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Viruses are the most abundant biological entities in the ocean and are believed to contribute to nutrient cycling, bacterial diversity, and horizontal gene exchange. However, little is known about the relationship between temperate phages and their hosts in marine environments. In this thesis, phage-host systems from the Gulf of Mexico were used to study the influence of temperate phages in bacteria. PhiHAP-1 is a temperate myovirus induced with mitomycin C from Halomonas aquamarina isolate. The genome of this phage was 39,245 nucleotides long and contained 46 predicted genes. Besides genes involved in lysogeny, PhiHAP-1 contained a protelomerase, which is responsible for resolution of telomeric ends in linear plasmid-like phages. Hybridization studies and PCR analysis indicated not only a lack of integration of the prophage in the host chromosome, but differences in genome arrangement between the prophage and virion forms of PhiHAP-1. These results suggest that PhiHAP-1 exists as a non-integrating linear phage with telomeric ends. Eleven pigmented Bacillus spp. isolates were examined for the occurrence of lysogeny and sporulation through induction with mitomycin C and decoyinine, respectively. The results from these experiments suggested a variety of interactions can occur between phages and their hosts, some of which may influence sporulation. The lysogenic strain B14905 had high frequency of sporulation and was selected for further analysis. The genome of B14905 contained 4 prophage-like regions, one of which was independently sequenced from an induced lysate. PCR and TEM analysis of a mitomycin C induced lysate indicated that two of these regions were inducible prophage, one was a defective phage, and one was a non-inducible phage remnant. One of the inducible prophages contained a transcriptional regulator that is hypothesized to be involved in regulation of host sporulation. The diversity of prophage and prophage-like elements found in B14905 suggest that the genetic diversity of phages in the oceans is vast. The studies of the temperate phages from H. aquamarina and Bacillus spp. isolates illustrates that integration of molecular, genomic, and function studies can be used to provide insight into the influence of prophage on host bacteria.

Thesis (Ph. D.)--Illinois State University, 1995. / Title from title page screen, viewed May 12, 2006. Dissertation Committee: Radheshyam K. Jayaswal (chair), Brian J. Wilkinson, Alan J. Katz, Herman E. Brockman, Anthony J. Otsuka. Includes bibliographical references (leaves 112-118) and abstract. Also available in print.

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.

Cells switch phenotypes or behaviors to adapt to various environmental stimuli. Often there are multiple alternative phenotypes, hence a cell chooses one phenotype among them, a process which we term a ``decision switch'. At the cellular level, decision switches are governed by gene regulation, hence they are intrinsically stochastic. Here we investigate two aspects of decision switches: how copy number of genetic components facilitates multiple phenotypes and how temporal dynamics of gene regulation with stochastic fluctuations affect switching a cell fate. First, we demonstrate that gene expression can be sensitive to changes in the copy number of genes and promoters, and alternative phenotypes may arise due to bistability within gene regulatory networks. Our analysis in phage-lambda-infected E. coli cells exhibit drastic change in gene expression by changing the copy number of viral genes, suggesting phages can determine their fates collectively via sharing gene products. Second, we examine decision switches mediated by temporal dynamics of gene regulation. We consider a case when temporal gene expression triggers a corresponding cell fate, and apply it to the lysis-lysogeny decision switch by phage lambda. Our analysis recapitulates the systematic bias between lysis and lysogeny by the viral gene copy number. We also present a quantitative measure of cell fate predictability based on temporal gene expression. Analyses using our framework suggest that the future fate of a cell can be highly correlated with temporal gene expression, and predicted if the current gene expression is known.

