recombination with the chromosome. We planned to isolate abortively transduced DNA (ATD) from transduced cells, which should have TPA attached, in order to characterize it. To increase the packaging of transducing DNA and TPA, a pac site was inserted into the “donor” chromosome which was labelled with BU to make it heavy. This method proved technically difficult and I did not succeed in purifying sufficient ATD-TPA to characterize. I decided instead to study P1 packaging of transducing DNA further. The argB gene was replaced with the P1 EcoRI-20 fragment, which contains pac. Transduction and UV irradiation showed that transduction frequencies of chromosomal markers close to pac were increased several hundred fold and could be further increased by UV irradiation, indicating that TPA was likely to be bound as normal. Southern blotting demonstrated that packaging is unidirectional; that increase in transduction frequency decreases with distance from pac and that the packaging of markers up to 30min from pac was increased. 4 further pac sites were placed at intervals on the chromosome. Strains containing different numbers of pac sites were analyzed by transduction, titration, and hybridization. Some novel phenotypes were observed. First, the time when cells start to lyse after P1 infection is delayed in multi-pac-containing strains, suggesting that in WT strains P1 starts packaging viral DNA earlier than in multi-pac strains, perhaps because the pac sites on the chromosome compete for recognition and cleavage by Pacase. Furthermore, strains which have more than 2-pac sites give very poor plate lysates, whose titers are 1000-fold lower than WT (or one-pac-containing strain).
14
OBRINGER, JOHN WILLIAM. "GENETIC EXCLUSION IN BACTERIOPHAGE-T4 (EXONUCLEASES)." Diss., The University of Arizona, 1987. http://hdl.handle.net/10150/184090.
Повний текст джерелаАнотація:
Genetic exclusion in phage T4 is the prime responsibility of the imm and sp genes. The map region containing imm does not allow sufficient bps to encode for proteins the size reported for the imm gp. After assaying 30 mutants of the genes adjacent to imm, I found 7 in gene 42 that were defective in the imm phenotype. Upon reverting amNG411(42), the mutant most defective exclusion, for its gene 42 phenotype the exclusion phenotype also changed. When assayed in UGA suppressor hosts, imm+ phage showed a decreased exclusion ability indicating that an opal codon was involved in production of the functional imm gp. I concluded that imm and gene 42 overlap in an out-of-phase orientation with the involvement of an opal readthrough. This overlap has implications in the genetic regulation of this region. This region of T4 also encodes several other genes important in phage intra- and interspecific competition. They are B-gt, 42 and sp. Using recombinant DNA techniques, I precisely located the sp gene to a region between 21.647 and 22.014 kbp on the T4 restriction map and determined its molecular weight as approximately 15 kDa. This same region of T4 was purported to contain gene 40. Complementation and marker rescue experiments with sp+ plasmids indicated that genes sp and 40 are the same. Gene 40 mutants also were found to be defective in sp function. Genes sp and 40 were redesignated gene sp/40 thus linking an early expressing gene with the morphogenic pathway of prohead assembly. Functionally, host enzymes exo III and exo V were found as participants in gp imm mediated exclusion. Presumably gp imm alters the pilot protein of the superinfecting DNA thus exposing it. Gp sp functions by an anti-lysozyme action. But the pleiotrophic effects of sp/40 are best explained by a temperature induced conformational rearrangement hypothesis. This work links molecular genetics to the ecological concept of competition and provides insights into the function and the evolutionary significance of the competition cluster genes. The competition cluster encodes fundamental adaptive strategies found universally in nature.
15
Smerk, Cari L. "P1 Bacteriophage and Tol System Mutants." Bowling Green State University / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1182462962.
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16
Bhandare, Sudhakar Ganapati. "Biocontrol of V. cholerae using bacteriophage." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/29904/.
Повний текст джерелаАнотація:
Cholera is a persistent threat to public health and is endemic in many countries. Of late, there is an emergence of antibiotic resistance in Vibrio cholerae and treatment is effective only if given early, thus there is a need for rapid and more effective treatment of cholera. One such treatment could be the use of bacteriophages. During infection, V. cholerae adheres to the surface of enterocytes but does not invade the host. They are therefore not protected from bacteriophage infection. The study presented in this dissertation evaluates the potential of bacteriophage being used as a biocontrol for V. cholerae. The aim of this project was isolation and in vitro characterisation of bacteriophages, selection of a candidate bacteriophage for biocontrol and its use in an infant rabbit model to assess its therapeutic efficacy. Seven phages were isolated in China, attempts to isolate in the UK environments were unsuccessful and five more phages were obtained from various sources. In total twelve phages were characterised for the one step growth curves following their host strain growth curves, their lytic spectra, electron microscopy, PFGE, restriction analysis and annotation of sequenced genomes. These in vitro characterisations could help in selecting the candidate bacteriophage for in vivo phage therapy trials. Amongst the phages studied, the phage Φ1 most nearly fitted the selection criteria. Its burst size was 43 ± 5.5; while the latent period was 12 ± 0.0 and it had broad host range as it could lyse 67 % of the total 91 strains; while its genome did not show any undesirable genes associated with lysogeny/antibacterial resistance or any cholera toxin genes upon genome annotation. In therapeutic trials using an infant rabbit model, Φ1 reduced the bacterial numbers significantly (4.7 log10 reduction with P < 0.001) and treated animals showed no symptoms of disease.
