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.

Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.

Errata sheet pasted opposite Table of Contents. / Includes bibliographies. / 142, [171] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis investigates the UV-induced inhibition of oriC initiation function and the nature of the phenotype of the mutant origin of replication of Escherichia coli, oriCc. The specific aims of this study are to delineate the source of the UV-induced trans-acting inhibition of oriC function, utilizing the phage vector [lambda]poriCc, and to demonstrate the oriCc allele as an enhanced DNA replication initiator from oriC. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997?

Ff filamentous bacteriophage infect male (F+) strains of Escherichia coli and are assembled at the cell membranes, by a secretion-like, non-lethal process. The pIII protein, located at one end of the virion-filament, is required at both the beginning and the end of the phage life cycle. During infection, the N-terminal domains of pIII, N2 and N1, bind to the primary and secondary host receptors, F pilus and TolA protein, respectively. At the end of the life cycle, the pIII C-domain mediates the termination and release of virions. Thus, both entry and release involve structural transitions of the virus coupled to membrane transactions of the virion proteins. "Unlocking” of the highly stable virion presumably results in membrane integration during entry, whereas a reverse event, “locking” of the virion, occurs upon detachment from the membrane at termination step of assembly/secretion. Recently, it was shown that the pIII C-domain plays an active role at the step of entry. This finding implicates the C-domain of pIII in “unlocking” of the virion, presumably resulting in the exposure of the membrane anchor at the very C-terminus of pIII (Bennett & Rakonjac, 2006). To further this work, this thesis has mapped the portion of the pIII C-domain required for infection, by constructing a set of nested deletions of the C-domain fused to the receptor binding domains N1 and N2, and then determined the infectivity of phage carrying the mutant proteins. This mapped the portion of the C-domain required for phage infection is different to that required for termination of assembly. The different requirement for entry and release suggests that the two processes are carried out by distinct mechanisms and/or depend on different sets of accessory proteins. In addition, a system was designed for the efficient production and purification of very short virions, the length of which is 1/20 that of the wild-type f1. These short virions, called microphage, are the first step towards the structural analyses of the phage termini cap structures, of which one contains pIII in the “locked” conformation.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 152-161).

Thesis (Ph. D.)--Cornell University, May, 1998. / Vita. Includes bibliographical references (leaves 157-164).

Thesis--Carleton University. / Also available in electronic format on the Internet.

The 2014 West Africa Ebola outbreak saw a substantial number of healthcare workers (HCWs) being infected, despite the use of personal protective equipment (PPE). PPE is intended to protect HCWs when caring for patients with Ebola virus disease (EVD), but PPE may play a role in the spread of Ebola in healthcare environments. Before the removal of PPE, chemical disinfection may prevent the transfer of pathogens to HCWs, but the efficacy of common disinfectants against enveloped viruses, such as Ebola, on PPE surfaces is relatively unknown. The purpose of this study is to assess the efficacy of two common disinfectants, chlorine bleach (Clorox® bleach) and quaternary ammonium (Micro-Chem Plus®), used in healthcare settings for inactivation of enveloped viruses on PPE. The virucidal activity of the two disinfectants were tested against bacteriophage Φ6, an enveloped, non-pathogenic surrogate for enveloped viruses, on Tyvek suit surfaces. Virus was dried onto Tyvek suit surface, exposed to the disinfectants at use-dilution for a contact time of one minute, and the surviving virus was quantified using a double agar layer (DAL) assay. The Clorox® bleach and Micro-Chem Plus® produced a >3.21 log10 reduction and >4.33 log10 reduction, respectively, in Φ6 infectivity. The results of this study suggest that chlorine bleach and quaternary ammonium are effective in the inactivation of enveloped viruses on Tyvek suit surfaces. Chemical disinfection of PPE should be considered as a viable method to reduce the spread of pathogenic, enveloped viruses to HCWs, patients, and other environmental surfaces in healthcare settings.

