Intracellular symbioses between bacteria and insects are numerous, and alter the ecology and evolution of host and symbiont alike. Long-term persistence results from either exploitation (e.g., reproductive manipulations) or mutually beneficial interactions (e.g., nutritional mutualisms). The endosymbiont Hamiltonella defensa, while not essential for growth or survival of healthy aphids, protects aphids from attack by parasitoid wasps. In this thesis, I have used a variety of sequenced-based techniques to illuminate the population and genome dynamics of H. defensa and to disentangle how these factors contribute to its ability to persist and protect its hosts.I characterized the phylogenetic relationships among H. defensa strains from aphids and a whitefly using a multilocus approach. Most loci evolve in a clonal manner, and one cluster of strains may have given rise to an obligate symbiosis. Some H. defensa strains were infected with the bacteriophage APSE, which encodes putative eukaryotic specific toxins and has been suggested to be involved in protecting aphids. I sequenced the toxin locus and the flanking regions from the APSE strains and found that although the phage genome backbone was highly conserved, strains contained non-orthologous toxin-cassettes. Sequenced cassettes contained one of three putative toxin families: Shiga toxin, cytolethal distending toxin, and YD-repeat toxins. A correlation was noted that of several genetically identical H. defensa strains, the one without phage APSE encoding the YD-repeat toxin failed to protect its aphid host. This APSE strain carrying the YD-repeat toxin has since been demonstrated to be essential for protection in several related H. defensastrains.To examine additional bacterial encoded loci that might facilitate the persistence in and protection of aphids by H. defensa, I sequenced the genome of one strain and obtained partial genomes of two additional strains. These genomes exhibit a streamlined metabolism, but are littered with mobile DNA and putative virulence factors. Horizontal gene transfer, recombination and rearrangements are common, and phage and plasmids have played an important role in resorting genes. Thus, although H. defensa benefits its host, its facultative lifestyle has resulted in a pattern of genome evolution associated with reproductive parasites rather than long-term mutualists.

Staphylococcal phage 80α can serve as a helper bacteriophage for a family of mobile genetic elements called Staphylococcus aureus pathogenicity islands (SaPIs). The prototype island, SaPI1, is able to hijack the 80α capsid assembly process and redirect capsid formation to yield smaller, phage-like transducing particles carrying SaPI DNA. Capsid size redirection is accomplished through two SaPI1-encoded gene products, CpmA and an alternate scaffold protein, CpmB. The normal 80α scaffold and the SaPI1 CpmB scaffold share a small block of conserved residues at their C-termini, several of which had been shown to be essential for CpmB function. This led to the hypothesis that the C-termini of both the phage and SaPI scaffolds interact in similar ways with the major capsid protein. The goal of this study was to test this hypothesis and to identify the amino acid residues at the capsid-scaffold interface, using a genetic approach.

Thesis advisor: Gregory R. Chiklis / Thesis advisor: Kathleen Dunn / In molecular diagnostics, the polymerase chain reaction (PCR) is used to amplify small amounts of nucleic acids found in patient samples, allowing for detection of diseases within hours of infection. This early detection allows medical professionals to diagnose and treat patients with greater success. It is crucial that internal controls, such as NATtrol™-treated microorganisms, are used in these PCR assays to avoid false-negative results and ensure accurate diagnosis of patients. NATtrol™ treatment renders microorganisms non-infectious while leaving them fully intact with their complete RNA or DNA genomes. Therefore, NATtrol™-treated microorganisms can be used in PCR as full-process internal controls that are spiked into patient samples and co-extracted and co-amplified within the sample. If the spiked NATtrol™ control returns expected results on the test, then the patient sample result can also be trusted. Here, we performed studies to validate the use of NATtrol™-treated MS2 virus as a universal full-process internal molecular control. In these studies, a quantitative, real-time, reverse-transcription PCR (qRT-PCR) assay was performed on the Roche LightCycler 480 instrument. Studies included working range validation, limit of detection, within-run precision, between-run precision, real-time stability, freeze-thaw (transport) stability, and open-vial (use-life) stability. All studies demonstrated the precision and stability of the MS2 NATtrol™ molecular control. / Thesis (BS) — Boston College, 2014. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology Honors Program. / Discipline: Biology.