17
Jie, Zexin. "Engineering bacteriophage to overcome antibiotic resistance." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/28877.
Повний текст джерелаАнотація:
Antimicrobial resistance is a global health issue that is endangering millions of lives and has been seen as a major threat to public health. Due to the rapid evolution of drug resistance, clinically available antibiotics are unsustainable, and thus, new antibiotics are urgently demanded to combat this crisis. Some novel antibacterial agents have been demonstrated as potential alternatives to replace commonly used antibiotics, but evidence has indicated that bacteria still can produce resistance to these antimicrobial agents. Thus, it is essential to develop new strategies for effectively controlling and overcoming antibiotic resistance.
As a natural process, antibiotic resistance is caused by different mechanisms, including gene mutation and biofilm formation. Therefore, using a single agent relying on a monomodal antibacterial mechanism could be challenging to eliminate a well-established bacterial community. This research aims to develop a multimodal antibacterial system containing antibiotics, bacteriophage and silver nanoparticles to overcome antibiotic resistance, including drug-resistant strains and biofilm-covered colonies. A polymer linker poly(oligo(ethylene glycol) methyl ether acrylate)-block-poly(3-vinyl-benzaldehyde) (POEGA-b-PVBA) was used to conjugate an antibiotic (gentamicin) and silver nanoparticles (AgNPs) onto BAA47B1 phage. Antibacterial and antibiofilm activities were evaluated using Pseudomonas aeruginosa PAO1 (wild-type) and Gentamicin-resistant Pseudomonas aeruginosa. The planktonic cell growth was assessed using bacterial optical density measurements, and biofilm biomass was determined using crystal violet staining.
Results showed a significant decrease in cell growth and biofilm biomass for both wild-type and gentamicin-resistant P. aeruginosa strains at lower concentrations treated with the triple component formulation compared to the single agent. The treatment of three components formulation was found to inhibit the biofilm formation as well as to induce biofilm dispersal, leading to a significant reduction in biofilm biomass. The results suggested that the combined formulation of gentamicin, phage, and AgNPs can be promising in effectively controlling P. aeruginosa biofilms.
18
Luan, Weisha. "Structural studies on bacteriophage portal proteins." Thesis, University of York, 2013. http://etheses.whiterose.ac.uk/4800/.
Повний текст джерелаАнотація:
In tailed bacteriophages and evolutionarily related herpes viruses, the portal protein is a central component of the DNA packaging molecular motor, which translocates viral genomic DNA into a preformed procapsid. The motor is the most powerful molecular machine discovered in nature, generating forces reaching ~50 pN and translocating DNA with a speed of several hundred bp/sec using ATP as an energy source. The oligomeric portal protein ring is situated at a unique vertex of the procapsid forming a conduit for DNA entry and exit. Although the three-dimensional structure has already been determined for portal proteins from bacteriophages P22, SPP1 and phi29, several important questions about the role of individual protein segments in DNA translocation and their interaction with other components of the motor remain unanswered. Structural information on portal proteins from other bacteriophages, like T4 for which a wealth of biochemical information is already available, will help to answer at least some of these questions. The portal protein of bacteriophage SPP1 (gp6) can form circular oligomers containing 12 or 13 subunits. It is found as a 12-subunit oligomer when incorporated into the viral capsid and as a 13-subunit assembly in its isolated form. The X-ray structure of the SPP1 portal protein is available only for the isolated 13-subunit assembly of the N365K mutant form. Because this mutation results in a reduction in the length of packaged DNA, determining the structure of the wild type portal protein would shed light on the mechanism of DNA translocation. Elucidation of the mechanism of DNA packaging depends also on the availability of accurate structural information on the SPP1 portal protein in its 12-subunit biologically active state. Such structural knowledge would be particularly useful in future, for designing a stable molecular machine that can function in vitro. In this thesis, experiments were designed to promote the formation of the dodecameric gp6: viz fusing gp6 with TRAP protein that forms a stable circular dodecamer as well as the co-expression of gp6 with the SPP1 scaffolding protein gp11. The protein targets were cloned, expressed and purified, and the oligomeric state of gp6 was characterised by a combination of biochemical, biophysical and structural approaches. The structure of the wild type gp6 was solved at 2.8 Å resolution, revealing a 13-fold symmetrical molecule. The protein’s fold is the same as for the N365K mutant, with most significant conformational differences observed in the tunnel loop and in segments of the clip and crown domains. Comparison with the structure of N365K mutant reveals significant differences in subunit-subunit interactions formed by tunnel loops, including different hydrogen bonding and van der Waals interactions. It is likely that these differences account for the different amount of packaged DNA, indicating involvement of tunnel loops in DNA packaging. The portal protein of bacteriophage CNPH82, cn3, was also successfully cloned, expressed and purified. Promising crystallisation conditions have been identified that yield crystals diffracting to 4.2 Å. Further optimisation should lead to determination of the X-ray structure of this protein in not too distant future. Self-rotation function calculations and SEC-MALLS analysis indicate that the cn3 protein forms 13-subunit assemblies, in common with the SPP1 portal protein. Foundation work has been carried out for the bacteriophage T4 portal protein, aimed at identifying suitable production and purification conditions. In addition, the full-length bacteriophage SPP1 scaffolding protein gp11 has been cloned, purified and crystallised. Degradation was observed in the full length gp11 protein and therefore a series of truncations were designed, cloned and purified aiming to improve the stability. Further studies on limited proteolysis of the full-length gp11 should lead to a stable gp11 tuncation that will form crystals with better diffraction.