Phages are the most abundant components of the marine environments and can control host abundances. The severity of viral infections may depend on whether phages are lytic, lysogenic, or chronic, which can be influenced by host activity and by environmental conditions. Lysogeny remains the least understood process. Knowledge of virioplankton dynamics and their life strategies in the Red Sea remain unexplored. In this Ph.D. research we aimed to quantify virioplankton abundance, the variability on viral and bacterial dynamics, and to investigate the occurrence of lytic and lysogenic phages in the Red Sea. Accordingly, we used the flow cytometric technique to enumerate viral and bacterial abundances in the coastal pelagic area during two years of sampling and in the coastal lagoon waters for one year, together with water column distribution in open Red Sea waters. We conducted incubations of natural microbial communities in the laboratory to induce lysogenic bacteria by using the chemical mutagenic mitomycin C. We also explored the influence of host abundance, temperature, and ultraviolet radiation on viral dynamics and lysogeny. Our results showed that abundances of virses in the Red Sea ranged from 106 to 107 virus-like particles per mL, and bacteria ranged from 104 to 105 cells per mL. We observed a large variability i the values of virus-to-bacterium ratios, and lower values of viral production to those for temperate coastal waters and relatively close to values reported in other oligotrophic areas. Although the lytic phase was prevalent, lysogeny was detected when bacterial abundances decreased. We determined inducible lysogenic bacteria from undetectable to ~56% in the coastal Red Sea, although we found a lower maximum of 29.1% at a eutrophic coastal lagoon. The decay rates of viruses were influenced by UVB exposure, suggesting their susceptibility to solar radiation. Exposure to UVB radiation-induced prophage varied between 4 and 34%. Our findings identified the significant role of viral infections in controlling bacterial abundance and the importance of both lytic and lysogenic phases in the Red Sea waters. This study contributes to the understanding of lysogeny in marine phages.

Les bactériophages sont de puissants prédateurs bactériens. Leur développement est généralement redouté dans les industries agro-alimentaires mettant en oeuvre des fermentations, car les phages sont responsables d’accidents de fabrication affectant la qualité finale du produit. Leur impact lors de la vinification est moins bien défini. Le procédé comporte une étape de fermentation malolactique (FML), qui est assurée par la bactérie lactique Oenococcus oeni. La maîtrise de la FML est un moyen efficace pour protéger la qualité et la typicité des vins, vecteurs de commercialisation. Jusqu’à présent, cette étape fondamentale du processus de vinification n’est pas toujours maîtrisée. L’explication majeure réside dans l’insuffisance de la biomasse bactérienne endogène, liée aux conditions physico-chimiques difficiles du milieu. Des solutions visant à conduire rapidement la FML sont disponibles, comme l’inoculation de souches commerciales de O. oeni. Ces stratégies n’offrent toutefois pas une totale garantie de succès, et des retards ou non déclenchements de FML sont toujours observés. Ces situations inexpliquées amènent à s’interroger sur l’impact d’autres paramètres sur la fermentescibilité malolactique. L’objectif de cette thèse était d’évaluer la présence et la diversité des bactériophages antagonistes de la bactérie lactique O. oeni présents dans l’écosystème. La diversité des prophages présents dans le pangénome de O. oeni a été explorée. La lysogénie est fréquente dans l’espèce. Quatre groupes de prophages ont été identifiés sur la base de la séquence de l’intégrase, et du site de recombinaison site-spécifique utilisé. La pertinence de la classification établie a été vérifiée sur un panel de 40 phages isolés de moûts et de vins. Nos travaux suggèrent que la lysogénie est un moyen pour O. oeni de résister aux stress et aux phages, grâce à la présence de mécanismes de résistance sur les génomes prophagiques. La stabilité de la lysogénie lors de l’inoculation de souches lysogènes dans le vin et la possible libération de dérivés lytiques sont deux paramètres à prendre en compte lors des FML spontanées, et lors de l’inoculation de levains malolactiques. Ils sont susceptibles de moduler quantitativement et qualitativement la population. / Bacteriophages are responsible for the predation of bacteria. They pose an ever-present threat to the food industries because they invade and destroy the starter and affect production. Their destructive potential is currently difficult to establish during spontaneous production of wine. Malolactic fermentation (MLF) represents a main stage in the winemaking process, and is essentially driven by the lactic acid bacterium (LAB) Oenococcus oeni. A rapid and efficient FML is essential to optimize wine quality and typicity, as sales rely on top-quality products. Up to now, this essential step is not controlled, and this results from the limited growth of MLF bacteria in wine, due to stressing conditions. Inoculation of commercial bacterial starter cultures is a strategy to improve MLF control, and will allow a rapid and complete fermentation. However, despite these evolutions, sluggish or complete failures of MLF are still reported by wine farmers, either during spontaneous or directed fermentations. Such cases suggest that additional factors need to be identified. The outline of this thesis was to demonstrate the occurrence of phages infecting O. oeni in the ecosystem and provide essential information regarding phage diversity. We analyzed prophage diversity through comparative genomics and demonstrated that lysogeny is widespread. Four prophage groups were identified according to the integrase gene sequence and attachment site used for site specific recombination. The relevance of the classification scheme was verified through the analysis of a panel of 40 phages isolated from wine and must. We suggest that lysogeny helps O. oeni to cope with stress and phage attack, through the presence of specific anti-phage mechanisms harbored by the prophages. The stability of lysogens during inoculation in wine, and the possible release of lytic particules have to be considered during spontaneous or directed MLF. They are expected to shape the population.

