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

Bacteriophage genomes show pervasive mosaicism, indicating that horizontal gene exchange plays a crucial role in their evolution. Phage genomes represent unique combinations of modules, each of them with a different phylogenetic history. Thus, a web-like, rather than a hierarchical scheme is needed for an appropriate representation of phage evolutionary relationships. Part of the virology community has long recognized this fact and calls for changing the traditional taxonomy that classifies tailed phages according to the type of genetic materials and phage tail and head/capsid morphologies. Moreover, based on morphological features, the current system depends on inspection of phage virions under the electron microscope and cannot directly classify prophages. With the genomic era, many phages have been sequenced that are not classified, calling for development of an automatic classification procedure that can cope with the sequencing pace. The ACLAME database provides a classification of phage proteins into families and assigns the families with at least 3 members to one or several functions. In the first contribution of this work, the relative contribution of those different protein families to the similarities between the phages is assessed using pair-wise similarity matrices. The modular character of phage genomes is readily visualized using heatmaps, which differ depending on the function of the proteins used to measure the similarity. Next, I propose a framework that allows for a reticulate classification of phages based on gene content (with statistical assessment of the significance of number of shared genes). Starting from gene/protein families, we built a weighted graph, where nodes represent phages and edges represent phage-phage similarities in terms of shared families. The topology of the network shows that most dsDNA phages form an interconnected group, confirming that dsDNA phages share a common gene pool, as proposed earlier. Differences are observed between temperate and virulent phages in the values of several centrality measures, which may correlate with different constraints to rampant recombination dictated by the phage lifestyle, and thus with a distinct evolutionary role in the phage population. To this graph I applied a two-step clustering method to generate a fuzzy classification of phages. Using this methodology, each phage is associated with a membership vector, which quantitatively characterizes the membership of the phage to the clusters. Alternatively, genes were clustered based on their ‘phylogenetic profiles’ to define ‘evolutionary cohesive modules’. Phages can then be described as composite of a set of modules from the collection of modules of the whole phage population. The relationships between phages define a network based on module sharing. Unlike the first network built from statistical significant number of shared genes, this second network allows for a direct exploration of the nature of the functions shared between the connected phages. This functionality of the module-based network runs at the expense of missing links due to genes that are not part of modules, but which are encoded in the first network. These approaches can easily focus on pre-defined modules for tracing one or several traits across the population. They provide an automatic and dynamic way to study relationships within the phage population. Moreover, they can be extended to the representation of populations of other mobile genetic elements or even to the entire mobilome. Finally, to enrich the phage sequence space, which in turn allows for a better assessment of phage diversity and evolution, I devise a prophage prediction tool. With this methodology, approximately 800 prophages are predicted in 266 among 800 replicons screened. The comparison of a subset of these predictions with a manually annotated set shows a sensitivity of 79% and a positive predictive value of 91%, this later value suggesting that the procedure makes few false predictions. The preliminary analysis of the predicted prophages indicates that many may constitute novel phage types. This work allows tracing guidelines for the classification and analysis of other mobile genetic elements. One can foresee that a pool of putative mobile genetic elements sequences can be extracted from the prokaryotic genomes and be further broken down in groups of related elements and evolutionary conserved modules. This would allow widening the picture of the evolutionary and functional relationships between these elements.