19
Marion, William R. "Bacteriophage P22 scaffolding protein functions and mechanisms in procapsid assembly /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2009r/marion.pdf.
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20
Jakutyte, Lina. "Bacteriophage SPP1 entry into the host cell." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00669654.
Повний текст джерелаАнотація:
The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB - GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.
21
Grew, Edward Nicholas Delung. "Individual Based Modelling of Bacteria-Bacteriophage Interactions." Thesis, University of Exeter, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.486781.
Повний текст джерелаАнотація:
This thesis presents a novel individual based model capable of simulating the types of behaviour observed between bacteria and phage. Parasite-host relationships in bacterial ecology have commonly been modelled using a top-down differential ~quation approach tnat describes populations as single entities. These biological systems are intrinsically based on individuals whose behaviour relies on the interactions of many. A bottom-up, individualbased modelling approach has been used to simulate the behaviour observed between a bacterium and its corresponding bacteriophage. The use of individual based models has gained in popularity with the development of more powerful computers and is firmly established within current literature. The object-oriented programming language Java has been used to create Single, Co and Super infection models and the agent-based software Netlogo used to create a spatially explicit model. All the models were able to successfully recreate the real-life dynamics of bacteria and bacteriophage. Parameter space was explored within the models to investigate the effect of resource enrichment and flow rate within· a chemostat environment and to predict the persistence of phag~ within a bacterial population. An emergent individual based methodology founded on solid experimental data has forged a novel model that can highlight specific areas for further research and provide support for the outcome of investigational studies. Predicted future improvements In computing power will allow for the boundaries and possibilities of ecological modelling to be expanded and this work provides a basis from which to examine this pote~tial. These models provide scope for the investigation of a wide range of organisms as well as studies of parasite virulence, spatial dynamics and adaptive responses to selection pressures.
22
Pieroni, Peter. "Application of bacteriophage typing to Klebsiella pneumoniae." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1996. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq23909.pdf.
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23
Korehei, Reza. "Encapsulation of T4 bacteriophage in electrospun biopolymers." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44398.
Повний текст джерелаАнотація:
Packaging foods with antibacterial electrospun fibrous mats, in particular the incorporation of bacteria specific viruses such as bacteriophages may address concerns triggered by recent waves of bacterial food contamination. To this end several methods for incorporating or encapsulating T4 bacteriophage into electrospun fibres were investigated. The incorporation of T4 bacteriophage using simple suspension electrospinning lead to major losses in T4 bacteriophage activity, with more than five-orders of magnitude decrease in activity being observed. Improved T4 bacteriophage viability was obtained using two newly developed electrospinning processes, emulsion and coaxial electrospinning. In emulsion electrospinning, T4 bacteriophage was pre-encapsulated in an alginate reservoir via an emulsification process and subsequently electrospun into fibres. The emulsion electrospun fibres exhibited only two-orders of magnitude decrease in T4 bacteriophage activity. By contrast, complete T4 bacteriophage activity was maintained when coaxial electrospinning was employed. In the coaxial electrospinning process, a core/shell fibre structure was formed where the T4 bacteriophage was allocated to the fibre core, protected by the outer polymer shell. Depending on the polymer system used the rate of T4 bacteriophage release from the coaxial electrospun fibres varied. When hydrophilic poly(ethylene oxide) (PEO) was used as the polymer shell layer, immediate release of T4 bacteriophage was observed upon exposure to buffer. Increasing the PEO molecular weight increased the electrospun fibre diameter and viscosity of the releasing medium, which resulted in relatively slower T4 bacteriophage release pro-files. Similarly, the blending of cellulose diacetate (CDA) with PEO dramatically decreased the release behaviour. Depending on the PEO/CDA ratio, post-release electrospun fibre morphology varied from discontinuous fibres to minimally swollen fibres, consistent with the release profiles.