Nous avons analysé par cryomicroscopie électronique la morphologie et la structure de longues chaines d’ADN condensées par un polycation tétravalent, la spermine (polyamine). Les expériences ont été réalisées i) avec des solutions de chaînes diluées et ii) avec des chaines isolées confinées dans la capside d’un virus.Les expériences ont été réalisées avec de l’ADN Lambda (48kbp) en solution diluée (0.03 mM Ph) et à faible concentration ionique (10 mM Tris HCl, 1 mM EDTA, pH 7.6). Nous avons exploré une large gamme de concentrations en spermine, allant du seuil de précipitation (0.05 mM sp) jusqu’à la limite de re-solubilization et au-delà (400 mM sp). Seize minutes après mélange de l’ADN et de la spermine, les échantillons sont piégés en film mince et vitrifiés à basse température pour garder intactes les conditions ioniques, puis imagés à basse température sous faibles doses d’électrons (cryoMET). La plupart des chaînes d’ADN forment des agrégats de tores de structure hexagonale avec des interdistances entre hélices de 2.93, 2.88, et 2.95 nm pour des concentrations en spermine respectivement égales à 0.05, 1 et 100 mM spermine, ce qui est en bon accord avec les données collectées précédemment par diffraction des rayons X. A concentration plus élevée en spermine (200mM), les tores hexagonaux sont remplacés par des faisceaux cholestériques de structure plus lâche (3.32 nm entre hélices). Nous en déduisons que la forme comme la structure des condensats cristallins liquides ADN-sp sont liées aux interdistances entre hélices et déterminés par les conditions ioniques i.e. par l’énergie cohésive entre chaînes d’ADN. En dehors du domaine de précipitation (400mM sp), les molécules d’ADN forment un réseau soluble de fines fibres (4-6nm de diamètre) qui nous amènent à reconsidérer l’état de ces chaiînes en présence de spermine. Nous avons également conçu des expériences pour visualiser les agrégats formés 6 à 60 sec après addition de la spermine dans les mêmes conditions de tampon. Parmi les nombreuses formes originales que nous avons observées (absentes après 16 min), la présence de fibres étirées ou en hélice, visibles seulement après 9sec, nous conduit à proposer que les chaines d’ADN soient immédiatement étirées après addition de spermine puis relaxent sous forme de fibres hélicoïdales qui donnent naissance à de petits toroids (comprenant quelquefois moins d’une chaine) qui grandissent et fusionnent. Nous avons également analysé les dimensions de l’ensemble des tores observés et montré l’existence de contraintes géométriques qui restent à élucider. Puisqu’il était généralement impossible de prévenir l’agrégation des chaines d’ADN, nous avons choisi une autre approche pour analyser le collapse de chaines d’ADN individuelles. Nous avons utilisé une population de virus T5 contenant une fraction de leur génome initial (12-54 kbp). La molécule d’ADN, initialement confinée dans le petit volume de la capside (de de 80nm diamètre) est collapsée par addition de spermine. Par comparaison avec le premier jeu de données, nous avons travaillé à concentration plus élevée en ADN (0.45 mM Phosphates dans l’ensemble de l’échantillon) et la concentration en spermine a été ajustée entre 0.05 et 0.5 mM (ce qui correspond à des rapports de charges +/- bien inférieurs). Ces expériences ont donc été réalisées au voisinage de la ligne de précipitation, dans la « région de coexistence », entre le domaine où les chaines sont en condition de pelote et le domaine ou les chaines sont toutes collapsées sous forme de tores. Nous avons montré l’existence de formes intermédiaires entre ces deux états que nous appelons « tores chevelus » dans lesquels une partie de la molécule est condensées dans le tore alors que l’autre partie reste non condensée. Les distances entre hélices ont également été mesurées. Elles sont plus grandes dans ces structures intermédiaires que dans les tores formés à plus forte concentration en spermine. Ces deux séries d’expériences montrent l’intérêt des méthodes de cryo-microscopie pour étudier la structure locale des phases condensées de l’ADN. Nous avons montré comment le confinement modifie le comportement de l’ADN en solution et l’intérêt d’étudier ces effets compte tenu de son importance dans le contexte biologique. / By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these “hairy toroids”, part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

Le bactériophage T5 est un virus infectant E. Coli. L’assemblage et la maturation de sa capsidecomportent plusieurs étapes critiques pour la formation des virions lors du cycle infectieux.Parmi ces étapes, j’ai étudié les processus d’expansion et de décoration de la capside de T5.L’expansion implique d’importantes réorganisations conformationnelles des 775 sous-unités de laprotéine pb8 composant la capside, conduisant au doublement du volume de la capside qui peutalors contenir le génome du phage. J’ai déterminé des conditions physico-chimiques permettantd’induire l’expansion de la capside in vitro, puis j’ai effectué des expériences de SAXS résoluesdans le temps montrant que l’expansion est un processus hautement coopératif, qui conduit, en uneétape, à un état final remarquablement stable. D’autre part, j’ai réalisé une étude fonctionnelle de laprotéine de décoration pb10, montrant que sa fixation est un marqueur de l’expansion. Enfin l'étudestructurale de pb10 menée par SAXS a permis de déterminer un modèle à basse résolution de sonenveloppe moléculaire. / Bacteriophage T5 is a virus infecting E. Coli. Its capsid assembly and maturation include severalcritical steps leading to the formation of new virions during the infectious cycle.During my thesis I focused on the expansion and decoration of T5 capsid. Expansion consists inlarge conformationnal reorganizations of the 775 capsid protein subunits, yielding a two-foldincrease of the capsid volume and allowing it to accomodate the full-length genome. I haveascertained physicochemical conditions that trigger in vitro expansion of the capsid, and using atime-resolved SAXS study, I showed that expansion is a higly cooperative two-state process leadingto a remarkably stable final state. I have also carried out a functional study of the decoration proteinpb10, showing that it only binds to expanded proheads. Finally, a low resolution model of pb10 wasdetermined by SAXS.