Mu is a temperate bacteriophage which infects Escherichia coli and several other Gram negative enteric bacteria. It is an extraordinary phage in several respects and has carved a special niche for itself both as a genetic tool and a paradigm in phage biology, almost rivaling phage lambda. It is also a predator that has adapted its hunting skills well in order to have an extraordinarily wide host range. While phage Mu finds a mention in almost every genetics textbook for several of its unique and well-studied characteristics, there are a few aspects of its biology that are far from understood. In this thesis, light has been shed on one such less understood feature of Mu biology, namely its anti-restriction function. The enigmatic mom gene of bacteriophage Mu is the center of this thesis work. Bacteriophages, through their sheer number and versatility of attack tactics, constitute an overwhelming threat to bacteria in the natural environment. While it is not always possible to completely prevent the entry of foreign DNA into the cell, it is in the interest of the bacterium to tame the xenogeneic DNA, whose expression may have adverse effects on bacterial fitness. Bacterial nucleoid associated proteins (NAPs) participate in chromosome structuring and global transcriptional regulation. Besides this canonical role, they furnish the job of regulating xenogeneic DNA as well. NAPs are known to regulate the expression of toxin-antitoxin modules, pathogenicity islands and other horizontally transferred DNA and have a profound role in regulating transposon dynamics and the lifestyle of many phages. Chapter 1 introduces the role of bacterial NAPs in silencing foreign DNA, especially after the DNA establishes itself in the host. This thesis examines the role of a bacterial NAP namely Fis in fine-tuning an immune evasion function of bacteriophage Mu. A general introduction to phage Mu and its host expansion strategies, with special focus on its DNA modification function is also presented. Owing to the various immune evasion strategies, phages often have an upper hand on their hosts in the ongoing evolutionary arms race. One such strategy is DNA modification which bacteriophages have evolved as a means to protect their genomes from restriction enzymes of the host. While most phages employ the commonplace methylation modification for their anti-restriction function, phage Mu employs an unusual acetamido modification, catalysed by its protein Mom. Mom modified DNA is refractory to several restriction enzymes from different bacterial species. However, the modification is toxic to the host and thus mom expression needs to be precisely regulated to prevent untimely expression. A crowded multifactorial regulatory circuit has evolved to ensure the expression of mom without jeopardizing the welfare of the bacterial host. Chapter 2 uncovers a new player in mom regulation. The study shows that the bacterial chromatin architectural protein Fis is a transcriptional repressor of mom promoter and that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. Two distinct roles of Fis have been known previously in Mu biology. In addition to bringing about the overall downregulation of transposition events and transcription of early genes of phage Mu, Fis also stimulates tail fiber flipping by aiding the activity of a site-specific recombinase. The present study thus presents a novel facet of Fis function in Mu biology. While the regulation of mom has been a matter of intense investigation over the past few decades, most biochemical and structural aspects of the Mom protein per se have remained mysterious owing to the difficulties in cloning this toxic gene. Chapter 3 describes the expression, purification and biophysical characterization of Mom. A variety of techniques show Mom to be folded and dimeric in solution. SPR studies with Mom indicate its high affinity binding to DNA. Chapter 4 deals with the attempts to identify the elusive co-factor of Mom. To begin with, the in vivo activity of Mom was demonstrated by employing a simple plasmid cleavage assay based on the resistance of Mom modified DNA to certain restriction endonucleases. A variety of disparate in silico structure prediction tools such as I-TASSER, Robetta and PHYRE indicate Mom to be related to the GCN5-related N-acetyltransferase superfamily. Mutation of residues deemed important from