Viruses have an estimated global population size of 10 to the 31st, with a significant proportion found in the marine environment. Viral lysis of bacteria affects the flow of carbon in the marine microbial food web, but the effects of lysogeny on marine microbial ecology are largely unknown. In this thesis, factors that influence the control of lysogeny were studied in both the phiHSIC/Listonella pelagia phage-host system and in bacterioplankton populations in the Gulf of Mexico. Using macroarrays dotted with phiHSIC amplicons, viral gene expression over the course of a synchronous infection experiment was measured. Early, middle, late, and continually expressed genes were identified, and included open reading frames 45, 28, 18 and 17, respectively. Viral gene expression in cultures of the HSIC-1a pseudolysogen grown in low and normal salinity media was also analyzed. Overall, levels of viral gene expression were higher in the 39 ppt treatment as compared to the 11 ppt tre atment for most ORFs. In the 11 ppt treatment, free phage concentrations were one to two orders of magnitude lower than the 39 ppt treatment while intracellular phage concentrations were one-fold lower. Therefore, at low salinities, expression of phiHSIC genes is repressed resulting in a lysogenic-like state, while at 39 ppt, lytic interactions dominated. Few viral genes were highly expressed at low salinity, suggesting that repression of viral genes was controlled by host genes. Samples from the eutrophic Mississippi River Plume and the oligotrophic Gulf of Mexico were analyzed for lytic phage production and occurrence of lysogeny. Significant lytic viral production was only observed three stations, none of which were located within the MRP. This signifies that system productivity is not an accurate predictor of viral productivity. The lysogenic fraction was also inversely correlated to bacterial activity, which decreased with depth. These findings support the hypothesis that lysogeny is a survival mechanism for phages when host cell density is low or when conditions do not favor growth. A unifying theme from these experiments was that lytic processes dominated when bacterial growth conditions were optimal, while lysogeny was observed at unfavorable growth conditions or environmental stress (low salinity).

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, prodigiosin and a carbapenem, and the plant cell wall degrading exoenzymes, pectate lyase and cellulase. A complex regulatory network controls production of prodigiosin, including a quorum sensing (QS) system, and the role of post-transcriptional regulation was investigated. It was hypothesized that Hfq-dependent small regulatory RNAs (sRNAs) might also play a role. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in other bacterial species. An S39006 ∆hfq mutant was constructed and in the mutants production of prodigiosin and carbapenem was abolished, while production of the QS molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, it was found that Hfq regulated the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in the ∆hfq mutant, and virulence was attenuated in potato and C. elegans. It was also shown that the phenotype of an hfq mutant is independent of its role in regulating the stationary phase sigma factor, rpoS. In order to define the complete regulon of Hfq and identify relevant potential sRNAs, deep sequencing of strand-specific cDNAs (RNA-seq) was used to analyse the whole transcriptome of S39006 WT and the ∆hfq mutant. The regulon of another post-transcriptional regulator, RsmA, also involved in regulating prodigiosin production, was investigated by performing RNA-seq on an rsmA mutant. Moreover, global changes in the proteome of the hfq mutant was analysed using an LC-MS/MS approach with isobaric tags for relative and absolute quantification (iTRAQ). This study confirms a role for Hfq in pathogenesis and the regulation of antibiotic production in S39006, and begins to provide a systems-level understanding of Hfq and RsmA regulation using a combination of transcriptomics and proteomics.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21687号 / 医博第4493号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 清水 章, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Die genomische Insel (GI) FhaGI 1 des Stammes Francisella hispaniensis (Fhis) AS02 814 kann sowohl in die tRNAVal integriert als auch als episomale Form vorliegen und kodiert für einen putativen Prophagen. Im Rahmen dieser Arbeit konnte durch Verwendung synthetisch hergestellter, verkürzter Varianten von FhaGI-1 gezeigt werden, dass die GI auf andere Francisella Spezies übertragbar ist. Die ortsspezifische Integration und Exzision der GI sind Integrase-abhängige Prozesse, die durch weitere regulatorische Gene beeinflusst werden. Die Identifizierung der GI FphGI 1 in drei F. philomiragia-Stämmen zeigt, dass die tRNAVal als Integrationsort für GIs in Francisella dient. Die vermutlich nicht funktionale Integrase von FphGI 1 ist wahrscheinlich die Ursache für das Fehlen einer episomalen Form der GI. Das Vorhandensein von GIs in Francisella liefert einen Hinweis darauf, dass horizontaler Gentransfer zwischen verschiedenen Francisella Spezies möglich ist. Auf Grundlage von FhaGI 1 wurden zwei Varianten eines Francisella- Phagenintegrationsvektors (pFIV1-Val und pFIV2 Val) generiert. Der FIV Teil der Vektoren bildet eine zirkuläre, episomale Form, die nach der Transformation in verschiedene Francisella Spezies ortspezifisch in die tRNAVal integriert. Es konnte gezeigt werden, dass die Vektoren für die Expression von Reportergenen sowie die Komplementation von Francisella Deletionsmutanten geeignet sind. Sie sind sowohl in vitro als auch während der Infektion von Wirtszellen ohne Selektionsdruck stabil und zählen zu den low-copy-Vektoren. Damit erweitern die FIV-Vektoren das Repertoire der vorhandenen Werkzeuge zur genetischen Manipulation von Francisellen. Da der Stamm Fhis AS02 814 für Untersuchungen nicht zur Verfügung stand, wurde FhaGI 1 synthetisch in zwei Hälften hergestellt, die jedoch bisher nicht zusammengeführt werden konnten. Damit ist eine Aussage darüber, ob es sich bei FhaGI 1 tatsächlich um einen funktionalen Prophagen handelt, bis jetzt nicht möglich / The genomic island (GI) FhaGI 1 of strain Francisella hispaniensis (Fhis) AS02 814 can exist as a circular episomal form or integrated into the tRNAVal gene and codes for a putative prophage. In this work small-sized variants of FhaGI 1 were used to show that the GI can be transferred to other Francisella species. The site-specific integration and excision of the GI are integrase-dependent processes that are influenced by further regulatory genes. The identification of the GI FphGI 1 in three F. philomiragia strains shows that the tRNAVal gene serves as an integration site for GIs in Francisella. The integrase of FphGI 1 is probably non-functional and hence presumably the reason for the missing episomal form of the GI. The presence of GIs in Francisella might be an indication that horizontal gene transfer between different Francisella species could be possible. Two variants of a Francisella phage integration vector (pFIV1 Val and pFIV2 Val) were successfully constructed based on FhaGI 1. The FIV Val part of the vectors integrates site-specifically into the tRNAVal after transformation into different Francisella species. It was demonstrated that the vectors can be used for the expression of reporter genes as well as for the complementation of Francisella deletion mutants. They remain stable without selective pressure during in vitro growth and during the infection of host cells and fall into the group of low-copy-vectors. The FIV Val vectors expand the repertoire of tools that can be used for the genetic manipulation of Francisella. As strain Fhis AS02 814 could not be obtained for further analysis, FhaGI 1 was synthetically generated in two halves which could not be joined so far. Consequently, it is not possible to state whether FhaGI 1 actually codes for a functional prophage.