The causative agent of the most devastating honeybee disease, American foulbrood (AFB), is the spore-forming bacterium Paenibacillus larvae. To prevent AFB outbreaks beekeepers prophylactically treat their hives with antibiotics even though it decreases the overall health of uninfected hives. A new treatment for AFB is needed due to recent legislation against using antibiotics, antibiotic resistance developing in P. larvae, and the resilience of P. larvae spores. Bacteriophages, or phages, are an attractive alternative to traditional antibiotics because of their specificity and ability to evolve alongside their target bacterium. In this study, two phage cocktails were developed for the treatment of AFB. The first cocktail was comprised of Brevibacillus laterosporus phages. B. laterosporus is a commensal microbe in most honeybee guts. When treated with B. laterosporus phages, B. laterosporus is induced to produce an antimicrobial toxin to which P. larvae is highly sensitive. Treating AFB infected hives with B. laterosporus phages was able to clear active infections at a rate of 75% as opposed to untreated hives that did not recover. However, B. laterosporus phages did not clear latent P. larvae spores and recovered hives relapsed after treatment. The second cocktail was comprised of P. larvae phages and hives treated with the second cocktail recovered at a rate of 100%, protected 100% of at-risk hives, and treated hives did not relapse with AFB suggesting neutralization of P. larvae spores. A P. larvae phage used in the second cocktail was examined to identify any spore-phage interactions. Results from modified plaque assays, fluorescence from FITC-labeled phages bound to spores, and electron microscopy images all confirm that phages bind to P. larvae spores. Phage therapy for the treatment of AFB is an exciting avenue not only as an alternative to chemical antibiotics, but rather a treatment that can neutralize P. larvae spores.

Brevibacillus laterosporus (B. laterosporus) is a pathogen difficult to distinguish from Paenibacillus larvae (P. larvae), and contributes to Colony Collapse Disorder (CCD) of honeybees. To develop a biocontrol agent to limit its presence, bacteriophages were isolated from Utah County soil samples and used to infect B. laterosporus isolated from Utah County honey and larvae samples. Since CCD is prevalent in Utah beehives, bacteriophage that infect and lyse B. laterosporus may be isolated and characterized. Pathogens were isolated from soil samples, and 16S rRNA gene tests initially identified the strains as P. larvae. Bacteriophages were isolated, purified, and amplified sufficiently to obtain images by electron microscope and genome sequencing by 454 pyrosequencing. Genomes were annotated with DNA Master, a Multiple Document Interface (MDI) program. Open reading frames (ORF's) were compared to the National Center for Biotechnology Information's (NCBI) database of primary biological sequence information via the Basic Local Alignment Search Tool (BLAST) algorithm. Later testing determined the pathogen to actually be B. laterosporus. Plaques demonstrated lytic activity, and electron microscopy revealed bacteriophages of the myoviridae family. The five sequenced genomes were composed of linear dsDNA ranging from 45,552 to 58,572 base pairs in length, 92 to 100 genes per genome, and a 38.10% to 41.44% range of G + C content. Discovering and describing new bacteriophages is a reasonably reproducible process and contributes to appreciating the diverse relationships between bacteriophage, bacteria, and eukaryota. Scientific facilitation of the bacteriophages role in limiting detrimental bacteria may contribute as an adjunctive therapy for CCD.