Les bactériophages (ou phages) sont des prédateurs de bactéries et sont redoutés dans les productions agro-alimentaires mettant en œuvre des fermentations. En œnologie, la transformation du jus de raisin en vin fait appel à différents types de fermentation. La fermentation alcoolique est réalisée par des levures, et peut être suivie par une fermentation malolactique (FML), notamment pour les vins rouges, afin d’améliorer la stabilité microbiologique et les qualités organoleptiques du produit. La bactérie lactique OEnococcus oeni (O. oeni) appartient à la famille des Leuconostocaceae et est l’acteur principal de la FML. Des souches d’O. oeni sont utilisées comme levain malolactique, et inoculées dans le vin pour mieux maitriser les fermentations. O. oeni rencontre dans son environnement des phages spécifiques appelés oenophages. Toutefois, bien que la présence de ces oenophages ait été constatée, leur diversité reste à ce jour peu explorée, tout comme leurs implications dans l’élaboration du vin. Une fréquence élevée de la lysogénie a été observée dans l’espèce et parmi les levains commercialisés. Les risques associés a la présence de phages ou à la lysogénie sont des paramètres peu abordés dans la filière. Afin de répondre à ces interrogations, dans un premier axe, la diversité des oenophages a été étudiée en isolant des phages à partir d’une large collecte d’échantillons œnologiques menée dans le sud-ouest de la France. L’analyse d’échantillons de différents types de vin, de différents cépages, collectés à différentes étapes de la vinification nous a permis de mettre en lumière une diversité génomique des oenophages non suspectée. Nous avons initié le développement de nouveaux outils moléculaires pour étudier la dynamique des populations bactériennes et phagiques dans le contexte œnologique. Ainsi, une première approche par Digital Droplet PCR a été utilisée pour détecter et quantifier les populations lysogènes. Dans un deuxième axe, l’interaction phage-hôte en présence de composés phénoliques du vin a été étudiée. Les travaux suggèrent que la croissance d’O. oeni en présence de certains flavonols et acides phénoliques réduit la capacité d’adsorption des phages sur leur hôte. / Bacteriophages are viral predators of bacteria and a major concern in fermentations involved in food processing industry. In winemaking, transformation of grape juice into wine involves different types of fermentations. Alcoholic fermentation is driven by yeasts, and can be followed by malolactic fermentation (MLF), especially for red wines, which can improve microbial stability and sensorial quality of wine. The lactic acid bacterium OEnococcus oeni (O. oeni), member of Leuconostocacae family, is generally responsible for the MLF process. Strains of O. oeni are also used as starters to master MLF. O. oeni encounters specific phages called oenophages. Even though the presence of oenophages has been observed, their diversity remains poorly investigated, just like their implications in winemaking. However, high frequency of lysogeny has been observed among O. oeni strains and starters. Risks linked with the presence of phages and lysogeny are questioned in the sector. In the first part of this thesis, oenophages diversity has been studied with the isolation of a large number of phages during the collection of a broad range of oenological samples in South West France. Analysis of samples coming from different wine types and varieties, collected at different steps of winemaking enabled us to highlight an underestimated genomic diversity. We also initiated the development of new molecular tools to study population dynamics among phages and hosts in the winemaking context. Thus, a first approach by Digital Droplet PCR has been used to detect and quantify lysogenic strains. In the second part, phage-host interactions in the presence of wine phenolic compound were investigated. Our results suggest that growth of O.oeni cells in the presence of particular flavonols and phenolic acids reduces adsorption capacities of phages on their host.