In the PEO fibres the mechanism of T4 bacteriophage release is likely through solvent activa-tion/polymer dissolution, while in the PEO/CDA blends a more diffusion control mechanism likely prevails. The Encapsulation of T4 bacteriophage within the electrospun fibres also improved T4 bacteriophage storability; full T4 bacteriophage activity was maintained for more than a month at +4 °C, as compared to only hours for non-encapsulated phage. These results are significant in the context of controlling and preventing bacterial infections in perishable foods during refrigerated storage.
24
Rooks, David John. "Molecular ecology of shiga-toxin encoding bacteriophage." Thesis, University of Liverpool, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548790.
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25
Brockhurst, Michael. "The evolutionary ecology of bacteria-bacteriophage interactions." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.509898.
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26
Atterbury, Robert J. "Bacteriophage control of Campylobacters in retail poultry." Thesis, University of Nottingham, 2004. http://eprints.nottingham.ac.uk/14066/.
Повний текст джерелаАнотація:
Food-borne disease continues to be a major cause of human morbidity and mortality. During the past few decades, Campylobacter jejuni has ascended to become the greatest cause of bacterial enteric disease worldwide. Anecdotal evidence suggests the majority of human campylobacteriosis in industrialised countries is caused by the consumption of undercooked chicken. Campylobacter continues to frustrate current control strategies throughout the food chain and in 2001 was responsible for over 56, 000 cases of food poisoning in the U.K. alone. The work presented in this thesis examined the potential of host-specific bacteriophage as a novel measure to control the population of Campylobacter in poultry production. Several surveys in this thesis revealed that campylobacters and their bacteriophage permeate the entire poultry meat supply chain, from chickens in the broiler house to packaged retail products. Characterisation of the bacteriophage recovered from such sources showed that retail poultry isolates exhibited greater similarities in host range than those originating from broiler houses, implying poultry processing selected for a subpopulation of phage. Additionally, broiler chickens harbouring bacteriophage in their gastrointestinal tract generally contained fewer campylobacters. All of the phage isolates studied belonged to the Myoviridae virus family as they possessed dsDNA genomes encapsulated in an icosahedral head with a rigid, contractile tail. Fragments of the phage genomes exhibited significant sequence homology with a number of genes involved in DNA replication from phage T4. Studies of the attachment and replication of the phage isolates in vitro suggested that adsorption to the host cell was efficient but the burst size was low (˂10 virions per cell). Campylobacter jejuni was found to produce membrane vesicles but these did not significantly affect bacteriophage replication in vitro. A series of trials using 'phage therapy' in broiler chickens revealed that Campylobacter colonisation can be reduced by ≥log[subscript]10 8.0 cfu g[superscript]-1 caecal contents by dosing with specific bacteriophage. However, both the timing and extent of the reduction in Campylobacter colonisation showed considerable variation. Additionally, the ability of bacteriophage to infect their host in vitro was not a reliable indicator of their efficacy in vivo. The direct application of bacteriophage to the surface of chicken skin artificially contaminated with Campylobacter led to a significant reduction in the number of recoverable host cells. Host resistance to bacteriophage infection was not detected in either the in vivo trials or when recovering Campylobacter cells from chicken skin treated with phage. The work presented in this thesis demonstrates that bacteriophage have considerable potential in the control of Campylobacter in poultry production. They already appear to constitute a limiting factor in Campylobacter colonisation of the chicken gastrointestinal tract and can be detected with their host on retail products. However, further research is required to fully realise their potential and optimising the timing, level and type of bacteriophage used in dosing will be important for their efficacy in vivo.
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Green, Michael. "Studies on Bacillus bacteriophage populations in compost." Thesis, University of Liverpool, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307668.
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Loc-Carrillo, Catherine M. "Bacteriophage control of campylobacters in poultry production." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416725.
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29
Leggate, Daniel Richard. "Characterisation and mutagenesis of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621894.
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30
Stocking, Kristin 1959. "Adsorption of MS-2 bacteriophage to silica." Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/277019.
Повний текст джерелаАнотація:
Batch and column experiments were performed to investigate the adsorption of bacteriophage MS-2 to silica at pH 5. Linear isotherm analysis of batch experiment data gave partition coefficient (KP) estimates of 270 cm³/g and 580 cm³/g for 4°C and 24°C, respectively. Breakthrough-type column experiments indicated that sorption and desorption were slow, as evidenced by a slow approach to breakthrough and tailing of the desorption limb. A non-equilibrium advection-dispersion model with all adsorption sites on the silica assumed to be kinetically controlled was used to model the column data. The model-generated parameters yielded Kp estimates 1000-fold smaller than those given by isotherm analysis and indicated that the time scale for desorption is on the order of 1-6 hours.