Mass spectrometry has emerged as having a vital role in various applications to biochemical fields. In this thesis, we have utilized a variety of mass spectrometry techniques for both bacteriophage proteomics and colostrum and milk lipidomics studies. Our first study was the proteome characterization of Great Salt Lake bacteriophage NS01 with SDS-PAGE GEL to separate the viral proteins and high performance liquid chromatography (HPLC) coupled with an LTQ Orbitrap to identify the proteins after in-gel digestion. In this project, we have successfully identified 11 proteins with high confidence, p-values < 0.01, including coat protein gp88 with a coverage of 91% and tail protein gp86 with a coverage of 40.96%, which facilitated the classification of NS01 as a T7-like phage. Our second study was the discovery of colostrum and milk biomarkers that can be used to predict the likelihood of development of production-related metabolic diseases (PRMDs) in dairy cows through a lipidomics approach. In this study, an electrospray ionization, time-of-flight mass spectrometer was applied to lipid profiling, quantification and significant biomolecule selection. A Q-Star quadrupole, orthogonal time-of-flight mass spectrometer and an Agilent 6530 accurate-mass quadrupole/time-of flight mass spectrometer were both used for lipid biomarker fragmentation and identification. According to linear discriminative statistical modeling, three panels of biomarkers were defined. A combination of 2 milk lipid predictors, including DG18:0/18:0 and TG 18:0/18:0/18:1, provided PRMD predictions with 75.0% sensitivity at 90.0% specificity. A combination of 3 colostrum lipid predictors, including TG16:0/18:1/18:3, DG16:0/16:0 and C40H60NO, provided PRMD prediction with 90.0% sensitivity at 86.4% specificity. Furthermore, a combination of 7 colostrum and milk biomarkers, including calculated differences between 'shared' markers found to be significantly different in both colostrum and milk, provided a predictive sensitivity of 87.5% at a specificity of 100%. Thus, three panels of lipid biomarkers have been discovered in 1-4 day postparturient dairy cow colostrum and milk that can be used to predict resistance or susceptibility prior to onset of clinically apparent PRMDs. These novel lipids could be used as important diagnostic predictors in the future. Therefore, mass spectrometry based proteomics and lipidomics approaches have been efficient tools in the biochemical research described in this thesis.

A whey product has been formulated for use as a lactic starter medium. Phosphate-treated whey medium (PWM) has been shown to support growth of lactic cultures and prevent phage proliferation. Comparisons were made of a commercial phage inhibitory medium (PIM), reconstituted non-fat dry milk (NDM) and PWM. PWM inhibited all phages tested and stimulated starter growth. PWM was not as stimulating as PIM but was better than NDM. Good Cheddar cheese has been made using PWM. PWM is more economical than PIM but NDM is the most economical one if cheese yield is considered.

Marine animals live and thrive in a literal sea of microorganisms, yet are often able to maintain specific associations that are largely dictated by the environment, host immunity and microbial interactions. Animal-associated microbiomes include bacteria and viruses that vastly outnumber host cells, especially in the gut environment, and are considered to be integral parts of healthy, functioning animals that act as a metaorganism. However, the processes underlying the initial establishment of these microbial communities are not very well understood. This dissertation focuses on the establishment of a well-known developmental animal model, Ciona intestinalis (sea squirt), to study the establishment and maintenance of a stable gut microbiome. Generation of a new model for studying microbial colonization of the gut requires the ability to rear Ciona in the absence of microbes (i.e., germ-free). This dissertation describes the establishment of a germ-free technique for rearing Ciona and the methods utilized for bacterial exposure and colonization. Additionally, to determine the spatial structure of the gut microbiome, viral and bacterial communities within the three main gut compartments (stomach, midgut, hindgut) of Ciona from San Diego, CA, were assessed. The viral community was dominated by phages (viruses infecting bacteria), and numerous prophages (phages integrated into bacterial genomes) matching sequences found in bacteria belonging to the Ciona microbiome were detected within the active viral fraction. To determine the prevalence of prophages within the Ciona microbiome, a total of 70 bacteria cultured from the gut were tested, and 22 isolates were found to possess inducible prophages. When co-cultured with other bacteria, these induced prophages were capable of lytic infection of other members of the microbiome, often exhibiting broad host ranges. The dynamic interactions of gut bacteria and phages were explored further with the isolation and characterization of a novel Shewanella phage-host system from the adult Ciona gut. Lytic phage infection resulted in an increase in biofilm formation correlating with the release of extracellular DNA, a process that was also observed to a lesser degree in control cultures as a result of spontaneous prophage induction. Furthermore, addition of the Ciona immune protein VCBP-C to static cultures of this Shewanella sp. 3313 also enhanced biofilm formation; a similar phenomenon was noted in another bacteria, a Pseudoalteromonas sp. 6751. Interestingly, both of these isolates contained inducible prophages and binding of the VCBP-C protein to these lysogenic strains was found to influence prophage induction in vitro. Colonization of the gut in vivo also correlated with differential up-regulation of VCBP-C expression in germ-free animals and a subsequent induction of prophages. This dissertation makes an important contribution to the symbiosis field by developing a new model system in which novel aspects of host-microbe interactions can be investigated. The discovery that an innate immune effector can influence bacterial biofilms and result in the induction of prophages capable of lytic infection of other co-occurring bacteria reveals a previously unrecognized intersection between secretory immune molecules and phages in shaping the microbiome. These findings establish Ciona as a relevant and tractable model for studying trans-kingdom interactions during colonization of the gut epithelium.