Les prophages sont des vecteurs majeurs de l’évolution des génomes bactériens et ont des rôles divers dans le processus adaptatif de leurs hôtes et peuvent leur apporter un avantage sélectif. Au cours de l’évolution, certains gènes prophagiques peuvent être perdus, notamment ceux codant pour des protéines du cycle lytique. Cependant, alors que certains de ces prophages défectifs sont capables de s’exciser, ils sont maintenus dans le génome de l’hôte, suggérant une pression sélective pour les conserver. C’est le cas du prophage défectif KplE1 chez E. coli K12. Dans l’équipe, des travaux ont mis en évidence que le maintien en lysogénie de différents prophages était sous le contrôle du terminateur de la transcription bactérien Rho. Afin d’identifier de nouveaux facteurs de l’hôte impliqués dans le maintien des prophages, j’ai développé un crible génétique qui m’a permis d’identifier plusieurs candidats impliqués dans le métabolisme général, la détoxification du NO ou qui appartiennent à un autre prophage défectif. Mon travail a été de discriminer lesquels de ces candidats jouaient un rôle significatif dans le maintien des prophages. Sur les trois gènes impliqués dans la détoxification du NO, seule l’expression de norV ou norW permet le maintien de KplE1. NorV réduit le NO et cette réduction nécessite l’utilisation d’un électron généré par l’oxydation du NADH par NorW. J’ai pu également montrer que l’expression du gène norV permettait le maintien d’un autre prophage fonctionnel (HK620) partageant le même module de recombinaison spécifique de site que KplE1. L’ensemble de mes résultats montre qu’il existe un lien co-évolutif important entre les prophages et leurs hôtes. / Prophages play recognized roles in their host genomes evolution and adaptation to variable ecosystems. They can provide to their host selective advantages that increase their competitiveness. Upon evolution, some prophage genes can be lost, especially those coding for lytic cycle capacity. While some of the defective prophages are perfectly competent for excision, they prove to be maintained in bacterial genomes, suggesting the involvement of a selective pressure. This is the case for our defective prophage model: KplE1 in E. coli K12. Previous work in our laboratory demonstrated that lysogeny maintenance of various prophages was controlled by Rho which is the bacterial transcription termination factor. In order to identify new host factors involved in prophage maintenance, I developed a genetic screen. This screen allowed me to identify candidate genes involved in bacterial general metabolism, in NO detoxification and also some genes that belong to another defective prophage. I determined which candidate genes actually played a role in KplE1 maintenance. Among the three genes involved in NO detoxification, I showed that norV or norW individual expression allowed KplE1 maintenance. NorV reduces NO and this reduction needs an electron produced by NorW NADH oxidation. I also showed that norV expression allowed the maintenance of another functional prophage (HK620) that shares the same site specific recombination module as KplE1. Together, my results illustrate the coevolution between prophages and their hosts.