31
Palasingam, Kampan. "Homologous Recombination in Q-Beta Rna Bacteriophage." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc500683/.
Повний текст джерелаАнотація:
Q-Beta phage RNAs with inactivating insertion (8 base) or deletion (17 base) mutations within their replicase genes were transfected into Escherichia coli spheroplasts containing QB replicase provided in trans by a resident plasmid. Replicase-defective (Rep~) Q3 phage produced by these spheroplasts were unable to form plaques on cells lacking this plasmid. When individual Rep~ phage were isolated and grown to high titer in cells containing plasmid derived Q3 replicase, revertant Q3 phage (Rep'), with the original mutation (insertion or deletion) repaired, were obtained at a frequency of ca. 1 x 108. RNA recombination via a "template switching" mechanism involving Q3 replicase, the mutant phage genome, and the plasmid-derived replicase mRNA was shown to be the primary means by which these mutant phages reverted to wild type.
32
Grayson, Paul Politzer David. "The DNA ejection process in bacteriophage lambda /." Diss., Pasadena, Calif. : California Institute of Technology, 2007. http://resolver.caltech.edu/CaltechETD:etd-05252007-103551.
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33
Kroeger, Thomas William 1952. "Hydrophobic partitioning of the bacteriophage MS-2." Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/276963.
Повний текст джерелаАнотація:
In batch experiments at pH's 5 and 7, the partitioning of MS-2 between water and silica (unbonded) was compared with the partitioning between water and silica with 6.5 percent of the surface covered by hydrophobic C18 chains (bonded). The roles of double-layer and van der Waals forces in partitioning were explored by modeling the potential energies of interaction. MS-2 adsorption to unbonded silica was negligible at pH 7, but did occur at pH 5. Adsorption was independent of pH with the bonded silica and approximately 2.6 orders of magnitude greater than the unbonded at pH 5, suggesting the importance of hydrophobic partitioning. The total potential energies of interaction, which closely approach the pH-independent van der Waals potentials, are similar in magnitude for all pH's or silica types, and have no positive (repulsive) values. The insignificant contribution of the double-layer potentials suggests that these pH-dependent forces may not account for the pH-dependent adsorption observed with the unbonded silica.
34
Ranganathan, Srikanth. "Genetic and biochemical characterization of suppressors in the self-splicing nrdB intron of bacteriophage T4." Thesis, Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/25333.
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Kwon, Heajoon Yim. "Genetic and biochemical studies of the self-splicing nrdB intron of bacterical virus T4." Diss., Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/27591.
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36
Bedford, David Jonathan. "Molecular genetic analysis of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)." Thesis, University of East Anglia, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238840.
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37
Finnis, Christopher J. A. "A DNA packaging system from the Streptomyces phage #phi#C31." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250469.
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38
Silva, de Siqueira Regina. "An evaluation of the phage amplification assay for detection of salmonella." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288778.
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39
Sandegren, Linus. "Group I Introns and Homing Endonucleases in T-even-like Bacteriophages." Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-211.
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40
Fàbrega, Ferrer Montserrat. "Structural characterization of the T7 bacteriophage portal protein." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457690.
Повний текст джерелаАнотація:
The Escherichia coli infecting T7 bacteriophage shares a common dsDNA packaging mechanism with other bacteriophages of the Caudovirales order, Herpesviruses and Adenoviruses. The packaging machinery comprises the portal protein and the terminase complex. The portal protein is a channel located at a unique portal vertex that provides a conduit for DNA translocation, while the terminase complex recognizes a long concatemer of DNA, performs the nuclease catalytic activity and hydrolyses ATP. Available structural information about portal proteins describes them as oligomeric rings with an axial channel. High quality samples suitable for structural characterization of the portal protein of T7 bacteriophage were obtained and characterized. Both X-ray crystallography and cryo-electron microscopy (cryo-EM) data were collected, and an initial model built on the 5.8Å cryo-EM map was used to phase the crystallographic data, which allowed the building of a model of a tridecameric particle at 2.8Å resolution. The T7 portal particle is 170Å tall and 110Å wide toroidal protein with a central channel that ranges from 23Å to 95Å in diameter. Four domains have been identified in the structure: the wing, the stem, the clip and the crown. The a10-tunnel loop valve is proposed to play an important functional role. During packaging, it may adapt while DNA is translocated and rotated, and once the genome has been packed the side chain of tunnel loop residue Arg368 may be able to seal the channel and stabilize the DNA inside the capsid before tail assembly. Interestingly, these mechanisms would not only imply the flexibility of a loop region, but also the kink of the longer helix of the portal structure, a10.