Gene Transfer Agents (GTAs) are phage-like pthesiss that are produced by many alpha proteobacteria in late stationary growth phase and are capable of transferring chromosomal genes (termed "constitutive transduction"). Examination of alpha proteobacterial genomic sequences indicated widespread occurrence of GTA-like elements. The goal of this study was to investigate gene transfer potential of GTAs of marine alpha proteobacteria in culture as well as in natural marine environments. Another goal was to determine the potential of bacterial symbionts from zooxanthellae and coral to genetically transfer beneficial properties between symbionts. Ruegeria mobilis (ID 45A6) was isolated from cultures of the coral endosymbiotic dinoflagellate, Symbiodinium spp. A goal of the research was to determine if GTAs from this isolate have the capability of transferring genes to environmental recipients and have an impact on settlement of coral larvae. Little is known about coral settlement cues, yet there may be contributions from the extensive symbiotic relationship of coral reef-associated bacteria. Several gene transfer experiments in different environments were performed using transformed isolates of Ruegeria mobilis containing a transposon marker gene. Experiments were also performed using GTAs from the Ruegeria mobilis isolate to observe any impact GTAs have on coral larval settlement, using larvae from the brooding coral, Porites astreoides, and from the reef building coral, Montastraea faveolata. Gene transfer frequencies from statistically significant gene transfer experiments resulted in an average of 2.92 × 10-1 (transfer recipients to total viable population). Coral settlement experiments resulted in a statistically significant increase in larval settlement with the addition of GTAs for 80% of the executed experiments. The entire study has demonstrated that GTA-mediated gene exchange is much higher than any other mode of horizontal gene transfer and it has been established that these genes can be exchanged between bacterial taxa. GTAs can also have an impact on coral larval settlement mechanisms that are not yet completely understood. GTA-mediated beneficial gene exchange may be an important driver in adaptation to an evolving planet.

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.<p>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. <p>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. <p>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. <p>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.<p>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.<p>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.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

No description available.