this analysis indeed abolished or reduced Mom activity in vivo, validating the bioinformatics based prediction and shed light on the possible active site of Mom. However, acetyltransferases are not known to transfer acetamido groups. It was also necessary to establish beyond doubt, the chemical structure of the Mom modified nucleoside. High resolution mass spectrometry data showed the modification to be acetamido, corroborating the earlier sole report on this aspect. Based on the biochemical reactions that acetyl coenzyme A is known to participate in, it is difficult to explain the involvement of acetyl coenzyme A in acetamido addition. Notwithstanding the converging predictions of different bioinformatics tools, caution is recommended when inferring function from structurally similar family members. It is possible that a different chemistry might have converged on the same (acetyltransferase) fold, given that none of the known pathways utilizing acetyl coenzyme A can explain the Mom modification. Several likely candidates such as carboxy-SAM, glyoxylic acid and glycine were also tested for being donors of the two carbon entity transferred on adenine by Mom. Since these candidates tested negative in our genetic assays, a genome-wide genetic screen was subsequently devised to identify the host genes involved in mom modification. The assay exploited the phenotype of lethality associated with overexpression of Mom in E. coli in order to screen for mutations in the host genome that rescued the toxicity. However, the survivors which were obtained in this assay had emerged through mutations in the mom gene rather than abrogation of the co-factor synthesis pathway of the host. The results point at two possibilities: (i) utilization of essential gene(s) or (ii) existence of redundant pathways for the Mom modification reaction. Chapter 5 is an account of our attempts to trace the lineage of mom and its regulatory region, employing updated DNA and protein sequence databases. Despite the selective advantage conferred on the phage by the anti-restriction function of mom, in many Mu-like phages, mom is either absent or substituted with methyltransferases. However, in Mu-like genomes that do encode mom, in spite of a significant overall sequence divergence from Mu, the core elements of the mom regulatory circuit seem to have either co-evolved or have been selectively conserved. Although Mu appears to be unique in the possession of a regulatory circuit tailored for the purpose of mom regulation, recently discovered Mu-like genomes show that different types of regulatory features evolved several times in closely related genomes. It is very likely that a toxic gene like mom has earned its place in the phage genome by carrying along with itself a baggage of regulatory elements. Failure to sustain sufficient regulatory pressure may trigger the loss or replacement of the advantageous but potentially lethal mom function.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aggregatibacter actinomycetemcomitans (Aa) é uma bactéria associada à Periodontite Agressiva (PA). Ela invade tecidos moles, com ocorrência de lisogenia e bacteriófagos presentes em até 69% das subespécies. Estudos in vitro sugerem que a indução do bacteriófago (Aa17) ocorre numa co-cultura de Aa lisogênico com fibroblastos humanos. Se esta interação ocorre in vivo, com liberação do vírus, uma reação imunológica contra o Aa17 aconteceria. O objetivo deste estudo é constatar se anticorpos (AC) contra proteínas do Aa17 existem e estão associados à doença periodontal. Um objetivo adicional foi testar a resposta de AC contra os sorotipos do Aa. 52 indivíduos participaram: 31 com PA, 5 com Periodontite Crônica (PC) e 16 com Periodonto Saudável (PS). Soro foi coletado após a classificação clínica. As proteínas do Aa17 foram obtidas de preparações purificadas. As subespécies do Aa utilizadas para amostras de proteínas através de sonicação foram: 43717(American Tissue Culture Collection - ATCC) sorotipo A, 43718 (ATCC) sorotipo B, 33384 (ATCC) sorotipo C, IDH781 sorotipo D, NJ9500 sorotipo E and CU1000 sorotipo F. As proteínas foram separadas em géis de poliacrilamida e transferidas para membranas de nitrocelulose. As reações de Western-blotting ocorreram com o AC primário sendo o soro de cada indivíduo. Todas as membranas foram lidas pelo sistema Odyssey que captura sinais no