KplE1 est l’un des dix prophages présents sur le chromosome de la souche Escherichia coli K12. Nous avons montré in vivo que ce prophage est compétant pour s’exciser du chromosome bactérien bien qu’il soit incapable de former des particules virales et de lyser son hôte. Au laboratoire, nous avons identifié les protéines IntS (intégrase) et TorI (RDF), codées sur le prophage KplE1, et la protéine IHF (NBP) de l’hôte comme seules impliquées dans le mécanisme de recombinaison spécifique de site (RSS). Nous avons cartographié sur les régions attL et attR, les sites de fixations des protéines de recombinaison permettant l’assemblage de l’intasome, le complexe nucléoprotéique compétant pour la RSS. L’ensemble de ces sites ainsi que les gènes intS et torI qui chevauchent respectivement les régions attL et attR, ont permis de définir un module de recombinaison de type KplE1. Ce module est très conservé et se retrouve chez des phages infectant différentes souches d’E. coli et de shigella. Le modèle en terme de RSS est celui décrit pour les bactériophages de type λ. Cependant, le nombre et l’organisation des sites de recombinaison suggèrent que l’architecture de l’intasome de type KplE1 diffère de celle de λ. Nos résultats renforcent ainsi l’idée que l’assemblage de l’intasome est spécifique du module de RSS considéré même si, in fine, la réaction catalysée demeure similaire.En ce qui concerne l’expression des gènes intS et torI, le fait que ces gènes soient localisés à chacune des extrémités du prophage, rend ainsi impossible leur couplage transcriptionnel à partir d’un promoteur commun au moment de la commutation lyse/lysogénie, tel qu’il est connu pour les phages lambdoïdes. De part son orientation atypique sur attL, la présence de sites de fixations des protéines IntS et TorI au niveau du promoteur du gène intS, nous ont logiquement amené à étudier sa régulation. Nous avons ainsi montré que le gène intS est négativement régulé par son propre produit ainsi que par la protéine RDF TorI. Nos résultats in vivo et in vitro indiquent que l’efficacité de la réaction de recombinaison excisive est intimement liée à la quantité d’intégrase présente, pouvant alors justifier la raison d’être de ce contrôle strict de l’expression du gène intS. En parallèle, une approche in silico a révélé que cette orientation atypique du gène codant pour l’intégrase est largement répandue sur les génomes des prophages, nous amenant à généraliser ce mécanisme atypique de régulation négative de l’intégrase. / KplE1 is one of the 10 prophage region present on the Escherichia coli K12 chromosome. We showed in vivo that this prophage is fully competent to excise from the bacterial chromosome, although it is unable to form viral particles and lyse its host. In the laboratory, we have identified Ints (integrase) and TorI (RDF) proteins, encoded on the KplE1 prophage, and the host protein IHF (NBP) only involved in the mechanism of site-specific recombination (SSR). We have mapped on attL and attR regions, the binding sites of recombinant proteins for the assembly of the intasome, the nucleoprotein complex competent for SSR. All of these sites as well as intS and torI genes that overlap respectively attL and attR regions, have permit to define a KplE1 recombination module. This module is highly conserved and is found among phages infecting different E. coli and shigella strains. The model in terms of RSS is that described for λ bacteriophage. However, the number and organization of recombination sites suggests that the architecture of the KplE1 intasome differs from that of λ. Our findings reinforce the idea that the intasome assembly is specific to the SSR module considered even if ultimately the catalyzed reaction is similar.Regarding the intS and torI gene expressions, the fact that these genes are located at each end of the prophage, prevented the transcriptional coupling of these genes from a common promoter when the lysis/lysogeny switch occurs. Because of its atypical orientation on attL, and the presence of IntS and TorI protein binding sites that overlap its promoter region, we have logically studied the regulation of the intS gene. We have shown that intS is negatively regulated by both IntS and TorI proteins. Our in vivo and in vitro results suggest that the efficiency of the excision recombination reaction is closely related to the amount of this integrase, which can justify the strict control of the intS gene expression. In parallel, an in silico approach has revealed that the atypical orientation of the integrase gene is widespread in prophage genomes, leading us to generalize this atypical mechanism of negative regulation of integrase

