The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection. Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

Bacteriophages affect bacterial evolution, pathogenesis and global nutrient cycling. They are also the most numerous and diverse group of biological entities on the planet [1, 2, 3, 4, 5, 6]. Members of the Lambda phage family share a similar genetic organization and control early gene expression by suppressing transcription, a process known as antitermination. Transcription antitermination in Lambda is mediated by a phage-encoded protein whereas in lambdoid phage HK022, antitermination is mediated by a phage-encoded RNA molecules. Recent results suggest that another bacteriophage called HK639 also appears to use RNA-mediated antitermination. To characterize this newly identified phage we generated site directed mutations and identified where the phage integrates into the chromosome of its bacterial host.

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types. Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection.Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate. This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

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 les virus spécifiques des bactéries. Ils sont considérés comme les entités biologiques les plus abondantes de la biosphère (1031 au total). Une grande partie des bactériophages sont dits tempérés de part leur propriété à intégrer leur génome dans celui de leur hôte et à s'y maintenir en état de réplication passive appelé lysogénie. Les gènes de prophages apportent de nouvelles propriétés à l'hôte via la conversion lysogénique. De nombreux prophages défectifs et fonctionnels sont maintenus dans les génomes bactériens. Nous avons émis l'hypothèse que des stratégies de maintien aient été sélectionnées pour maintenir cette source de gènes, même si elle est potentiellement dangereuse car les prophages peuvent être induits dans des conditions de stress.Nos résultats suggèrent que le maintien de la lysogénie d'une catégorie de prophages, qui présente une organisation génétique atypique du module de recombinaison spécifique de site, est sous le contrôle du facteur de terminaison de la transcription Rho. Pour ces prophages, qu'ils soient défectifs ou fonctionnels, leur induction par inactivation de Rho, fait intervenir une nouvelle voie d'induction lytique indépendante de la voie classique via la réponse SOS.Ces interactions hôtes-virus reflète la coévolution de ces microorganismes, qui permet l'acquisition de gènes via le transfert horizontal tout en contrôlant l'expression des gènes délétères. Ceci permet l'acquisition de nouvelles propriétés et l'adaptation de l'hôte à différentes conditions environnementales. / Bacteriophages are the most abundant biological entities in the biosphere. A majority of them are temperate phages that are able to integrate their genome into the host and replicate passively in a lysogenic state. Hosts frequently benefit from such massive gene acquisition through lysogenic conversion. As prophages may be beneficial to their hosts, we hypothesize that hosts adapted strategies for maintaining that gene source. Since prophages integrate into and excise from the host chromosome through site-specific recombination (SSR), we investigated whether regulation of SSR at the level of gene expression could be involved in the maintenance process. Our results suggest that lysogeny maintenance of a class of prophages, which all share a same unusual genetic organization, are controlled by the transcription termination factor Rho. Rho is not only involved in horizontally acquired gene silencing but also in prophage maintenance, which can be seen as an adaptation of the host to maintain prophage genes. For these prophages, whether defective or functional, their induction by the inactivation of Rho, involves a new pathway of lysogeny escape, which is independent of the classical pathway via the SOS response. This newly characterized interaction reflects the coevolution of host and viruses, which allows the acquisition of genes, and thus new properties, via horizontal transfer, while controlling the expression of deleterious genes.

<p>Ever since the invention of antibiotics nearly a century ago,the threat of antibiotic resistance has been gradually increasing. As antibiotics are continually prescribed, the rate at which bacteria are becoming resistant to antibiotics is increasing as well. It is projected that antibiotic resistance is one of the largest threats to overall world health, and bacteriophage therapy is one of the leading strategies to combat it. Bacteriophages are viruses that infect and kill specific host bacteria andcan potentially be utilized to kill desired bacteria causing infections that are resistant to antibiotics.</p> <p><br></p> <p>The purpose of this research project is to learn more about the bacteriophage-host interaction through mass spectrometry and bioinformatic tools. This is done through the analysis of proteins and lipids that are produced when the bacteriophage infects the host bacteria. The growth curve of a Passage One From Frozen (P1FF) and a Passage Two From Frozen (P2FF) sample of Mycobacterium smegmatiswas calculated to determine to optimum time for bacteriophage infection. Twobacteriophages were chosen, PotatoSplit and Zalkecks, the Mycobacterium smegmatis samples were infected, samples collected, and mass spectrometry performed. A large portion of this research project is based on the analysis of the proteins and lipids that are produced during each bacteriophage’s infection. Proteomic and lipidomic strategies can be implemented to understand more about the bacteriophage-host interaction and discover any proteins and lipids that are produced at varying timepoints throughout the inoculation process. Bioinformatic tools can then be used to understand the potential functions of each protein or lipid and potential functions or applications of the bacteriophage in general, including the pathogenicity of each bacteriophage.</p> <p><br></p> <p> Determined from proteomic and lipidomic analysis, a list of all proteins and lipids found within each phage infected sample was made. An important trend discovered is that more phage proteins were expressed at later times during the phage infection –Hour 7 and Hour 10, whereas more bacterial proteins were expressed initially –Hour 0 and Hour 3. A case study to investigate the usage of different intensity types produced from mass spectrometry was completed. Overall, it was determined that both the number of phageproteins and bacterial proteins can differ depending on if LFQ or iBAQ intensity type data was used. Correlation between proteins and lipid ontology classes was performed and shows whether groups of lipids are upregulated or downregulated at 14each time point. Understanding the function of lipid ontology groups and the type of regulation provides insight into how the phage or bacteria are potentially using the lipids produced. Some of the main findings include lipids that are involved in bacterial defense mechanisms/energy usage increase over time. Some correlation trends were not consistent across the different bacteriophages, which can be contributed to the different phage life cycles  and therefore different phage-host interactions. Further investigation should be performed to determine the specific biological function of proteins and lipids to confidently make claims about potential applications for each phage. Also, further investigation should be performed to understand if the differences in results between bacteriophage PotatoSplit and Zalkecks are due to the varying life cycles.</p>

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