AC secundário (antihumano). A resposta de AC contra ao menos uma proteína do Aa17, assim como pelo menos um sorotipo do Aa foi observado em todos, com exceção de dois indivíduos (com PS), participantes. Um indivíduo do grupo PC e três do PA tiveram resposta de AC contra alguns, mas não todos os sorotipos do Aa. A resposta de AC contra todos os sorotipos foi o achado mais comum nos grupos PA (28/31), PS (14/16) e PC (4/5). A resposta de AC contra o complexo de proteínas do Aa17 foi observado em 7 indivíduos com PA, 2 com PC e 6 com PS. A presença de AC contra qualquer proteína do Aa17 tem significância estatística (p= 0,044), assim como a resposta de AC contra o sorotipo C (p= 0,044). Reações intensas foram vistas quando o soro reagiu contra proteínas do sorotipo C; em alguns casos um sinal tão forte que cobriu a maioria da faixa. Essa resposta intensa esteve presente em 17, 3 e 1 dos indivíduos com PA, PC e PS e tem significância estatística entre os grupos PA e PS (p= 0,001). A resposta de AC contra uma proteína do Aa17 ou seu complexo foi observado em todos os grupos. Esse achado sugere que a indução in vitro do Aa17 poderia também ocorrer in vivo, embora não sendo necessariamente associada à periodontite. A resposta de AC contra vários sorotipos do Aa foi um achado comum e não associado com a doença. Entretanto, a presença e a intensidade da resposta de AC contra o sorotipo C está associada à PA. / Aggregatibacter actinomycetemcomitans (Aa) is a bacteria associated with Aggressive Periodontitis (AP). It invades soft tissues, with occurrence of lysogeny and bacteriophage presence up to 69% of Aa subspecies. In vitro studies suggested that bacteriophage (Aa17) induction occurs upon co-culture of Aa lysogens subspecies with human fibroblasts. If such an in vivo interaction resulted in Aa17 induction and release of virions, an immunologic reaction to Aa17 proteins could ensue. The purpose of this investigation was to learn whether serum antibodies (AB) to Aa17 proteins are found in human sera, and whether they are associated with periodontal disease. An additional purpose was to test the AB response against known Aa serotypes.52 individuals took part: 31 with AP, 5 with Chronic Periodontitis (CP) and 16 with a Healthy Periodontium (HP). Serum was collected after clinical classification. Aa17 proteins were obtained from purified Aa17 preparations. The Aa strains used for protein sampling through sonication were: 43717(American Tissue Culture Collection - ATCC) serotype A, 43718 (ATCC) serotype B, 33384 (ATCC) serotype C, IDH781 serotype D, NJ9500 serotype E and CU1000 serotype F. Proteins were separated by SDS-PAGE gels and then transferred to nitrocellulose membranes. Western-blotting reactions were carried out with the primary AB being each subjects serum. All membranes were read through the Odyssey system which captures signals from a dye in a secondary (antihuman) AB. AB response against at least one Aa17 protein, as well as a response to at least one Aa serotype, was observed in all but two individuals (with HP) who participated in the study. Serum from one individual from the CP group and three from the AP group had AB response to some, but not all Aa serotypes. AB response against all Aa serotypes was the most common finding in AP (28/31), HP (14/16) and CP (4/5) groups. AB response to the full complex of Aa17 proteins was observed in 7 individuals with AP, 2 with CP and 6 with HP. Chi-square tests comparing the AP with HP groups indicated that the presence of AB against any Aa17 protein is statistically significant (p= 0,044), as well as AB response against serotype C (p= 0,044). Intense reactions were seen when sera reacted with proteins from serotype C; in some cases the signal was so strong that it covered much of the lane. This intense response was present in 17, 3, and 1 of the AP, CP and HP subjects, respectively and was statistically significant (p=0.001) between the AP and HP groups. AB response against a Aa17 protein or its whole complex was observed in all groups. This finding suggests that the in vitro Aa17 induction could also be occurring in vivo, although it is not necessarily associated with periodontitis. AB response against multiple Aa serotypes was a common finding and is not associated with disease. However, the presence and intensity of AB response against Aa serotype C, is associated with AP.