41
Wright, Alice Ann. "The Genomic Sequence and Annotation of Bacteriophage HK239." TopSCHOLAR®, 2010. http://digitalcommons.wku.edu/theses/208.
Повний текст джерелаАнотація:
Bacteriophages are viruses that infect bacteria and they are the most numerous biological entities on Earth. Temperate phage can adopt two different lifestyles. In the lytic lifestyle, a phage injects its genome into the host and a controlled developmental program ensues. The phage DNA is replicated, phage genes are expressed and new viral particles are assembled. Ultimately, the host cell lyses and the phage particles are released into the environment. In the lysogenic lifestyle, a phage integrates its genome into the host chromosome, creating a prophage. The cell containing the prophage is known as a lysogen. Most prophage genes are not expressed. However, those that are encode a wide variety of functions. One function is exclusion, or the prevention of a different phage type from successfully infecting the lysogenic cell. Most exclusion systems are limited to a specific phage. Bacteriophage HK239 is unique in that it has a wide range of exclusion including Lambda, P1vir, P2, HK022, and T4rII. To learn more about HK239, the genome was sequenced and annotated. The genome is 41,538 bp in length and there are 71 open reading frames. It has a genomic organization similar to other lambda phage and is most closely related to bacteriophage HK022. No additional genes that share homology with known exclusion functions were identified through the sequence analysis of the HK239 genome. It is possible that an open reading frame for which no database matches were found may indeed encode an exclusion function.
42
Karunaratne, Desiree Nedra. "Structural investigation and bacteriophage degradation of bacterial polysaccharides." Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/25832.
Повний текст джерелаАнотація:
Seventy eight serologically distinct strains of Klebsiella bacteria are known to exist. The capsular polysaccharide surrounding the bacterial cell of these pathogenic Enterobacteria is of immunological significance. Structures of the capsular polysaccharides of nearly sixty seven strains of Klebsiella have been established, and each one found to be unique. The structures of the K antigens from Klebsiella, serotypes K67 and K80 are presented as a contribution to the continuing program of elucidation of the chemical structures of these antigens in an attempt to explain their immunological responses.
Chemical methods of structural elucidation were employed and the following two structures were obtained. [formula omitted] The polysaccharide from K67 was unique among the Klebsiella K antigens in having a four-plus-two-plus-one repeating-unit (indicating a branch on a side chain), while K80 was unique as it was the first instance that a pyruvic acetal was found on a terminal rhamnose unit.
The importance of bacteriophage-borne enzymes in the generation of single repeating-units containing labile substituents is demonstrated. Klebsiella K44 polysaccharide was degraded using a crude solution of Φ44 bacteriophage. The oligosaccharides obtained were crucial in the determination of the position of the 0-acetate group. In the case of the polysaccharide from Klebsiella K26, the degradation performed using a crude solution of Φ26 bacteriophage resulted in the isolation of a single repeating-unit containing a pyruvic acetal together with an oligosaccharide corresponding to a single repeating-unit devoid of its terminal pyruvate containing sugar. The structures of these compounds which are as follows, were useful in further confirmation of the structures of the original polysaccharides. [formula omitted] Escherichia coli. another pathogenic Enterobacteria possessing immunologically significant K antigens, has been found to contain capsular polysaccharides bearing a strong resemblance to those of Klebsiella. Recently it was discovered that some strains of E. coli contained K antigens comprising amino sugars. A preliminary study on the chemical behaviour of amino sugars, and chemical methods of structure elucidation of such polysaccharides have been included in an appendix as this has been a new area of research in this laboratory. An appendix containing compilations of the cross-reactions, known structures, and chemotypes of the Klebsiella K antigens has also been included.Science, Faculty of
Chemistry, Department of
Graduate
43
Lau, Mathew Thye Ngak Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilms." Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences, 2007. http://handle.unsw.edu.au/1959.4/40491.