<p>These studies are based on the isolation of ë replication defective mutants that had acquired multiple point mutations within ë replication initiation genes O and P in a cryptic prophage (Hayes et al., 1998). Each mutant cell arose after shifting wild type cells with a cI[Ts] cryptic ë prophage deleted for int-kil, and from ren into E. coli, from 30oC to 42oC. Derepression of the trapped cryptic prophage kills the host cells (designated as RK+). Rare colony forming units survive and were designated as RK- mutants. This led to a hypothesis that ë replication-triggered cell stress provokes mutator activity, i.e., increases the frequency of replication errors within the simultaneously replicating chromosome of the host E. coli cells. We tested this hypothesis by asking three questions: (1) Do unselected, untargeted (with no link to ë fragment) auxotrophic mutations appear within the RK- mutant population selected from RK+ culture cells? (2) Is replication initiation from the cryptic ë fragment, or, alternatively, just expression of one or more ë genes required for the appearance of the unselected auxotrophic mutations? (3) Do E. coli functions participate in the appearance of unselected auxotrophic mutations within the RK- mutant population? Our results indicate that auxotrophic mutations unlinked to the ë fragment appeared at high frequency within RK- mutants. RK- auxotrophs arising on rich medium were identified by screening the survivor clones for growth on minimal medium. The appearance of RK- auxotrophic colonies at high frequency (>1 per 100 RK- mutants) leads us to conclude that auxotrophic mutations arise during the independent selection for RK- mutants. Conditions that inhibited ë fragment induction fully suppressed the mutator phenotype. Mutation of host dnaB such that the helicase does not support replication initiation from the induced ë fragment completely suppressed host cell killing, but not the appearance of auxotrophic mutations. We asked if E. coli error-prone polymerases IV and V, or gene functions regulated as part of the host SOS response contributed to the provoked mutator phenotype and observed no close correlation. We demonstrated that the RK+ starting cells did not have a distinct intrinsic mutator activity in several ways, including moving the cryptic ë fragment to different E. coli host cells, blocking ë fragment induction by the addition of a cI+ plasmid to eliminate ë gene expression at high temperatures, and independent assays for spontaneous rifampicin resistance. We found that the induced mutator phenotype associated with the appearance of untargeted auxotrophs was linked to the expression of lambda gene P, and did not require replication initiation from the cryptic ë prophage. We also found that the mutator phenotype of the induced cryptic ë fragment increased the frequency of rifampicin resistant colonies among the RK- mutant population. </p>

<p>These studies are based on the isolation of ë replication defective mutants that had acquired multiple point mutations within ë replication initiation genes O and P in a cryptic prophage (Hayes et al., 1998). Each mutant cell arose after shifting wild type cells with a cI[Ts] cryptic ë prophage deleted for int-kil, and from ren into E. coli, from 30oC to 42oC. Derepression of the trapped cryptic prophage kills the host cells (designated as RK+). Rare colony forming units survive and were designated as RK- mutants. This led to a hypothesis that ë replication-triggered cell stress provokes mutator activity, i.e., increases the frequency of replication errors within the simultaneously replicating chromosome of the host E. coli cells. We tested this hypothesis by asking three questions: (1) Do unselected, untargeted (with no link to ë fragment) auxotrophic mutations appear within the RK- mutant population selected from RK+ culture cells? (2) Is replication initiation from the cryptic ë fragment, or, alternatively, just expression of one or more ë genes required for the appearance of the unselected auxotrophic mutations? (3) Do E. coli functions participate in the appearance of unselected auxotrophic mutations within the RK- mutant population? Our results indicate that auxotrophic mutations unlinked to the ë fragment appeared at high frequency within RK- mutants. RK- auxotrophs arising on rich medium were identified by screening the survivor clones for growth on minimal medium. The appearance of RK- auxotrophic colonies at high frequency (>1 per 100 RK- mutants) leads us to conclude that auxotrophic mutations arise during the independent selection for RK- mutants. Conditions that inhibited ë fragment induction fully suppressed the mutator phenotype. Mutation of host dnaB such that the helicase does not support replication initiation from the induced ë fragment completely suppressed host cell killing, but not the appearance of auxotrophic mutations. We asked if E. coli error-prone polymerases IV and V, or gene functions regulated as part of the host SOS response contributed to the provoked mutator phenotype and observed no close correlation. We demonstrated that the RK+ starting cells did not have a distinct intrinsic mutator activity in several ways, including moving the cryptic ë fragment to different E. coli host cells, blocking ë fragment induction by the addition of a cI+ plasmid to eliminate ë gene expression at high temperatures, and independent assays for spontaneous rifampicin resistance. We found that the induced mutator phenotype associated with the appearance of untargeted auxotrophs was linked to the expression of lambda gene P, and did not require replication initiation from the cryptic ë prophage. We also found that the mutator phenotype of the induced cryptic ë fragment increased the frequency of rifampicin resistant colonies among the RK- mutant population. </p>

No description available.