Повний текст джерелаАнотація:
Pseudomonas aeruginosa is a ubiquitous environmental microorganism that opportunistically colonizes immune-compromised hosts. P. aeruginosa is capable of establishing complex. matrix-encased biofilms during colonization of both environmental and living host surfaces. Biofilms formed by P. aeruginosa are physiologically very different from free-living P. aeruginosa cells, and exhibit increased resistance to environmental stresses, including antibiotic treatment. While the development and establishment of P aeruginosa biofilms has been extensively studied in vitro, several new behaviours of P. aeruginosa in biofilms have recently been observed that may greatly impact on the spread, recolonization and function of biofilms. These processes include bacteriophage mediated lysis and dispersal of P. aeruginosa biofilms, and the generation of phenotypic and genetic variation among bacterial cells that disperse from the biofilm. In this project, the role of bacteriophage activity and phenotypic variation in the development of P. aeruginosa biofilms has been investigated. Induction of a Pf1-like prophage of P aeruginosa (here named Pf4), during biofilm formation was characterized and was shown to increase over the progression of biofilm development. It was observed in this study that the activity of Pf4 caused the emergence of small colony variant (SCY) phenotypes in the effluent run-off from P. aeruginosa biofilms. Computational analysis of the genome ofPf4 resulted in the identification of a novel Toxin-Antitoxin (TA) gene pair, not previously identified within the genome of P. aeruginosa, of which the putative toxin gene product was determined here to play a role in growth-inhibition and the small colony phenotype of P. aeruginosa. TA gene pairs are proposed to induce stress responses in host cells and therefore play a role in survival during periods of environmental stresses such as oxidative or starvation stress. To study the effects of the Pf4 toxin and its possible role in the stress response of P aeruginosa, the Pf4 toxin gene was cloned and placed under the regulation of an inducible arabinose promoter. The proteome expression and biofilm formation as a result of toxin over expression were compared. The proteomic studies performed here indicated that P. acrllginosu biofilms do respond to expression of the toxin component of this putative TA element by increased expression of stress related proteins. Many stress-related groups of proteins were found to be over expressed during induction of the toxin indicating a possible role in stress survival of P. acrllginosa. Homology studies of the Pf4 toxin indicated a strong structural sequence relationship with the toxin ParE of the ParDE TA system. The mode of action of ParE toxin had previously been determined and showed the ParE toxin to be a strong gyrase inhibitor. The Pf4 antitoxin was, however, found to have homology to the Phd antitoxin of the Phd-Doe system of bacteriophage PI. The mode of action of the Doc protein remains to be clearly determined. To better understand the interaction between the Pf4 antitoxin and its cognate toxin protein interaction studies were performed. Peptide fragments of the Pf4 antitoxin were generated for an SPR binding assay and this study identified putative peptide sequences that are responsible for binding of the Pf4 antitoxin to its cognate toxin. Further investigation of a selected strong binding peptide showed that there were 3 key amino acids that were important in binding to the Pf4 toxin, namely His65, Ser67 and A 69 sp. Overall, this study has identified a key role for bacteriophage Pf4 in biofilm development and phenotypic variation in P. aeruginosu, and has provided initial insight into the molecular mechanisms by which this bacteriophage influences growth and gene expression in this organism.
44
Mosaico, Maria Luisa. "The utility of bacteriophage lambda in gene targeting." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0021/MQ55228.pdf.
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45
Sturino, Joseph Miland. "Bacteriophage Defense Systems and Strategies for Streptococcus thermophilus." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-12152003-034319/.
Повний текст джерелаАнотація:
The genomes of six Streptococcus thermophilus bacteriophages were compared to identify genes that could be targeted by engineered phage defense systems with potentially widespread efficacy. The genes associated with the S. thermophilus phage Sfi21-prototype genome replication module, including a putative primase and a putative helicase, were found to be among the best candidates due to their frequency of distribution in industrial phage isolates, striking sequence conservation between independent isolates, and intrinsic strategic importance in early phage development. Fourteen antisense RNA cassettes targeting the phage k3-derived helicase (hel3) or primase (pri3) genes were expressed in S. thermophilus NCK1125. These constructs consistently reduced the efficiency of plaquing (EOP) of phage k3 to between 5 x 10-1 and 2.0 x 10-3 depending on the (i) gene targeted and (ii) region of the gene that was targeted. The largest antisense RNAs were generally found to confer the largest reductions in EOP, however shorter antisense RNAs designed to the 5' region of the gene retained much of the inhibitory function, especially if they contained sequences complementary to the ribosome binding site. Expression of antisense RNAs correlated with decreased levels of phage encoded primase transcripts, likely due to increased degradation of the dsRNA complex. This, in turn, correlated with diminished phage genome replication and aborted phage development. In a separate study, invariant and highly conserved amino acids within a primase consensus sequence were targeted by site-specific mutation within the S. thermophilus phage k3-encoded putative primase. PCR products containing the desired mutation(s) were cloned and expressed in S. thermophilus NCK1125. The majority of the examined constructs remained sensitive to phage k3, however four constructs conferred strong phage resistance to the bacterial host. The mutated residues resided within a putative ATPase/helicase domain suspected to be critical for primase function in vivo. The co-expression in trans of the K238(A/T) or RR340-341AA mutant proteins suppressed the function of the native, phage-encoded primase protein in a dominant negative fashion via a proposed subunit poisoning mechanism. According to this model, the plasmid-encoded mutant primase subunits are structurally intact and form stable interactions with the native, phage-encoded primase subunits, thus inhibiting their activity. These constructs completely inhibited phage genome synthesis and reduced the efficiencies of plaquing more that nine log cycles. Given the magnitude of the resistance conferred, it was concluded that the putative primase is essential for genome replication in S. thermophilus Sfi21-type phages. Further, it was also clear that host-encoded factors were unable to complement the resultant deficiency. Amber mutations introduced upstream of the transdominant RR340-341AA and K238(A/T) mutations restored phage genome replication and phage sensitivity of the host, indicating that translation was required to confer phage resistance. Residues within a critical oligomerization domain were also identified through genetic analysis. Introduction of an E437A mutation downstream of the transdominant K238T mutation completely suppressed phage resistance, indicating that the E437A mutation precluded the association of the mutant and native subunits. To our knowledge, this is the first use of subunit poisoning to inhibit phage replication in the lactic acid bacteria.