Part 1: Prophage induction has been demonstrated to be a sensitive indicator for a wide variety of toxic and mutagenic compounds and, as a consequence, has been utilized for biologically based carcinogen screenings. Fourteen marine bacterial isolates were screened for development into the Marine Prophage Induction Assay (MPIA), for marine samples. The selected isolate (P99-4S3) was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. This isolate demonstrated a log-linear response to increasing dose of mutagens, and sensitivity to known environmental contaminants. Field-testing of the assay over two years demonstrated the MPIA would be a useful screening tool for environmental contamination. Part 2: The observed resistance of natural populations of Synechococcus to viral infection may be due to lysogeny with associated homoimmunity. A thirteen-month study of lysogeny in natural populations of Synechococcus demonstrated that lysogeny does occur and exhibits a seasonal pattern. Experiments were performed along a transect of the Mississippi River plume, which provided a variety of ambient nutrient regimes for comparison of lysogeny in Synechococcus. Nutrient amendments did not enable induction and often led to a decrease in viral production. Lysogeny in Synechococcus was primarily correlated with ambient host and cyanophage abundance. Cross-infectivity studies demonstrated cyanophage isolates possess variable virulence. The 35 isolates were examined by transmission electron microscopy (TEM), with 33 identified as myoviruses and two as podoviruses. This dominance of myovirus lytic cyanophage is consistent with prior observations. Twenty-five Synechococcus isolates were screened for prophage induction utilizing the inducing agent Mitomycin C. Eleven isolates demonstrated a statistically significant increase in virus-like particles (VLP’s) in treatment samples. No correlation was observed between their resistance to lytic viral infection and prophage induction. Isolate P99-14, with consistently high levels of prophage induction, was investigated further. In contrast to lytic cyanophage, the induced cyanophage is non-tailed. Differential staining and nuclease digestion experiments indicate that the induced particle contains single-stranded DNA. Environmental conditions potentially leading to prophage induction were investigated with Synechococcus cultures and natural populations. The isolate P99-14 demonstrated that high, continuous light caused prophage induction. Natural populations determined that shifts in salinity, temperature and phosphate are not triggers of prophage induction.

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.

Le but de ce travail était d'identifier les fonctions régulées par quorum-sensing (QS) chez la bactérie phytostimulatrice Azospirillum. Les effets phytobénéfiques in vitro des souches B518 et TVV3 (isolées du riz) ne sont pas altérées par l'inactivation des molécules signal impliquées dans le QS. La combinaison d'une approche ciblée et d'une approche globale par protéomique montre que le QS régule des fonctions liées à l'adaptation à la plante, notamment à la colonisation racinaire chez B518. Chez TVV3, aucune fonction régulée par QS n'a pu être identifiée mais les gènes impliqués dans le QS sont localisés dans un environnement atypique, constitué de gènes prophagiques. La mise en évidence d'un prophage chez TVV3 a conduit à la caractérisation de phages tempérés chez dix autres souches et au séquençage du premier génome d'un bactériophage isolé d'Azospirillum. Ce travail montre que la régulation de type QS est souche spécifique et révèle la prévalence des phages chez Azospirillum.

3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce prophages, cultures of Clostridium scatologenes ATCC 25775 were exposed to an effective concentration of mitomycin C at 2μg/ml and 5μg/ml. Induction with temperature was performed at 42ºC and 55ºC. Bacteriophage liberation, determined by a decrease in optical density was not observed in response to mitomycin C or by different growth temperatures. Nineteen environmental samples were tested for the presence of a bacteriophage that could infect Clostridium scatologenes ATCC 25775. The first cycle of enrichments suggested a decrease in cell density, consistent with the presence of a bacteriophage but this was not observed in further iterations. Plaque assays were performed to confirm the presence of phage, but no plaques were observed. Although, different experimental conditions were tested, a transducing bacteriophage capable of infecting Clostridium scatologenes ATCC 25775 was not isolated. Transposons have been successfully used to generate mutants in Clostridium difficle. Therefore, we attempted to introduce transposons Tn5 and Tn916 into Clostridium scatologenes ATCC 25775 using electroporation. Transposon mutagenesis using Tn916 did not yield antibiotic resistant colonies. In contrast, commercially available transposon Tn5 gave antibiotic resistant colonies. However, further screening of the colonies using transposon specific primers in PCR reactions, did not yield any PCR product. We were unsuccessful in developing a genetic modification system in Clostridium scatologenes ATCC 25775 using bacteriophage or transposons.