46
Gunneriusson, Elin. "Display of affinity proteins on bacteria and bacteriophage /." Stockholm : Department of Biotechnology, Royal Institute of Technology, 1999. http://www.lib.kth.se/abs99/gunn0521.pdf.
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47
Marks, James D. "Making human antibody fragments in bacteria and bacteriophage." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306239.
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48
Short, Nicholas J. "The DNA sequence of the filamentous bacteriophage Pf1." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305822.
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49
Bryant, J. M. "Expression, mutagenesis and properties of bacteriophage K1E endosialidase." Thesis, University of Cambridge, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597038.
Повний текст джерелаАнотація:
A full length endoE gene construct has been generated from the partial clones used to sequence the gene. Recombinant endoE has been expressed and purified as a fusion protein that is catalytically active and possesses the same kinetic characteristics as the bacteriophage enzyme. The mature, active endoE is 76 kDa in size and is probably produced by the post-translational cleavage of a 90 kDa primary translation product. Recombinant endoE has been purified to homogeneity, in milligram quantities, with the fusion protein partner removed for further studies of the enzyme. The role of the C-terminus of endoE was studied by expression of C-terminally truncated proteins. These were expressed from 3' truncated endoE gene constructs generated either by exonuclease III-mediated nested deletion or by PCR. The site of proteolytic processing has been located to within 18 amino acids and further truncation beyond this cleavage site renders the enzyme inactive and more susceptible to proteolytic degradation. The endoE sequence contains two copies of the sialidase motif and one P-loop motif. The importance of these motifs in enzyme activity was studied by site-directed mutagenesis, replacing part or all of the motifs with alanine residues. These indicated that the P-loop motif was not involved either in binding or hydrolysis of polysialic acid and that the sialidase motifs appear to be necessary to allow production of correctly folded, soluble protein but are not involved directly in catalysis. The kinetic characteristics of the binding interaction of the endoE fusion protein and polysialic acid have been studied and compared to those of C-terminally truncated endoE fusion proteins. As a first step in investigating the use of endoE as a reagent in cell biological studies, the subcellular localisation of polysialic acid and the effects of chloroquine and brefeldin A treatment on its localisation in cultured MCF-7 breast tumour cells have been studied.
50
Reed, Patricia. "Function of bacteriophage Orf recombinases in genetic exchange." Thesis, Durham University, 2006. http://etheses.dur.ac.uk/4917/.
Повний текст джерелаАнотація:
Recombination events in bacteriophages frequently occur by illegitimate exchange at short tracts of sequence homology, enabling these viruses to acquire novel genes and serve as vehicles for horizontal gene transfer. The emergence of new pathogenic organisms due to the acquisition of virulence determinants from bacterial viruses has stimulated considerable interest in the mechanisms of phage recombination. Bacteriophage λ encodes its own recombination system, consisting of Exo, β and γ proteins. An additional λ recombinase, Orf, participates in the early stages of exchange, supplying a function equivalent to the Escherichia coli RecFOR complex. The host enzyme complex promotes the loading of the RecA strand exchange protein onto SSB-coated ssDNA. This thesis describes the purification and biochemical analysis of the λ Orf protein, in parallel with two distantly related homologs from E. coli cryptic prophage DLP12 and Staphylococcus aureus phage ɸETA.X Orf was found to belong to a family of proteins originating from diverse lambdoid phage and prophage sources. Members of this family reside within a conserved genetic module located between phage replication and cell lysis functions. Orf exists as a homodimer, arranged as a toroid with a shallow cleft running perpendicular to the central cavity. K binds preferentially to DNA containing single- stranded regions, and associates with E. coli SSB protein in the presence of ssDNA. The Orf homolog from E. coli DLP12 displayed similar properties. This work suggests that members of the Orf family function as recombination mediator proteins, stimulating the assembly of strand exchange proteins onto ssDNA, and highlights the importance of overcoming the barrier presented by SSB proteins during lambdoid phage recombination.